The Molecules that Play Dr. Jeckel and Mr. Hyde in Aging, Alzheimer’s Disease, and Type II Diabetes

By James P Watson, with summary and contributions from Vince Giuliano

Introduction – GSK-3s – The “Dr. Jeckel and Mr. Hyde” Molecules

Have you ever heard of Glycogen Synthase Kinase 3 alpha or beta?  If you haven’t, don’t feel bad…..they do so much more than just inhibit glycogen synthesis.  If you are interested in what causes aging or Alzheimer’s disease, however, you better learn about GSK-3, because this is big….really big!  Why hasn’t GSK-3s received more press?  Maybe it is because no PR agent has been hired to “pitch” this poorly named enzyme.  So I am going to pretend I am a PR firm hired to develop an advertising campaign for GSK-3α and GSK-3β.  The first thing that PR firms do is to develop a catchy “tag line” to describe the GSK-3s.  One possibility would be “GSK-3: We control much more than Glycogen Synthesis” (That isn’t that catchy, but GSKs do control over 50 molecular pathways).  Most people don’t even know what glycogen is, so maybe a better tag line would be “GSK-3: the Master Molecular Multi-tasker” (every modern woman can relate to that!).  Another one that might get attention is the Great Satan Kinase, but actually that slights it’s good side.  For the engineers or science geeks who may read this blog, we have another PR slogan for GSK-3: “The Multipurpose Signal Amplifier and Multipurpose Signal Dampener”.  Even that technical mumbo-jumbo doesn’t really reflect how powerful the GSK-3 enzymes are, however.  For this reason, our PR firm has finally decided to give GSK-3s a “human face” -

The Molecular Dr. Jeckel and Mr. Hyde!  Image may be NSFW.
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They are your worst enemy and your best friend. They can throw a “monkey wrench” in all of the major molecular signaling cascades (like the Insulin/IGF pathway, the mTOR pathway, etc.).  They can keep you healthy or they can make you age. They can cause Alzheimer’s disease and Diabetes or they can protect you from these diseases.  In fact, because the way they function as amplifiers, GSK-3s can kill your cells by activating apoptosis pathways.  This is why they are both “good” and “bad” for you.

This is the first of what we expect to be a series of blog entries  on less-familiar but very important  and well-studied  molecules encountered in humans which have major impacts on aging and health – the Strange But Powerful Molecules Series.  GSK-3s have been studied in the cancer research community for over 30 years, but have largely been ignored in the aging research community despite their profound impacts on aging processes.  Additional posts in the series will be concerned with other lesser-known candidates of similar great importance, starting with the P66shc protein and with the non-coding gene known as ANRIL.  We characterize GSK-3 kinases, what they do, their impacts on biological pathways, why they are of critical importance for certain disease processes and aging, and about the ways they can be activated or turned off.

KEY POINTS

As is often the case, this blog gets quite complicated. So, we summarize some of the most important points here.

1.  The GSK-3s are important because they “signal amplifiers” that turn on genes or up regulate enzymes, but are also “signal dampeners” that turn off genes or down regulate enzymes. They have about 50 targets, approximately half of which are transcription factors and half are other (non-transcription factor) enzymes within the cell.  They have so many different impacts on the cell that they are best referred to as “molecular multi-taskers”.  They play important roles in multiple disease processes and in aging itself.
2. “In the pathways in which GSK-3 acts, it serves as a key regulator of normal cellular functions. However, when GSK-3 is dysregulated, it also has a key role in causing diseases such as diabetes, Alzheimer’s disease, bipolar disorder and cancer(ref).”  GSK-3s now are well documented to play key roles also in Chronic Traumatic Encephalopathy, Fronto-temporal dementia, Multiple Sclerosis, Diabetes type II, colitis, and arthritis.  “Aberrant activity of GSK-3 has also been implicated in the pathologies of many diseases and disorders such as metabolic disorders (diabetes, atherosclerosis, heart disease), neurological disorders (Parkinson’s, Alzheimer’s, amyotrophic lateral sclerosis, schizophrenia, bipolar disorder, mood disorders), cancer and aging (cellular senescence, cancer stem cells, control of stem cell pluripotency and differentiation), immune disorders and other maladies among others(ref).^{3, 4, 5, 6, 7}
3. The GSK-3s exercise regulatory functions which impact on cell cycle and division dynamics and a DNA repair pathway.
4. GSK’s directly impact aging since they can function as molecular mediators of the Insulin/IGF pathway and the mTOR pathway.  However, when GSK-3s are inhibited by compounds such as lithium, they can “bypass” the normal methods of blocking these “pro-aging pathways” and can therefore make a direct impact towards reducing aging by inhibiting the Insulin/IGF pathway and inhibiting the mTOR pathway.  This mechanism may be why GSK-3 inhibitors are such powerful compounds.
5. The GSK’s act by phosphorylating key molecules.  They prefer molecules which are already phosphorlated, so they can be thought of as being phosphorylation amplifiers.  They can increase phosphorylation, by a factor of 100 or 1,000.  With this kind of amplifier muscle, they can activate or shut down important signaling cascades.
6. In normal resting tissues GSK-3s are active, turned on,  Many kinds of signals can turn GSK-3s off, for examples, insulin signaling and growth factor signaling.  The molecular process of turning GSK-3s off and degrading them is also phosphorylation, this time of the GSK-3 itself..
7. GSK-3 plays important roles in both the innate and adaptive immune systems.  For this reason, GSK-3 inhibition may help treat some types of autoimmune diseases where there is a hyperactive innate or adaptive immune system.
8. GSK-3 plays a major role in inflammatory processes and in neuroinflammatory diseases in particular. Among its functions are control of various triggers of inflammation. And the production of pro and anti-inflammatory cytokines.
9. There are two major isoforms of GSK-3, the alpha isoform which regulates longevity-related processes and he beta isoform which regulates aging and “shortivity”-related processes.  The main molecular bodies of the two isoforms are very similar, but they have different molecular “tails.” What they do biologically is quite different.
10. GSK-3 alpha when active negatively regulates at least three pro-aging pathways: mTOR, Wnt and P53. Basically, in normal quiescent cells GSK-3α restrains runaway aging due to activation of these pathways.  If GSK-3α is not functioning normally, accelerated aging can occur.
11. GSK-3β, on the other hand can promote the formation of both beta-amyloid plaques in the brain and tau tangles in nerve cells, the two main “usual suspect” causes of Alzheimer’s disease.  Unlike GSK-3α, its expression increases with aging.  It is thought to be an important long-term contributor to age-related neuro-degeneration.  GSK-3β plays a role in signaling pathways like those involved in glycogen metabolism.  It appears that its aberrant inhibition may be relevant in diabetes and neurodegenerative disorders.  This isoform also promotes degradation of NF-kappaB and reduction of inflammatory conditions. So it is far from being all-bad.
12. Many health-beneficial effects are associated with inhibition of GSK-3β and activation of the beta-catenin pathway, including bone mass maintenance, less osteoporosis, reduced insulin signaling, less atherosclerosis and lowered blood pressure.  These might be limited however by competition for beta-catenin by pathways that respond to high states of oxidative stress, where beta-catenin is required as a cofactor.  Therefore, effective therapeutic inhibition of GSK-3β might require additional anti-oxidant interventions such as suppression of P66shc.
13. Blocking GSK-3β enhances expression of many proteins, an example being Nrf2 since GSK-3β phosphorylates Nrf2.  This fact alone could explain some  of the health benefits induced by lithium and a number of phytosubstances.
14. However, since GSK-3β also phosphorylates the inflammatory factor BF-kappaB, it appears that in the presence of inflammation there may be a cost trade-off to blocking expression of GSK3β.
15. There appears to be a number of effective GSK-3β inhibitors.  Lithium is a good one, and many its neurological impacts and others of its health-producing effects could be due to GSK-3 inhibition, lower cholesterol, less oxidized LDL signaling.
16. While beneficial effects can sometimes be realized by inhibiting GSK-3, care must be taken that such inhibition or activation does not also produce a negative effect via the other isoform or via a different pathway than that targeted.  As a phosphorylation amplifier, like a PA system amplifier GSK-3t does not necessarily pay attention to the message itself.

 THE SCIENCE OF GSKS

1.  INTRODUCTION TO THE GSKs – serine/threonine protein kinases

They are “Molecular Multi-taskers” that functions as “signal amplifiers” for kinase cascades and transcription factor activation of genes

Discovered in the 1980s,GSKs3s were at first thought to merely be a “negative regulator” of glycogen synthase, and thus got their name.  However, as more and more research has been done, the consensus emerged that GSKs exerts a broad, regulatory influence on 50+ targets and that glycogen synthesis inhibition was not the primary function of GSKs.  Today we know that at least 25 transcription factors are activated/inhibited by GSK-3s  and an additional 25+ other proteins are activated/inhibited by GSK-3s.  For this reason, GSK-3s are are now viewed as “multitasking molecules” that act as “signal amplifiers” for many molecular pathways involving inflammation, gene expression, cell mobility, and apoptosis.  Most recently, it has become clear that GSK-3s regulate lifespan as well.  GSK-3s now are well documented to play a role in Alzheimer’s disease, Chronic Traumatic Encephalopathy, Fronto-temporal dementia, Multiple Sclerosis, Diabetes type II, Cancer, colitis, and arthritis.

GSK-3  do their “work” by phosphorylating substrates.  A kinase phosphorylates substrates (a molecule on which an enzyme acts) by transferring phosphate groups to them from high-energy donor molecules.  This in turn can have a profound effect on the properties of the phosphorylated substrate.  “The phosphorylation state of a molecule, whether it be a protein, lipid, or carbohydrate, can affect its activity, reactivity, and its ability to bind other molecules. Therefore, kinases are critical in metabolism, cell signalling, protein regulation, cellular transport, secretory processes, and countless other cellular pathways(ref).” And, in its special role of being a kinase amplifier, GSK-3 can be considered to be “the mother of all kinases.”

The GSKs have a rather special property in that they only phosphorylate substrate proteins that are already phosphorylated, and in this respect function essentially as phosphorylation amplifiers. The literature speaks of already-phosphorylated substrates as being “primed,” and the GSKs impact differently on primed and unprimed substrates.

2.  Substrates phospherylated by GSK-3 These include the following:

A. Transcription factor phosphorylation – GSK-3s activate at least 13 transcription factors and inactivate 12 transcription factors by phosphorylation. This, of course, implies contributing to turning genes on and off. As mentioned, GSKs prefer phosphorylating these transcription factors if they are “primed”. That means that another kinase must phosphorylate the transcription factor first. Only then, GSK-3 will come along and further activate the transcription factor. In this sense, GSKs are “amplifiers” of another signaling kinase cascade. (sort of like how p66shc is a “ROS amplifier” for redox signaling).

B. Non-transcription factor proteins – GSK-3s activate another 10 non-transcription factor proteins and inactivate another 14 non-transcription factor proteins. For these proteins, the GSK-3s also prefer activating the “pre-primed” proteins that have already been phosphorylated on another residue by another kinase cascade. Thus GSK-3s also function as a “kinase cascade amplifier”, rather than an original stimulus for the pathway. Think, then, of GSK signaling being like mass-media signaling, in some cases indifferent to the messages being communicated.

Here is a diagram of the transcription factors that are substrates of GSK phosphorylation.  The yellow ones are activated and the black ones are inactivated.  The diagram has left off the STAT transcription factors, which include STAT1, 2,5, and 6.  The “big picture” here is that GSK-3s act as a “kinase signal amplifier” to increase gene expression or increase gene suppression by phosphorylating the transcription factor.  GSK-3 inhibitors would have the opposite effect on these transcription factors, meaning that the black ones would be “yellow” and the yellow ones would be “black” with a GSK-3 inhibitor such as Lithium, a non-selective  “ATP noncompetitive inhibitor” of GSK-3β, or maleimide derivatives, which are selective “ATP competitive inhibitors” of GSK-3β.

Reference: May 2014  GSK-3 as potential target for therapeutic intervention in cancer

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Next is a diagram of the other proteins that are activated or inactivated by GSK-3s. The yellow ones are activated by GSK-3s, whereas the black ones are inactivated by GSK-3 phosphorylation. As mentioned below, a GSK-3 inhibitor such as Lithium, a Maleimide derivative, or a Thiadiazolidindione would exert the opposite effect, turning the yellow proteins “black” and the black proteins “yellow”.

Illustration reference:   GSK-3 as potential target for therapeutic intervention in cancer

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Frequent readers of this blog can note that GSK-3 impacts on a great many of the “usual suspects” related to disease processes and aging. That is their importance.

3.  How GSK-3s are activated and how they are inactivated

Ser-9 or Ser-21phosphorylation inactivates GSK-3s.

GSK-3s are normally activated when the cell is resting, but are inactivated by external stimuli. For instance, GSK-3s are actively inhibiting glycogen synthase until insulin signaling occurs. Then insulin inhibits GSK-3 and thus the “inhibition of an inhibitor of glycogen synthase” results in the synthesis of glycogen within the cell (liver, muscle, or fat cell). Most all of the ways that GSKs are inactivated is via phosphorylation of GSK-3.  This “phosphoinactivation” pathway goes through PI3K.  Growth factors, amino acids, Toll-like receptors (TLR), T cell receptor (TCR), CD28, and cytokine receptors have all been shown to mediate the PI3K/Akt-mediated “phosphor-inactivation” of GSK-3α and GSK-3β.  GSKα is inactivated by the phosphorylation of Serine 21 (Ser-21).  GSK-3β is inactivated by the phosphorylation of Serine 9 (Ser-9). Activation of Akt occurs when it is phosphorylated at thr-308 by PDK1 and with phosphorylation of Ser-473 by mTORC2.

Upon activation by PDK1 + mTORC2, Akt can phosphorylate GSK-3β at Ser-9.  PKC, p70S6K, p90RSK, and PKA can phospho-inactivate GSK-3s as well.  Inactivation of GSK-3s results in the activation of transcription factors important for regulating the innate and adaptive inflammatory response. The following diagram illustrates  how GSK-3s are inactivated and what their downstream targets are:

Reference for illustration:2012 Glycogen Synthase Kinase 3: A Point of Convergence for the Host Inflammatory Response

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4.  Isoforms of GSK-3

There are two isoforms of GSK-3, alpha and beta.

a. The “alpha isoform” of GSK-3

- a longevity regulator – the mainly good guy

GSK-3α is encoded from a distinctly different gene than GSK-3β. GSK-3 isoforms also have different substrate preferences, especially in the brain (probably the same in peripheral tissues).  GSK-3α expression does NOT increase with aging, whereas GSK-3β expression increases with aging.  Whereas GSK-3β “knock out” embryos die by day 16 due to liver failure, GSK-3α knockout mice are viable and display increased insulin sensitivity and reduced fat mass.  However, these GSK-3α “knock outs” still have metabolic and neurological abnormalities in adulthood.

GSK-3α is most abundantly expressed in the brain with particular abundance in the hippocampus, the neocortex, and the cerebellum.    In the hippocampus, GSK-3β is more abundantly expressed than GSK-3α (GSK-3α is the “good GSK 3″, whereas GSK-3β is the “bad GSK-3,” though not always).  There is mounting data that GSK-3α is an “upstream cause” of longevity and that GSK-3α negatively regulates three pro-aging pathways – mTOR, Wnt, and p53.  However, GSK-3α does not seem to have that much of an effect on another pathway we would like to see suppressed in aging, the Insulin/IGF pathway (see below).

A.  Inhibition of the mTOR pathway – GSK-3α inhibits mTOR and this is the primary mechanism by which GSK-3α exerts its “anti-aging effects”.   In GSK-3α “knock out” mice, there is an increase in the phosphorylation of three mTORC1 targets:  4E-BP1, S6 Kinase, and ribosomal S6 protein.  This results in a decline in autophagy of these GSK-3α “knock out” mice. The three markers of autophagy reduced in these GSK-3α  “knock out” mice are ATG6, LC3-I/II ratio, and p62.

It also appears that GSK-3α is needed for autophagosome formation.

In summary, the “unrestrained” or “constitutive up regulation” of the mTOR pathway appeares to be the major mechanism by which GSK-3α “knock outs” exhibited accelerated aging.

B. Inhibition of Wnt signaling – The inhibition of Wnt signaling is a minor mechanism by which GSK-3α works.  WNT signaling can be a “pro-aging pathway”.  Klotho-deficient mice have accelerated aging due, in part, to increased Wnt signaling.  GSK-3α inhibits Wnt signaling by phophosphorylating beta-catenin, which targets it for ubiquitin proteasomal degradation.

C. p53/sestrin pathway – Sestrins are target genes of p53. Sestrins protect cells from various stressors by functioning  as antioxidants and as mTORC1 inhibitors.  GSK-3β can regulate p53 activity, but I am not sure if GSK-3α can do the same.

D. Insulin/IGF-1 pathway – Although GSK 3α “knock out” mice have an increased expression of IRS-1, this does not result in an increase in Akt phosphorylation or activation of downstream targets of the Insulin/IGF pathway.  For this reason, it does NOT appear that GSK-3α has that much of an effect on the Insulin/IGF pathway.

GSK-3α activity is increased by phosphorylation of tyrosine-279 and GSK-3α activity is decreased by phosphorylation of serene-21.  Thus upstream “phosphorylators” of GSK-3α  can regulate how active this protein is.   Unfortunately, I could not find any inhibitors or activators of GSK-3α Most of the work in GSKs has been focused on GSK-3β, which is the “bad GSK”.

Reference:May 2014  GSK-3 as potential target for therapeutic intervention in cancer

b. The “beta isoform” of GSK

- a pro-aging regulator, especially in the brain, the necessary but sometimes bad guy

The strongest data on the fact that GSK-3β is “upstream” in the cause of neurodegenration is the fact that GSK-3β induces both the formation of Amyloid-beta aggregates and also results in the hyperphosphorylation of tau.  The molecular mechanism for both of these GSK-3β effects are now well established.  No other explanation for AD has been shown to explain both the Amyloid beta part of this disease and the tau hyperphosphorylation part of this disease. GSK-3β expression increases with aging.  it appears to be a “pro-aging” pathway that is “upstream” and occurs prior to the formation of the following events and pathologies in the brain”

A. Synaptic dysfunction – Synaptic dysfunction is an “early feature” of AD.  It precedes and may even cause the neurodegeneration.  Both GSK isoforms are expressed in synapses, especially the hippocampal synaptosomes

B. GSK-3β and NF-kB – GSK-3β phosphorylates NF-kB and promotes the proteasomal degradation of NF-Kb NF-kB is the master of inflammation.  It is no surprise then to find out that there are many interactions between GSK-3β and the NF-kB pathway.  First of all, GSK-3β directly phosphorylates NF-kB at serene 468. This results in the proteasomal degradation of NF-kB. In addition, GSK-3β interacts with I-kappaB alpha in epithelial cells.  This results in the prevention of the degradation of I-kappaB alpha. Thus GSK-3β has an anti-inflammatory effect on the NF-kB pathway. This appears to be an exception to the idea that this GSK-3β isoform is “bad” for aging people.

C. GSK-3β and Wnt signaling – GSK-3β is a central figure in Wnt signaling – it can either increase the degradation of beta-catenin (in unstimulated cells) or increase Wnt/beta-catenin signaling in stimulated cells. The importance of this is treated below when we discuss how GSK-3β is involved in malignant transformations in certain cancers. In the Wnt pathway, scaffolding regulatory proteins control the specificity of GSK-3β mediated signaling.  In the absence of cell stimulation, CK1 phosphorylates beta-catenin at S45.  This “primes” beta-catenin for subsequent phosphorylation by GSK-3 at S41, S37, and S33. These phosphorylation targets target beta-catenin for ubiquination and proteasomal degradation.  The following diagram illustrates this.

Reference: May 2014  GSK-3 as potential target for therapeutic intervention in cancer

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 Wnt/beta-catenin induced gene expression is modulated by GSK-3.

However, once the cell is stimulated by Wnt, then the GSK-3 pathway can activate the Wnt-catenin signaling pathway, increasing its activity like an “amplifier”. The molecular mechanism here is the phosphorylation of LRP5/6, which stabilizes Axin. Here is an illustration of this:

Again from Reference: May 2014  GSK-3 as potential target for therapeutic intervention in cancer

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GSK-3 can enhance Wnt/beta-catenin signaling

D. GSK-3β and c-Myc

GSK-3 can regulate c-Myc activity. c-Myc controls cell division and the cell cycle via the CDC25 gene. c-Myc also influences the gene expression of GADD45, the DNA damage pathway that functions as a tumor suppressor by arresting the cell cycle.

Reference:Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7

E. GSK-3β and p53 – There is direct interaction between p53 and GSK-3β after DNA damage to induce apoptosis

After DNA is damaged, the tumor suppressor p53 is the key intermediate that induces either cell cycle arrest or apoptosis of the cell. Apoptosis by p53 after DNA damage is triggered by cysteine/aspartate proteases, such as Caspace-3. However, until recently, there was no explanation for how p53 activated Caspace-3. Then in 2002, Jope and colleagues from UAB showed that with chemotherapy induced DNA damage, the nuclear localized GSK-3β was activated. However, this GSK-3β activation was not due to the normal method of activation, which is the phosphorylation of Tyr-216 on GSK-3β. Instead, there was a direct protein-to-protein interaction between p53 and GSK-3β and this association actuated GSK-3β, making it an active protein that then would phosphorylate other proteins (such as tau).

Reference: 2003Direct, activating interaction between glycogen synthase kinase-3β and p53 after DNA damage

  5.  Differential GSK Inactivation and Differential Timing of GSK-3 induced activation/inactivation of Transcription Factors

The diagrams above did not include the STAT family of transcription factors.  These STAT factors are important because of their roles in oncogenic transformation [“Stats (for signal transducers and activators of transcription) are a family of transcription factors that regulate cell growth and differentiation. Their activity is latent until phosphorylation by receptor-associated kinases. A sizable body of data from cell lines, mouse models, and human tissues now implicates these transcription factors in the oncogenesis of breast cancer(ref). Because Stat activity is modulated by several posttranslational modifications and protein-protein interactions(ref), these transcription factors are capable of integrating inputs from multiple signaling networks. Given this, the future utilization of Stats as prognostic markers and therapeutic targets in human breast cancer appears likely(ref).”]  STAT3 and STAT5 are preferentially activated by GSK-3s, whereas STAT1 and STAT6 are not activated very much by the GSK-3s.  Although STAT1 is not activated early by certain signaling stimuli (Ex: IFGAR1/2), the same transcription factor can be activated late by GSK-3s. The diagram below illustrates how GSK-3s regulate the STAT family of transcription factors.

Reference for illustration: 2012 Glycogen Synthase Kinase 3: A Point of Convergence for the Host Inflammatory Response

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“Regulation of STATs by GSK3 — (A) In IFN-γ, GM-CSF, and IL-6 stimulated cells, GSK3 inhibition suppressed both STAT3 and STAT5 activity, but only minimally affected STAT1 and STAT6 activity. (B) GSK3-β inhibition differentially affects early and late STAT1 activation in IFN-γ-treated cells. Inhibition of GSK3 had no effect on early STAT1 activity (<1hr) but diminished late STAT1 phosphorylation by increasing SHP-2 activity. The ability of GSK3 inhibition to reduce STAT1 activity resulted in reduced levels of iNOS, TNF, and RANTES by IFN-γ-stimulated macrophages.”

  6.  The Four Ways that GSK-3 activity is regulated

The activity of GSK-3s is highly regulated. Four key mechanisms have been identified that regulate GSKs. They are

1)The phosphorylation of GSK3 itself, 2) the sub cellular localization of GSK3s, 3)The formation of protein complexes containing GSK3s, and 4)the phosphorylation state of the GSK3 substrates. The diagram below illustrates these 4 regulatory mechanisms:

Reference  2007 Glycogen Synthase Kinase-3 (GSK-3): Inflammation, Diseases, and Therapeutics

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7.  Primed vs Non-primed sustrates

Active GSK-3s exhibit a 100 to 1,000-fold increase in substate specificity for pre-primed substrates, compared to non-primed substrates.  N-terminal phosphorylation of GSK-3β (Ser9) acts as a “pseudosubstrate” for the phosphate binding site and thus competes for the binding of arginine 96 with pre-primed, vs non-primed substrates.  The diagram below shows what this effect does with pre-primed substrates.

Reference:  Glycogen Synthase Kinase 3: A Point of Convergence for the Host Inflammatory Response

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“ — it is first important to define GSK3’s preference to phosphorylate substrates that are pre-primed (pre-phosphorylated) on a serine or threonine located around a five amino acid consensus sequence corresponding to serine/threonine-X-X-X-serine/threonine-P, where the first serine or threonine is the residue to be phosphorylated by GSK3, X can be any amino acid, and the last serine- or threonine-P is the pre-primed (pre-phosphorylated) residue [42](Figure 1B). The preferential phosphorylation of pre-primed (pre-phosphorylated) substrates by GSK-3 has been shown to result in increasing substrate phosphorylation efficiency by more than 100 to 1000-fold, as compared to non-primed substrates [43].”

8.  GSK-3S and Aging – Is it Due to Alpha or Beta?

There is a lot of confusion as to whether GSK-3s are “pro-aging” or “anti-aging” factors.  The answer is “both”.  It is becoming clear that GSK-3s are both the activators of aging (GSK-3 beta) and the inhibitors of aging (GSK-3α).  GSK-3α and GSK-3β are 98% identical in their kinase domains, but due to their N-terminal and C-terminal ends, their effects on aging are the opposite.  The effect of GSK-3s on downstream target proteins is their ability to phosphorylate the “downstream target” protein. Phosphorylation of the GSK 3 target usually inactivates it and may in some cases, result in the proteasomal degradation of the down stream target protein (i.e. by the UPS system). Unlike most protein kinases, GSKs are typically active in unstimulated cells and when the cell is activated, the GSKs are inhibited.

 9.  Inhibition of GSK-3 β signaling by lithium

Most importantly, there is something that we can do about GSK 3β signaling (the pro-aging form).   Lithium has been known to increase activator protein 1 (AP-1) binding to DNA, but the exact mechanism of action eluded researchers for a long time.  Once GSK-3β was discovered, the mystery was solved.  GSK-3β phosphorylates c-Jun (JNK). This JNK phosphorylation prevents activator protein 1 (AP-1) from binding to DNA.  As a result, since Lithium inhibits GSK-3β, this inhibition resulted in an increase in AP-1 binding to DNA, thus activating many genes.  Lithium increases activator protein 1 (AP-1) binding to DNA by inhibiting GSK-3β, which then cannot phosphorylate JNK, which then cannot inhibit AP-1 binding to DNA.  In Item 10 below, we describe how lithium inhibition of GSK-3β also extends concentrations of Nrf2 by preventing phosphorylation of Nrf2 by GSK-3β.

References:

• 2009Validating GSK3 as an in vivo target of lithium action
• 2003 Lithium blocks the c-Jun stress response and protects neurons via its action on glycogen synthase kinase 3

Lithium carbonate or Lithium chloride, compounds that have been used since 1871 by psychiatrists for treating mania, bipolar illness, and depression.  The FDA approved Lithium in 1970 and although most patients have now been treated with other medications for depression and bipolar illness, it is still available and is inexpensive.  Lithium has already been proven to prolong lifespan in all lower animals.  Lithium is a safe, clinically effective inhibitor of GSK 3β.  At high levels used for treating psychiatric conditions, there are a lot of side effects.  However, at lower concentrations needed to inhibit GSK-3s, there are very few side effects.  The following facts support this emerging view that Lithium is a longevity agent that works via GSK-3 inhibition and promotes longevity and decreased neurodegeneration.

A. Low dose lithium uptake promotes longevity in humans and metazoans - A large Japanese cohort study was done and reported in 2011 which showed that tap water-derived lithium and a longevity effect. There was an inverse correlation between drinking water lithium concentrations and all cause mortality in 18 different Japanese municipalities with a total of 1,206,174 people (p = 0.003). These same authors also showed that low dose lithium exposure of lithium chloride extended life span in C elegans (p = 0.047).

Reference:  2011 Low-dose lithium uptake promotes longevity in humans and metazoans

B. Lithium administration increases brain gray matter - Several studies of patients with bipolar illness who were treated with Lithium showed dramatic, measurable, statistically significant increases in brain gray matter. (p = 0.0043)

References:

• 2009 A longitudinal study of the effects of lithium treatment on prefrontal and subgenual prefrontal gray matter volume in treatment-responsive bipolar disorder patients
• 2010 Lithium-induced gray matter volume increase as a neural correlate of treatment response in bipolar disorder: a longitudinal brain imaging study
• 2002 Increased gray matter volume in lithium-treated bipolar disorder patients
• 2010 Lithium-induced gray matter volume increase as a neural correlate of treatment response in bipolar disorder: a

C. Lithium in tap water reduces suicide mortality - A large study of 40 different municipalities in Japan showed that there was an inverse relationship between suicide and tap water lithium levels in the Aomori prefecture. The Aomori prefecture has the highest rates of suicide mortality in Japan. This study suggests that the low dose lithium in tap water may protect those who drink tap water from depression, which is the leading reason why Japanese commit suicide.

Reference: 2011 Low-dose lithium uptake promotes longevity in humans and metazoans

10.  Inhibibiting GSK-3β enhances expression of Nrf2 since GSK-3β phosphorylates Nrf2.

Many of the health effects of GSK-3β inhibition and the use of lithium may be due to this effect.  See the 2012 publication Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/β-TrCP axis “Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL.”  And see the 2013 publication Nrf2 is controlled by two distinct β-TrCP recognition motifs in its Neh6 domain, one of which can be modulated by GSK-3 activity, Supression of GSK-3β signaling could also explain why lithium works in part by activating Nrf2 signaling  See the just published report Antiarrhythmic effect of lithium in rats after myocardial infarction by activation of Nrf2/HO-1 signaling.  “In conclusions, lithium protects ventricular arrhythmias by attenuating NGF-induced sympathetic innervation via antioxidant activation of Nrf2/HO-1 axis.” We have written extensively in this blog about the important health benefits of the Nrf2 pathway(ref)(ref)(ref).

An important corollary of this could be that phytochemical activation of Nrf2 could be frequently or even generally mediated by inhibition of GSK-3β.  At least this appears to be the case for the phytochemical nordihydroguaiaretic acid.  See the 2012 publication  Signaling pathways activated by the phytochemical nordihydroguaiaretic acid contribute to a Keap1-independent regulation of Nrf2 stability: Role of glycogen synthase kinase-3.  “Our study demonstrates that NDGA activates Nrf2 through multiple signaling cascades and identifies GSK-3β as an integrator of these signaling pathways and a gatekeeper of Nrf2 stability at the level of the Neh6 phosphodegron.”  We want to do further research and check out whether this corollary is also true for the many other phyto substances which activate Nrf2.  In Vince’s treatise he identified 36 of these. If the corollary is generally correct, it might explain the puzzling issue of how phytochemicals upgrade the expression of Nrf2.

11.  GSK 3s and Inflammation – Diabetes, Cancer, Multiple Sclerosis, Alzheimers, and Colitis

It is now clear that Glycogen Synthase Kinase 3 plays an important role in several diseases via their ability to up regulate inflammation.  GSK-3s are powerful regulators of inflammation because of their ability to act as a “kinase cascade amplifier”.  As a result, GSK-3s have been found to play an important role in the CNS neuroinflammatory diseases such as multiple sclerosis, Alzheimer’s disease, and  mood disorders.  Here the cells that are involved include microglia and astrocytes.  GSK-3s also play a key role in peripheral inflammatory disease such as colitis and arthritis via macrophages/monocytes.  GSK-3s also appear to play a role in Diabetes via their local inflammation effect and in Cancer via their general inflammation effect.  The diagram below illustrates this:

Reference for diagram:2007 Glycogen Synthase Kinase-3 (GSK3): Inflammation, Diseases, and Therapeutics

Image may be NSFW.
Clik here to view.

A. GSK-3s and TLR-induced inflammation – Since GSK-3s have such a vital role in inflammation, GSK-3 inhibitors have been shown to have a very strong anti-inflammatory effect on a cellular level.  The brain normally is protected from the classical immune inflammatory response, however. This is probably one of the most important roles of the blood brain barrier (BBB) and is why the brain has been referred to as “immune privileged”.  Unfortunately, when the BBB is disrupted or inflamed, macrophages or monocytes invade the brain and activate resident glia cells, including astrocytes and microglia.  This is why with many neurodegenerative disorders, gliosis is a sine qua non of the disease on histology. The primary way that GSK-3s amplify inflammation is in the toll-like receptor (TLR) induced inflammation.  Here are the classic TLR receptor and what activates them:

TLR receptor subtype        What activates them

• TLR2                    lipoteichoic acid from Streptococcus pneumonia
• TLR4                    synthetic lipid A
• TLR5                    flagellin protein from Salm typhimurium
• TLR9                    human CpG

When any of the above TLR agonists are used to stimulate human peripheral mononuclear cells (PBMCs) in the presence of a GSK-3 inhibitor, there was a selective reduction of 50-90% in the pro-inflammatory cytokines IL-1β, IFN-γ, IL-12, and IL-6.  The following paragraph goes into more detail on this.

Reference:2012 Glycogen Synthase Kinase 3: A Point of Convergence for the Host Inflammatory Response

B. GSK-3s and cytokine-induced inflammation – Recent work has shown that GSK-3 activity is required for the full stimulation of the production of several pro-inflammatory cytokines.  This includes IL-6, IL-1beta, and TNFalpha, following stimulation by toll-like receptors in monocytes and peripheral blood mononuclear cells.  GSK-3s also modulate the production of the anti-inflammatory cytokine, IL-10.  When a GSK-3 inhibitor was used in vitro, the production of IL-10 increased by 3-5 fold in human monocytes activated with LPS.  On the contrary, GSK-3s increase the production of the pro-inflamatory cytokine, IL12.  When a GSK-3 inhibitor was used in vitro, it reduced the production of IL-12 by LPS-stimulated human monocytes by 70%.  With GSK-3 inhibitors, IL-6 and TNF-α production by LPS-stimulated monocytes was reduced by 60-80%.

C. GSK-3 isoforms are differentially activated with inflammation – Despite these harmful effects of GSK-3s, they are vital to life.  Deletion of the GSK-3β gene results in death of the embryo.  Adding GSK-3α is unable to “rescue” the GSK-3β “knock out” mice.  When probes were used to determine which GSK-3 was involved with inflammation, it was clear that GSK-3β was activated via the phosphorylation of Ser-9 by LPS-induced inflammation, whereas GSK-3α was not activated with LPS-induced inflammation.

References:

2007 Glycogen Synthase Kinase-3 (GSK3): Inflammation, Diseases, and Therapeutics

2005 Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3

 12. GSKs and Alzheimer’s disease

As we mentioned above, the GS-3s induce the phenomena thought to be the cause of Alzheimer’s disease according to both of the leading theories: the beta amyloid theory(ref) and the tau tangles theory(ref), since they promotes the expression of both beta-amyloid and tau tangles.  In fact,  GSK-3B is that it is the only protein that directly cause all of the following features of AD

1. GSK-3 is increased in white cells early in Alzheimer’s disease. 

Reference: Glycogen synthase kinase-3 is increased in white cells early in Alzheimer’s disease

1. GSK-3beta induces memory deficits in vivo, long before the appearance of Amyloid-beta aggregates and before the appearance of tau tangles.  Reference:  GSK-3 is essential in the pathogenesis of Alzheimer’s disease
2. GSK-3alpha induces Amyloid beta productionand GSK-3alpha inhibition reduces GSK-3alpha reduces Amyloid beta production.  Reference:  GSK-3 is essential in the pathogenesis of Alzheimer’s disease
3. Amyloid beta fragments (Abeta 25-35) activate tau protein kinase I(TPKI).   Reference: Activation of tau protein kinase I/glycogen synthase kinase-3beta by amyloid beta peptide (25-35) enhances phosphorylation of tau in hippocampal neurons
4. Tau protein kinase 1 (TPKI) is the same thing as Glycogen Synthase Kinase 3beta (GSK-3B). Thus, GSK-3b IS THE ENZYME THAT ACTUALLY DOES THE HYPERPHOSPHORYLATION!  References: Physiology and pathology of tau protein kinases in relation to Alzheimer’s disease,  Alzheimer’s disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Synaptic dysfunction – Synaptic dysfunction is an “early feature” of AD.  It precedes and may even cause the neurodegeneration.  Both GSK isoforms are expressed in synapses, especially the hippocampal synaptosomes.

A very interesting angle is the role of GSK-3 in the relationship of H. pylori to Alzheimer’s disease.  A July 2014 publication Helicobact er pylori Filtrate Induces Alzheimer-Like Tau Hyperphosphorylation by Activating Glycogen Synthase Kinase-3β draws a connection:  “Abnormal hyperphosphorylation of microtubule-associated protein tau is involved in the pathogenesis of several neurodegenerative disorders including Alzheimer’s disease (AD). Helicobacter pylori (H. pylori) infection has been reported to be related with a high risk of AD, but the direct laboratory evidence is lacking. Here we explored the effect of H. pylori infection on tau phosphorylation. The results showed that H. pylori filtrate induced significant tau hyperphosphorylation at several AD-related tau phosphorylation sites, such as Thr205, Thr231, and Ser404, both in mouse neuroblastoma N2a cells and rat brains with activation of glycogen synthase kinase-3β (GSK-3β). Application of GSK-3 inhibitors efficiently attenuated the H. pylori-induced tau hyperphosphorylation. Our data provide evidence supporting the role of H. pylori infection in AD-like tau pathology, suggesting that H. pylori eradication may be beneficial in the prevention of tauopathy.”

13.  GSK-3s and cancer

Aberrant activity of GSK-3 has been implicated in many types of cancers, especially those that are resistant to chemotherapy, radiation therapy, and targeted therapy. Targeting GSK-3 may be a means to overcome the resistance of these cancers to certain chemotherapeutic drugs, radiation, and small molecule inhibitors.  Here are some example cancers where GSK-3 is known to be involved with carcinogenesis:

A. GSK-3 and beta-catenin in liver cancer - decreased GSK-3 levels due to HBx and ERK, HCV core protein GSK-3β is known to be involved with the formation of liver cancer.  The dysregulation of GSK-3β phosphorylation and inhibition of GSK-3β activity and beta-catenin signaling is the molecular mechanism involved with this type of cancer. In a recent study of 80 patients with hepatocellular cancer, the expression of GSK-3β protein was noted to be decreased.  However, exceptions to this trend were also found.  In some cancers, GSK-3β was noted to have high basal levels of S9-phosphorylated GSK-3β.

B.  HBx and ERK – The molecular mechanism by which GSK-3β levels are decreased in hepatocellular carcinoma appears to be related to one of the hepatitis virus proteins which is expressed in liver cells in the precancerous phase of liver cancer. This virally produced protein is called HBx.  HBx activates ERK.  This “primes” ERK for GSK-3β phosphorylation.  ERK phosphorylates GSK-3β at T43.  Then p90Rsk phosphorylates GSK-3β at S9, which then results in the inactivation of GSK-3β.  This inactivation of GSK-3β results in beta-catenin stabilization and signaling.  Similar molecular mechanisms occur which stabilizes paxillin, a cytoskeleton regulator.

C, HCV protein – Another way that GSK-3s are involved with hepatocellular carcinomas involves the HCV core protein.  HCV core protein increases in hepatic cancers and stabilizes beta-catenin levels. This occurs via the inactivation of GSK-3β by the phosphorylation of GSK-3β at Ser-9.  Once beta-catenin is stabilized, then it can enhance canonical Wnt/beta-catenin targets, such as c-Myc, cyclin D1, Wnt 1-inducible signaling pathway protein 2 (WISP2), and connective tissue growth factor (CTGF). All of these genes are up regulated when beta-catenin stabilized by the GSK-3 mediated HCV core protein.

D.  SIRT2  upregulation  – GSK-3β and beta-catenin signaling plays a role in the Sirtuin 2 mediated transformation of liver cells into hepatocellular cancer.  SIRT2 is up regulated in about 50% of hepatocellular cancers.  In these cancers, there is a shorter over all survival.  SIRT2 regulates the activation of Akt by deacetylating Akt.  Once Akt is deacetylated, it can then phosphorylate GSK-3β and inactivate the protein.  This activates the beta-catenin pathway and promotes cell migration of hepato-cellular carcinoma cells.  For this reason, many have suggested that GSK-3β is a “tumor suppressor” and that the loss of GSK-3β expression or activity may contribute to hepatocellular cancer development.  Thus, there appears to be a very good side to GSK-3β in addition to all the evil sides we have been describing.

E.  Cancer treatment with GSK-3β inhibitors – increased survival in a Wnt/beta-catenin independent mechanism. Studies of GSK-3 inhibitor treatment of cancer cells have shown that cancers are inhibited with these compounds.  This is a paradoxical finding, since an “inhibitor of a tumor suppressor” should make the cancer cells grow. Instead, the cancer cells show a reduced survival and proliferation via a Wnt/beta-catenin independent mechanism.  Specifically, treatment of cancer cells with a GSK-3β inhibitor decreases telomerase expression (hTERT) and also reduces telomerase activity.  This occurs with both non-selective inhibitors such as Lithium and with selective inhibitors such as SB-415286.  Here their anti-cancer effects are mediated by apoptotic signaling (Caspace-8, caspace-3, and p53 gene activation).

Conclusion:  Hepatocellular cancer development can be explained in part by the down-regulation of GSK-3β activity by HBx/ERK mechanisms, by HCV protein mechanisms, and by SIRT2 dependent mechanisms.  These studies suggest that GSK-3β functions as a tumor suppressor and that an inhibitor would have an adverse effect on cancer, causing the cancer to proliferate.  However, the opposite effect occurs when GSK-3 inhibitors are used on cancer cells.  Instead, the cancers show reduced survival and proliferation via Wnt/β-catenin independent mechanisms involving hTERT and apoptotic proteins.  Again, an example of how interacting pathways sometimes work to produce unexpected results.

Thus, GSK-3 inhibitors show promise in the treatment of cancer.

Reference: May 2014  GSK-3 as potential target for therapeutic intervention in cancer.

14. GSK-3s and signaling in selected pathways

The GSK-3s impact on a number of pathways that are involved both in embryogenesis and cancer processes, We comment on some of these.

A. GSK-3 and mTORC1 signaling – AMPK can down-regulate mTORC1 via GSK-3 induced TSC2 degradation. However, Wnt signaling can up-regulate the mTORC1 pathway via GSK-3 via beta-catenin mediated inhibition of TSC2 degradation. GSK-3 always prefers “primed substrates.” This is true for the tuberous sclerosis complex 2 (TSC2) in the mTOR pathway. Specifically, AMPK can act as the “primer” and first phosphorylates TSC2. Then GSK-3 will phosphorylate TSC2 and thus activate the proteasomal degradation of TSC2. This results in the down-regulation of the mTOR pathway. In contrast, Wnt signaling suppresses the GSK-3 mediated phosphorylation of TSC2. This results in the activation of the mTOR pathway. Rapamycin inhibits mTORC1 and this then suppresses the Wnt/beta-catenin induced cellular proliferation of cancer. This is how GSK-3 plays a role in cancer via the mTORC1 pathway. In addition to GSK-3, three other proteins are involved in the modulation of the effects of Wnt signaling on cancer (DKK1, Dvl, and Axin).

B. GSK-3 and SMAD1 - MAPKs can “prime” SMAD1 and then GSK-3 can activate the “primed” SMAD1 transcription factor. The SMAD1 transcription factor is the downstream transcription factor that mediates the effects of TGF-beta superfamily ligands such as BMPs. SMAD1 signaling is often increased in cancer. As mentioned before, GSK-3 prefers “primed substrates”. In this case MAPKs such as ERK, JNK, and p38 can each phosphorylate SMAD1. Then GSK-3 will phosphorylate SMAD1 and thus induce its ubiquitinylation and proteasomal degradation. As seen with the other examples above (TSC2), Wnt signaling suppresses the GSK-3 mediated phosphorylation of SMAD1 and therefore prevents the proteasomal degradation of SMAD1. This system of regulation plays an important role in embryonic pattern formation during embryogenesis.

C. GSK-3 and Hedgehog Pathway - GSK-3 plays a role in segmental pattern formation in the embryo. The Hedgehog pathway is very important in development (embryogenesis), but is normally silenced in most adult tissues. However in cancer, the Hedgehog pathway can be re-activated. The classic cancers where the Hedgehog pathway is re-activated are basal cell carcinoma of the skin and medulloblastoma. Here constitutive Hedgehog pathway activation occurs due to mutations in various genes of the Hedgehog pathway such as the receptor, patched 1 (PTCH1) or smoothened (SMO). Other types of Hedgehog signaling occurs in cancer too. One of the most common is paracrine Hedgehog signaling. This occurs in a subset of epithelial cancers found in the colon, panaceas, and ovaries. Here the cancer cells secrete Hedgehog ligands which then activate Hedgehog signaling in the surrounding non-cancerous stroma cells, creating a “cancer favorable environment” for the cancer cells to grow and then for the cancer cells to invade the stroma (i.e. local recurrence of cancer).

Most of what we understand about the Hedgehog pathway comes from studying embryology.  During embryogenesis, the Hedgehog pathway regulates segmental pattern formation via three genes: desert hedgehog (DHH), Indian hedgehog (IHH), and sonic hedgehog (SHH).  Hedgehog ligands bind to their receptor, PTCH1.  This causes the internalization and degradation of smoothened (SMO), which then promotes the dissociation of SUFU-glioma associated oncogene homolog (GLI).  GLI3 can be phosphorylated by PKA, GSK-3 and CK1.  Once GLI3 is phosphorylated, it becomes a transriptional repressor.  Activatee GLI1 proteins stimulate the transcription of Hedgehog target genes, which includes IGF2.  IGF2 has been shown in some cancers to induce PI3K/PTEN/Akt/mTOR pathway and thereby increase cancer cell proliferation and survival.  In addition, IGF2 can also inhibit GSK-3β.  GSK-3 can positively regulate hedgehog signaling.  When GSK-3 is activated, the hedgehog signaling pathway is activated.  When GSK-3 is inactivated, then the hedgehog signaling pathway is promoted.  GSK-3 affects hedgehog signaling pathway via the phosphorylation of SUFU, a binding partner for GSK-3β.

D. GSK-3 and Notch Signaling Pathway – GSK-3 regulates notch signaling, EBV viral protein EBNA2 is a “biological equivalent” of a Notch receptor. Notch signaling plays an important role in a wide variety of cancers. The prototypical cancer where Notch signaling is unregulated is T cell acute lymphoblastic leukemia (T-ALL). In T-ALLs, 50% of cancers have Notch signaling up regulated. However, it is also up regulated in some breast cancers, cervical cancers, pancreatic cancers, endometrial cancers, renal cancers, lung cancer, colon cancer, head and neck cancer, Hodgkin’s lymphoma, and large cell anapestic lymphoma. The most common way that Notch Signaling is up regulated in cancer is the over-expression of the receptor gene.

The classic form of Notch 1 receptor over expression is seen in cervical cancer. 99% of cervical cancers are associated with high risk HPV virus. The viral oncogenes E6 and E7 disrupt the cell cycle regulation by targeting p53 and Rb. However, the E6 and E7 disruption of the cell cycle control by p53 and Rb is not enough to transform the cervical epithelial cells into cancer cells. To do this, the Notch 1 receptor must be over-expressed. As the pre-malignant cells progress farther and farther towards becoming cancer cells, the expression of Notch 1 receptor gradually increases. It is not surprising when researchers found out that the up regulation of the Notch 1 receptor was induced by E6 and E7 viral oncogenes.

There is another small class of tumors that appear to be caused by the Epstein Barr virus where Notch signaling has a very pivotal role. These cancers include Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma, and lymphomas in immune compromised patients. The EBV can “immortalize” B lymphocytes and the process of B cell immortalization is a EBV protein called the “transactivator EBNA2″. EBNA2 controls the expression of several viral genes and cellular genes. EBNA2 is tethered to promoter regions of these genes by a cellular repressor called CSL. CSL resembles the physiological activation of CSL-repressed promoters by intracellular Notch receptors. Thus, the EBNA2 protein is the “biological equivalent” of an intracellular Notch receptor (N-ICD). EBNA2 and N-ICD have been shown to be partially interchangeable.

The primary way that the Notch Signaling Pathway is activated is by direct cell-to-cell contact.  One cell has what is called a “ligand” and the other cell has a “receptor” that matches the ligand.  The ligand-receptor interaction is what triggers the Notch signaling pathway.  There are six ligands that have been identified in human cells (DII1, DII3, DII4, Dik, Jagged1, and Jagged 2).  There are four transmembrane receptors found in the Notch signaling pathway (Notch 1, Notch 2, Notch 3, and Notch 4.  The cell-to-cell contact and binding of a Notch ligand to a Notch receptor (DII2, etc.) then can trigger a number of intracellular pathways, such as the Ras/MAPK pathway, the PKC/NF-kB pathway, the Wnt signaling pathway, the TGF-beta pathway, and the Sonic Hedgehog pathway (Shh).  All of these pathways promote tumor growth.  In the Shh pathway, GSK-3β phosphorylates NICD, This phosphorylation event results in the prevention of the proteasomal degradation of NICD.  This results in the prolonged survival of the NICD protein by preventing its proteasomal degradation.

Reference: May 2014  GSK-3 as potential target for therapeutic intervention in cancer

E. GSK-3β and Tumor cell Exosome Signaling -Tumor cells secrete exosomes rich in lipid-raft containing membranes that interact with other tumor cells by using Notch signaling to increase PTEN and GSK-3β expression in the target cell that the exosomes fuse with. Another very interesting study of pancreatic cancer cells has recently been done, focusing on the role of secreted exosomes from the cancer cells. Exosomes are “nano particles” made by the endosomal compartment of the cell.  These secreted nano particles contain both cell surface ligands that allow for docking onto the surface of other cells and surface ligands that trigger endocytosis by the cell that does the “receiving”.  This exosome/cell interaction led to the decreased expression of a nuclear Notch receptor target called Hes-1.  Unexpectedly, blocking presenilin resulted in the activation of PTEN and GSK-3β.  Conversely, inhibiting either PTEN or GSK-3β resulted in the increase in He’s-1 expression.  This inhibition increased the apoptotic response and decreased the survival of the cancer cells.

Reference: 2009, Essential role of Notch signaling in apoptosis of human pancreatic tumor cells mediated by exosomal nano particles

Cancer and GSK-3β – Conclusions:  GSK-1s play a major role in cancer by their interaction with the Wnt/beta-catenin pathway, the mTOR pathway, SMAD1, the Hedgehog pathway, and the Notch pathway.  In most all of these pathways, another kinase first primes the protein by phosphorylating the GSK-1 target first, then GSK-1beta phosphorylates the target protein.  This results in the proteasomal degradation of the protein that has been phosphorylated.  However with cancer, there is a dysregulation in GSK-3β.

 15. GSK-3β inhibitors - compounds and drugs that may help prevent or treat Alzheimer’s disease

There are a large number of compounds that have been shown to inhibit GSK-3s. The way they have been found is by In vitro testing of compounds to see if they can activate  GSK-3 inhibitor, the effects of these other GSK-3 inhibitors is additive. This means that the two together have a greater effect than either one could achieve alone (i.e. insulin + a GSK inhibitor). The GSK-3 inhibitors that have been found so far include metal cations, aminopyrimidines, arylindolemaleimide, thiadiazolidindiones, halomethylketones, peptides, and natural products derived from marine organisms. Here is a partial list of these compounds:

A. Metal Cations - non-selective, ATP noncompetitive inhibitors

We have already discussed lithium, the most important of these.  Several other divalent metal cations also inhibit GSK-3 by their divalent positive charge.  These include Beryllium, Copper, Mercury, and Tungsten.  Lithium appears to be the most selective GSK-3β inhibitor of this group of metal cations, however.  Unfortunately, these metal cations are NOT selective inhibitors of GSK-3.  For instance, Lithium chloride or Lithium carbonate will also inhibit casein kinase-2, p38 regulated/activated kinase, and MAPK activated protein kinase-2.  Lithium is also an inhibitor of polyphosphate 1-phosphatase and inositol monophosphate, two enzymes that are required for the synthesis of inositol.   Paradoxically, Lithium chloride has also been reported to acutely elevate phosphatidylinositol 3-phosphate levels in rat cerebellar granule cells, thereby activating PKB. It is this “non-selectivity” problem that has prevented the widespread use of Lithium chloride for other diseases other than depression and bipolar illness.

Many of the other metal cations mentioned above also have serious toxicity, such as Copper and Mercury. For this reason, these other metal cations are not likely to become “drugs” anytime soon.

References:

• 2005 Opposite effects of lithium and valproic acid on trophic factor deprivation-induced glycogen synthase kinase-3 activation, c-Jun expression and neuronal cell death

• 2005 GSK-3 activity in neocortical cells is inhibited by lithium but not carbamazepine or valproic acid

• 1999  The mood-stabilizing agent valproate inhibits the activity of glycogen synthase kinase-3.

B. Valproic acid

Sodium valproate is also a weak inhibitor of GSK-3β.  Valproic acid increases activator protein 1 (AP-1) binding to DNA, but     only at much higher concentrations than lithium. The direct mechanism of how valproic acid works is similar to how lithium works.  At high concentrations, valproate can inhibit GSK-3β, and therefore activate SP-1 induced gene expression.  However, there is some debate as to whether clinical levels of valproate directly inhibits GSK-3β.  Clinically, It may be that Valproic acid inhibits GSK-3β by some indirect mechanism.

References:

• For general information on valproic acid see the 2010 blog entry Valproic acid – The phoenix drug arises again
• 1999 The mood-stabilizing agent valproate inhibits the activity of glycogen synthase kinase-3
•  2002 Valproate regulates GSK-3-mediated axonal remodeling and synapsin I clustering in developing neurons
• 2005 GSK-3 activity in neocortical cells is inhibited by lithium but not carbamazepine or valproic acid.
• 2005 Opposite effects of lithium and valproic acid on trophic factor deprivation-induced glycogen synthase kinase-3 activation, c-Jun expression and neuronal cell death

C. Aminopyrimidines

These are synthetic compounds:

• CT98014
•  CT98023
•  CT99021
•  TWS119

C. Thiadiazolidindiones

One of the FDA approved GSK-3β inhibitors is Rosiglitazone, a drug with a somewhat rocky history.

D. Maleimide derivatives – SB-213763, SB-415286

 Maleimide derivatives were identified as leads from a high throughput screen of SmithKline Beecham compound bank against  rabbit GSK-3α.  SB-213763 is an arylindolemaleimide with an IC50 of 34 nM. SB-415286 is a anilinomaleimide with an IC50 of 78 nM.  Both of these are compounds that inhibit GSK-3α in a ATP competitive manner.  The two compounds were equally effective at inhibiting human GSK-3α and human GSK-3β.  The additional good feature of these molecules is that they are very selective kinase inhibitors and showed little or no inhibition of 24 other kinases when tested against these other kinase compounds in vitro. The maleimide derivatives have an “additive effect” with insulin to stimulate glycogen synthesis.  Unfortunately, the maleimide derivatives do not have any synergistic effects with other GSK-3 inhibitors, such as Lithium chloride.

E. ATP-competitive compounds

F. Natural products from Marine organisms

Several compounds have been found from marine organisms that inhibit GSK-3. They include the following:

• 6-BIO
• Dibromocantharelline
•  Hymenialdesine
•  Indirubins
•  Meridianins

G. Halomethylketones

• HNK-32
• H. peptides

16. Inhibition of GSK-3β may remove the “ceiling” of the cardioprotective effects of certain phytochemicals in postconditioning after ischemia

At least, this appears to be the case for genistein, a soy derivative.  The 2009 publication The Ceiling Effect of Pharmacological Postconditioning with the Phytoestrogen Genistein Is Reversed by the GSK3β Inhibitor SB 216763 [3-(2,4-Dichlorophenyl)-4(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione] through Mitochondrial ATP-Dependent Potassium Channel Openingreports:  “In conclusion, the cardioprotective effect of pharmacological postconditioning with genistein and 17β-estradiol involves mitochondrial K_ATP channels and is limited by a ceiling effect of protection.   This loss of cardioprotection is associated with a loss of Akt phosphorylation capacity with increased durations of ischemia.  However, this ceiling effect can be reversed by administration of the GSK3β inhibitor SB 216763 through the opening of mitochondrial K_ATP channels.”  This publication raises several interesting issues such as: 1. The roll of the mitochondrial K_ATP channels in post and possibly pre-conditioning, 2. Whether other phytochemicals might exercise similar effects possibly employing  other GSK-3β inhibitors such as lithium, and 3. Whether an enhanced pre-conditioning and post-conditioning effect can thus be realized for other forms of trauma such as surgery, head injuries, etc.

17. On scarcity of beta-catenin, impact of this on GSK3s, and missed opportunities for healh enhancement

The 2013 publication “Gone with the Wnts: Beta-catenin, T-Cell Factor, Forkhead Box O, and Oxidative Stress in Age-dependent Diseases of Bone, Lipid, and Glucose Metabolism” introduces an additional complication regarding health and longevity interventions based on GSK-3.Here is the diagram from that article:

Image may be NSFW.
Clik here to view.

We mentioned above that GSK-3α inhibits Wnt signaling by phophosphorylating beta-catenin, which targets it for ubiquitin  proteasomal degradation.  Further that  WNT signaling can be a “pro-aging pathway,” and we discussed its role in cancer processes.

Normally, Wnt signaling induces positive effects on bone, the pancreas, and lipids.  Specifically, Wnt activation of the LRP5/6 Frizzled receptor induces inactivation of GSK-3β.  In addition, with plenty of beta-catenin in the nucleus, beta-catenin can act as a cofactor to activate the “downstream target genes of Wnt signaling”, which are mediated by the transcription factor,TCF/LEF.  This results in the activation of Axin2, OPG, ALP, and other genes that produce  the following phenotype:

Osteoblast stimulation and prevention of apoptosis => bone mass maintenance

1. Osteoclast inhibition => no osteoporosis
2. Beta-cell mass maintenance in the pancreas => normal insulin signaling
3. Decreased gluconeogenesis by the liver => less non-dietary, hepatic-induced hyperglycemia
4. Decreased LDL synthesis by the liver => lower LDL cholesterol levels => less oxidized LDL signaling via the Lectin-like LDL receptor of the cells => less p66shc activation
5. Less atherosclerosis
6. Lowered blood pressure

Unfortunately, there are other transcription factors that requires beta-catenin as a cofactor. These are the Forkhead Box transcription factors (FoxOs). FoxOs need beta-catenin as a cofactor for activation of the genes involving oxidative stress signaling. Thus FoxOs　”steal” beta-catenin away from TCF/LEF. This “shift to the left” of beta-catenin supply results in a change from the “beneficial effects” of Wnt signaling to the “adverse effects” of Wnt signaling.

The nuclear translocation of FoxO transcription factors (FoxO1, FoxO3a, FoxO4, and FoxO6) normally occurs by the inhibition of the Insulin/IGF-1 pathway, since Akt phosphorylation prevents the nuclear localization of FoxOs. However, nuclear translocation of FoxOs can all be triggered by ROS, by one of the MAPK kinases called c-Jun (JNK). In both scenarios, beta-catenin is required as a cofactor for the activation of genes required for cellular resistance to oxidative stress and DNA damage. (SOD, Catalase, Gadd45, etc.)

Conclusion: in the presence of oxidative stress, there can not be enough nuclear beta-catenin to “go around”. As a result, there is inadequate stimulation of the downstream targets of Wnt signaling and you develop the following phenotype:

1. Decreased osteoblast activity and increased osteoblast apoptosis
2. Increased osteoclast activity
3. Decreased bone mass due to #1 and #2
4. Decreased beta-islet cell mass in the pancreas and reduced insulin
5. Increased gluconeogenesis by the liver
6. Increased LDL cholesterol
7. Increased atherosclerosis
8. Increased blood pressure

All of the above phenotypes are classically seen with human aging. This is why GSK-3β inhibition alone will probably not “solve” aging.  Instead, we must do something about oxidative stress as well, probably something about P66shc expression.

We expect that the next blog in this Strange But Powerful Molecules Series will be about P66shc.

By Vince Giuliano

I rarely hesitate to talk about exciting research developments reported in this blog with my friends.  However, only a few people  in our local community of Wayland Massachusetts have known about the blog or our work on the aging sciences.    A few of my friends have suggested that I do a presentation on our work for the benefit of the local community. I gave such a talk on October 7, 2014 on The Prospects that Emerging Science Offers Us for Long Healthy Lifespans. The talk was the first in a 2014 seasonal series of lectures, “The Great Presenter Series” sponsored by the Wayland Public Library.

The PowerPoint presentation for that talk can be downloaded by clicking on the links that follow.  I had to divide the presentation into two parts so it could be handled by the blog software. You can download both parts and view them with your own versions of Microsoft PowerPoint or Open Office

Longhealhylife10-2-14PT1  Overview on the sciences of aging

Longhealhylife10-2-14PT2 Actions and interventions for extending healthspan and lifespans  – Focus on the personal and practical

Because most members of the Wayland library audience could not be presumed to have a biology science background, I strove to keep the presentation relatively non-technical and general.  The talk was well-attended and well-received, and followed by a long and lively discussion period.  Regular readers of this blog might find the presentation useful for kicking off discussions with their own non-scientist friends about some of the key facts relating to the longevity sciences and easy-to-apply interventions that offer the possibility of longer than average lifespans for those that pursue them.

Vince

By Vince Giuliano with inputs from by James P. Watson

Accelerated by the publication in December 2013 of a seminal paper by David Sinclair and his US and Australian colleagues(ref), there has been increasing interest and excitement about the prospects of discovering means for offsetting age–related declines in NAD+ as a strategy for disease prevention and life extension.  Actually, similar proposals have appeared in the literature for several years.  However, for lay people and even many scientists, the scientific discussions of NAD+ are so complicated that they seem to be virtually unfathomable.  In fact, understanding NAD+ and its roles requires fathoming a large number of related actors and their roles – complex metabolic pathways, an alphabet soup collection of enzymes and gene activation cofactors, the family of Sirtuins, biological processes in different cell compartments and organs, and multiple pathological and health-producing concomitants.  The complex of related actors has come to be called the NAD World.   It is not surprising that many intelligent  people are left confused about it.

We have already referred to NAD+ and its health concomitants several times in this blog.  In fact, Jim Watson characterized it as the most important scientific topic he encountered in 2013 and you might want to start out by reviewing  what he said about it in this blog entry.   The purpose of this current Part 1 blog entry is to provide an overview treatment of the NAD World and its nuances: to identify the major molecular entities involved, their roles, health and longevity ramifications, the reasons for the current excitement, and to begin to clarify what is actually known and what the remaining uncertainties are.  We also discuss a few important facets of the general topic which are ignored in many of the current publications, such as the high degree of circadian regulation of NAD+ .and about  proteins that regulate NAD+ and its cousins such as CD38 and CCR2.  There will be at least one more additional Parts in this series.  They will go into additional detail about how common disease processes relate to disfunctionalities in the NAD World, and will examine a number of strategies currently being researched or proposed for maintaining high constituent levels of NAD+.  They will also discuss the importance of developing a set of easily-detectible biomarkers to enable comparative evaluation of these strategies.  My perception is that a big new area of longevity science is opening up, one that will offer new practical interventions that could well lead to longer healthier lives.

INTRODUCTIONS TO THE MAIN ACTORS AND THEIR RELATIONSHIPS — INTRODUCTION THE NAD WORLD

It was suggested back in 2009 that NAD and the molecules and pathways it dances with constitute a world for explaining metabolism and aging.

The 2009 publication The NAD world: A new systemic regulatory network for metabolism and aaging-Sirt1, systemic NAD biosynthesis, and their importance related: “For the past several years, it has been demonstrated that the NAD-dependent protein deacetylase Sirt1 and nicotinamide phosphoribosyltransferase (Nampt)-mediated systemic NAD biosynthesis together play a critical role in the regulation of metabolism and possibly aging in mammals. Based on our recent studies on these two critical components, we have developed a hypothesis of a novel systemic regulatory network, named “NAD World”, for mammalian aging. Conceptually, in the NAD World, systemic NAD biosynthesis mediated by intra- and extracellular NAMPT functions as a driver that keeps up the pace of metabolism in multiple tissues/organs, and the NAD-dependent deacetylase Sirt1 serves as a universal mediator that executes metabolic effects in a tissue-dependent manner in response to changes in systemic NAD biosynthesis. This new concept of the NAD World provides important insights into a systemic regulatory mechanism that fundamentally connects metabolism and aging and also conveys the ideas of functional hierarchy and frailty for the regulation of metabolic robustness and aging in mammals.”

Here is a handy reference guide to some of the main molecular actors that will be showing up in the following discussions and their significance – a cast of main characters in the NAD World but by no means all of them.

NAD - Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells. The compound is a dinucleotide, since it consists of two nucleotides joined through their phosphate groups. One nucleotide contains an adenine base and the other nicotinamide. Nicotinamide adenine dinucleotide exists in two forms, an oxidized and reduced form abbreviated as NAD⁺ and NADH respectively.(ref)  As we will see, it is a molecule of central importance to metabolic and other key biological processes in humans.

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NAD+ - oxidized form of NAD, very important as a cofactor for health and longevity-related processes including DNA repair and mitochondrial operability as will be discussed.  The central actor in our current drama, it acts as a biological oxidizing agent.  Declines sharply with age negatively impacting on DNA repair and mitochondrial health and benefits conveyed by sirtuins.

NADH – reduced form of NAD. Acts as a biological reducing agent.  Cycled back in the body to NAD+ by what are known as the NAD salvage pathways described below.

NA  – Nicotinic acid (aka Niacin aka Vitamin B3) – Niacin –” is an organic compound with the formula C6H5NO2 and, depending on the definition used, one of the 20 to 80essential human nutrients.(ref)”  Important in the current drama as a precursor to Nicotinamide which is a precursor to NAD. Unfortunately, consuming niacin is generally not a good way to enhance body levels of NAD since its metabolite niacinamide inhibits the sirtuins.

 NAM – Nicotinamide (also known as niacinamide and nicotinic amide), “is the amide of nicotinic acid (vitamin B₃ / niacin). Nicotinamide is a water-soluble vitamin and is part of the vitamin B group. Nicotinic acid, also known as niacin, is converted to nicotinamide in vivo.”(ref).  It is a precursor of NAD but there is a catch to simply supplementing with it.  It inhibits the sirtuins and their health-producing properties.

NR – Nicotinamide riboside – a precursor of NAD, and is a source of Vitamin B3.   Available as a commercial dietary supplement thought to promote NAD+.  One of the possible approaches to enhancing body levels of NAD+(ref)

 NAD Salvage Pathways -  “Besides assembling NAD⁺ de novo from simple amino acid precursors, cells also salvage preformed compounds containing nicotinamide. Although other precursors are known, the three natural compounds containing the nicotinamide ring and used in these salvage metabolic pathways are nicotinic acid (Na), nicotinamide (Nam) and nicotinamide riboside (NR).^[2] These compounds can be taken up from the diet, where the mixture of nicotinic acid and nicotinamide are called vitamin B₃ or niacin. However, these compounds are also produced within cells, when the nicotinamide moiety is released from NAD⁺ in ADP-ribose transfer reactions. Indeed, the enzymes involved in these salvage pathways appear to be concentrated in the cell nucleus, which may compensate for the high level of reactions that consume NAD⁺ in this organelle.^[26]  Cells can also take up extracellular NAD⁺ from their surroundings^[27](ref).”

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Image and legend source is the 2002 publication Manipulation of a Nuclear NAD⁺ Salvage Pathway Delays Aging without Altering Steady-state NAD⁺ Levels:   ” Model for life span extension via increased flux through the NAD+ salvage pathway. – Type III histone deacetylases such as Sir2 and Hst1–4 catalyze a key step in the salvage pathway by converting NAD+ to nicotinamide. Additional copies of PNC1, NPT1, NMA1, and NMA2 increase flux through the NAD+salvage pathway, which stimulates Sir2 activity and increases life span. Additional copies of QNS1 fail to increase silencing. Unlike other steps in the pathway, its substrate cannot be supplied from a source outside the salvage pathway and is therefore limiting for the reaction. Abbreviations: NAD +, nicotinamide adenine dinucleotide; NaMN, nicotinic acid mononucleotide; NaAD, desamido-NAD+(ref).”  A number of important matters are not shown in this simplified diagram, such as ATP (required as an energy source in the lower loop to make NAD), the key roles of SIRT1, and CD38 which eats up NAD+.

NADPH – is the reduced form of NADP⁺. “NADPH provides the reducing equivalents for biosynthetic reactions and the oxidation-reduction involved in protecting against the toxicity of ROS (reactive oxygen species), allowing the regeneration of GSH (reduced glutathione).^[3] NADPH is also used for anabolic pathways, such as lipid synthesis, cholesterol synthesis, and fatty acid chain elongation. — The NADPH system is also responsible for generating free radicals in immune cells. These radicals are used to destroy pathogens in a process termed the respiratory burst.^[4] It is the source of reducing equivalents for cytochrome P450 hydroxylation of aromatic compounds, steroids, alcohols, and drugs(ref).”

NAADP – Nicotinic acid adenine dinucleotide phosphate “(NAADP) is one of the most potent stimulators of intracellular Ca²⁺ release known to date. The role of the NAADP system in physiological processes is being extensively investigated at the present time. Exciting new discoveries in the last 5 years suggest that the NAADP-regulated system may have a significant role in intracellular Ca²⁺ signaling. The NAADP receptor and its associated Ca²⁺ pool have been hypothesized to be important in several physiological processes including fertilization, T cell activation, and pancreatic secretion(ref).”

 NADPH oxidase – “Nicotinamide adenine dinucleotide phosphate-oxidase is a membrane-bound enzyme complex. It can be found in the plasma membrane as well as in the membranes of phagosomes used by neutrophil white blood cells to engulf microorganisms. — NADPH oxidase generates superoxide by transferring electrons from NADPH inside the cell across the membrane and coupling these to molecular oxygen to produce superoxide anion, a reactive free-radical. Superoxide can be produced in phagosomes, which contain ingested bacteria and fungi, or it can be produced outside of the cell. In a phagosome, superoxide can spontaneously form hydrogen peroxide that will undergo further reactions to generate reactive oxygen species (ROS)(ref).”  Plays important roles in generating ROS to deal with pathogens.

 NAMPT – “Nicotinamide phosphoribosyltransferase (NAmPRTase or NAMPT) also known as pre-B-cell colony-enhancing factor 1 (PBEF1) or visfatin is an enzyme that in humans is encoded by the PBEF1 gene.^[1] This protein has also been reported to be a cytokine (PBEF) that promotes B cell maturation and inhibits neutrophil apoptosis. — This enzyme participates in nicotinate and nicotinamide metabolism.(ref)”  NAMPT is the rate-limiting enzyme of the NAD(+) salvage pathway and enhances SIRT1 activity by increasing the amount of NAD+.

NMNAT – Nicotinamide/nicotinic acid mononucleotide adenylyltransferase  An enzyme that plays a role in NAD synthesis. “–  a rate-limiting enzyme present in all organisms, reversibly catalyzes the important step in the biosynthesis of NAD from ATP and NMN. NAD and NADP are used reversibly in anabolic and catabolic reactions(ref).”

SIRTUINS (Silent Information Regulators  SIRT1 – SIRT7)  Sirtuins extend the lifespans of lower ife forms including yeast, nematodes and drosphila, and appear to have pluripotent health effects in humans.  “Sirtuin or Sir2 proteins are a class of proteins that possess either mono-ADP-ribosyltransferase, or deacylase activity, including deacetylase, desuccinylase, demalonylase, demyristoylase and depalmitoylase activity.^[2][3][4][5] Sirtuins regulate important biological pathways in bacteria, archaea and eukaryotes. The name Sir2 comes from the yeast gene ‘silent mating-type information regulation 2′,^[6] the gene responsible for cellular regulation in yeast. Sirtuins have been implicated in influencing a wide range of cellular processes like aging, transcription, apoptosis, inflammation^[7] and stress resistance, as well as energy efficiency and alertness during low-calorie situations.^[8] Sirtuins can also control circadian clocks and mitochondrial biogenesis(ref).”  Sirtuins convert NAD(+) into nicotinamide (NAM).  The sirtuins vary in their functions and in general serve as deacetylases. We have written about them a number of times in this blog(ref), and we will comment further here on some of their specific functions.  SIRT1, in particular, the mammalian counterpart of SIR2, both depends on the presence of NAD+ for its activation and has important regulatory functions in the NAD salvage pathway.  “SIRT1 is required to regulate adaptive responses to acute and chronic energy limitations, such as fasting and dietary restriction.  In general, the evolutionary role of SIR2 ones is thought to be that they provide mechanisms through which an organism can adopt to changes in environment and diet. An example of the possible role of SIRT6 could be to allow animals to switch between vegetarian and meat diets. Sirtuins are completely dependent on the availability of NAD+ for their formation and thus are central actor’s in the NAD World. An important negative consequence of insufficient levels of NAD+ is insufficient levels of sirtuins and compromise of their important biological activities.”  A table showing the deacetylase and deacelase activities of the seven sirtuin sisters can be found on this site.

CCAR2 (Cell Cycle And Apoptosis Regulator 2, also known as DBC1) An important negative regulator of SIRT1 (ref) and a consumer of NAD+.

CD38  – “CD38 (cluster of differentiation 38), also known as cyclic ADP ribose hydrolase is a glycoprotein^[1] found on the surface of many immune cells (white blood cells), including CD4⁺, CD8⁺, B lymphocytes and natural killer cells. — CD38 is a multifunctional ectoenzyme that catalyzes the synthesis and hydrolysis of cyclic ADP-ribose (cADPR) from NAD⁺ to ADP-ribose. These reaction products are essential for the regulation of intracellular Ca^2+[5] (ref).”  CD38 can be a major consumer of NAD as a substrate.

MNA – Methylnicotinamide -  ” Methylnicotinamide is a metabolite of nicotinamide and is produced primarily in the liver. It has anti-inflammatory properties (PMID 16197374 ). It is a product of nicotinamide N-methyltransferase [EC 2.1.1.1] in the pathway of nicotinate and nicotinamide metabolism (KEGG). 1-Methylnicotinamide may be an endogenous activator of prostacyclin production and thus may regulate thrombotic as well as inflammatory processes in the cardiovascular system(ref).”

PARP – “Poly (ADP-ribose) polymerase (PARP) is a family of proteins involved in a number of cellular processes involving mainly DNA repair and programmed cell death. — The PARP family comprises 17 members (10 putative). They have all very different structures and functions in the cell.– PARP is found in the cell’s nucleus. The main role is to detect and signal single-strand DNA breaks (SSB) to the enzymatic machinery involved in the SSB repair. PARP activation is an immediate cellular response to metabolic, chemical, or radiation-induced DNA SSB damage. Once PARP detects a SSB, it binds to the DNA, and, after a structural change, begins the synthesis of a poly (ADP-ribose) chain (PAR) as a signal for the other DNA-repairing enzymes such as DNA ligase III (LigIII), DNA polymerase beta (polβ), and scaffolding proteins such as X-ray cross-complementing gene 1 (XRCC1). After repairing, the PAR chains are degraded via Poly(ADP-ribose) glycohydrolase(PARG).^[1]  — It is interesting to note that NAD+ is required as substrate for generating ADP-ribose monomers. The overactivation of PARP may deplete the stores of cellular NAD+ and induce a progressive ATP depletion and necrotic cell death, since glucose oxidation is inhibited. In this regard, PARP is inactivated by caspase-3 cleavage (in a specific domain of the enzyme) during programmed cell death. — PARP enzymes are essential in a number of cellular functions,^[2] including expression of inflammatory genes:^[3] PARP1 is required for the induction of ICAM-1 gene expression by smooth muscle cells, in response to TNF^[4] (ref).”

ATP – ” Adenosine triphosphate (ATP) is a nucleoside triphosphate used in cells as a coenzyme, often called the “molecular unit of currency” of intracellular energy transfer.^{[1]  —} ATP transports chemical energy within cells for metabolism(ref).”

ADP – Adenosine diphosphate, abbreviated ADP, is an important organic compound in metabolism and is essential to the flow of energy in living cells. — The cleavage of a phosphate group from ATP results in the coupling of energy to metabolic reactions and a by-product, a molecule of ADP.^[1] Being the “molecular unit of currency”, ATP is continually being formed from lower-energy molecules of ADP and AMP(ref).”.

AMP – Adenosine monophosphate,  NAD can be synthesized from it(ref),

“AMP, ADP, ATP – ATP consists of three phosphate groups attached in series to the 5’ carbon location, whereas ADP contains two phosphate groups attached to the 5’ position, and AMP contains only one phosphate group attached at the 5’ position. Energy transfer used by all living things is a result of dephosphorylation of ATP by enzymes known as ATPases. The cleavage of a phosphate group from ATP results in the coupling of energy to metabolic reactions and a by-product, a molecule of ADP.^[1] Being the “molecular unit of currency”, ATP is continually being formed from lower-energy molecules of ADP and AMP. The biosynthesis of ATP is achieved throughout processes such as substrate-level phosphorylation, oxidative phosphorylation, and photophosphorylation, all of which facilitating the addition of a phosphate group to an ADP molecule.”(ref)

AMPK - ” AMP-activated protein kinase (AMPK) plays a key role as a master regulator of cellular energy homeostasis. The kinase is activated in response to stresses tha`t deplete cellular ATP supplies such as low glucose, hypoxia, ischemia, and heat shock(ref).”

cAMP - “AMP can also exist as a cyclic structure known as cyclic AMP (or cAMP). Within certain cells the enzyme adenylate cyclase makes cAMP from ATP, and typically this reaction is regulated by hormones such as adrenaline or glucagon. cAMP plays an important role in intracellular signaling. (ref)

CREB – cAMP response element-binding protein^[1] – is a cellular transcription factor. It binds to certain DNA sequences calledcAMP response elements (CRE), thereby increasing or decreasing the transcription of the downstream genes.^[2] CREB was first described in 1987 as a cAMP-responsive transcription factor regulating the somatostatin gene^[3] (ref)”

 PGC1 alpha  – “PGC-1-alpha (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha)  “is a protein that in humans is encoded by thePPARGC1A gene.[1] The protein encoded by this gene is a transcriptional coactivator that regulates the genes involved in energy metabolism. This protein interacts with the nuclear receptor PPAR-gamma, which permits the interaction of this protein with multiple transcription factors. This protein can interact with, and regulate the activities of, cAMP response element binding protein (CREB) and nuclear respiratory factors (NRFs). It provides a direct link between external physiological stimuli and the regulation of mitochondrial biogenesis, and is a major factor that regulates muscle fiber type determination. This protein may be also involved in controlling blood pressure, regulating cellular cholesterol homoeostasis, and the development of obesity[2] (ref).  ”We have discussed PGC-1-alpha several times in this blog (ref).

TOP LEVEL POINTS

 My approach in this blog entry will be top-down, starting with general points that establish the importance of the topic to health and longevity – and finishing with some of the more-technical detail that conveys so much richness to the topic. Here is a list of top-level points that will be detailed in the following citations:

1.  NAD levels decline precipitously with aging in mammals and humans, and are strongly negatively affected by multiple disease and stress processes including obesity. At advanced ages these levels are a tiny fraction of what they are in young people.
2.  Maintaining a high level of constitutive NAD+ in the body is critical for maintaining health and functionality, for averting or reversing a number of deleterious disease phenomena, for energy and vitality, and possibly for extending life spans in humans.
3.  NAD+ is a key substrate for the production on SIRT1 and other sirtuins needed for proper histone deacetylation and gene regulation in response to changing conditions. Its availability is also critical for preventing a state of pseudohypoxia in cell nuclii and proper generation of mitochondrial proteins required for efficient electron transfer chain operation.
4.  Inadequate levels of NAD can negatively impact on DNA repair, runaway inflammation, normal metabolism, responses to oxidative stress, mitochondrial health and electron transfer chain efficiency in mitichondria, and lead to the Warburg Effect of pathological metabolism.
5.  NAD+ is needed for responsiveness to oxidative stresses and for control of inflammatory processes
6.  NAD+ deficiency thus impairs a. Proper DNA damage repair, b. Production of sirtuins and processes that depend on sirtuins for homeostasis and health, c. Control of gene activation, d. Mitochondrial functionality and viability, e. Capacity to deal with injuries and pathological situations, and f. ability to control excess inflammation.
7.  The rate of DNA damage increases significantly with aging, increasing demand for NAD+ for DNA repair. However in general, the reasons for age-related decline in NAD are not well understood.
8.  As a consequence of the above, inadequate levels of NAD can lead to a laundry list of disease processes, including type 2 diabetes, Alzheimer’s disease, and cancer. And to acceleration of aging.  Indeed, I venture an opinion that there is an unvirtuous cyclic process of interaction of the NAD-related feedback loops that manifests itself in the progressive acceleration of the processes of aging with aging itself – why we generally age faster and faster as we get older.
9.  There is a great deal of research knowledge about what generates NAD, what runs it down, its relationship to metabolism and other metabolic factors, the biological processes affecting it or affected by it, and how it’s expression relates to multiple disease processes.
10. It has long been known that many deleterious disease phenomena can be prevented or reversed by promoting higher levels of NAD in animal models. There appears to be ample evidence that this approach is generally safe. 
11.  For a number of years it has been proposed in research publications that promoting NAD levels in humans could be an effective preventative and/or therapeutic strategy.for multiple disease processes  Also, it is a provem strategy for extending the ifespans of c-elegans worms. However, this strategy has never entered our mainline medical or health maintenance system.
12.  Multiple strategies have been investigated for enhancing human levels of NAD, including enhancing its original synthesis in the body, downregulating biological processes that consume it, ingesting precursor molecules such as NMN or NR, and direct introduction of NAD into the bloodstream via IV.  At present, we do not know which of these approaches will be most efficacious and cost-effective. 

I will amplify on these points by citing selections from a great many relevant publications that describe aspects of the NAD World.  After this, I go on to second-level of selected key points that relate to the biological mechanisms involved – for example, the key systematic roles that NAD plays in regulating metabolism and aging,  and how NAD is consumed as an important substrate for DNA repair processes. Topics 11 and 12 above will mainly be discussed in further blog entries in this series.

RESEARCH RESULTS

 The 2013 publication The NAD⁺/sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling reports: “NAD⁺ is an important co-factor regulating metabolic homeostasis and a rate-limiting substrate for sirtuin deacylase. We show that NAD⁺ levels are reduced in aged mice and C. elegans and that decreasing NAD⁺ levels results in a further reduction in worm lifespan. Conversely, genetic or pharmacological restoration of NAD⁺ prevents age-associated metabolic decline and promotes longevity in worms. These effects are dependent upon the protein deacetylase sir-2.1 and involve the induction of mitonuclear protein imbalance as well as activation of stress signaling via the mitochondrial unfolded protein response (UPR^mt) and the nuclear translocation and activation of FOXO transcription factor DAF-16. Our data suggest that augmenting mitochondrial stress signaling through the modulation of NAD⁺ levels may be a target to improve mitochondrial function and prevent or treat age-associated decline. — Alterations in NAD⁺ levels have a powerful metabolic impact, since it serves as an obligatory substrate for the deacetylase activity of the sirtuin proteins (Guarente, 2008; Haigis and Sinclair, 2010; Houtkooper et al., 2010a). The best-characterized mammalian sirtuin is SIRT1, which controls mitochondrial function through the deacetylation of targets that include PGC-1α and FOXO (Chalkiadaki and Guarente, 2012; Houtkooper et al., 2012). The administration of NAD⁺ precursors, such as nicotinamide mononucleotide (Yoshino et al., 2011) or nicotinamide riboside (NR) (Canto et al., 2012), has proven to be an efficient way to increase NAD⁺ levels and SIRT1 activity, improving metabolic homeostasis in mice. Furthermore, the NAD⁺-consuming poly(ADP-ribose) polymerase proteins—with PARP1 and PARP2 representing the main PARP activities in mammals—were classically described as DNA repair proteins (Gibson and Kraus, 2012; Schreiber et al., 2006), but recent studies have linked these proteins to metabolism (Asher et al., 2010; Bai et al., 2011a; Bai et al., 2011b; Erener et al., 2012). Indeed, genetic or pharmacological inactivation of PARP1 increased tissue NAD⁺ levels and activated mitochondrial metabolism (Bai et al., 2011b). An association between PARPs and lifespan has been postulated (Grube and Burkle, 1992; Mangerich et al., 2010), but a causal role remained unclear. A final line of evidence in support of a role for NAD⁺ in metabolic control came from the deletion of an alternative NAD⁺-consuming protein, CD38, which also led to NAD⁺ accumulation and subsequent SIRT1 activation in mice, and proved protective against high-fat diet-induced obesity (Barbosa et al., 2007). — Considering the intimate link between metabolism and longevity (Guarente, 2008; Houtkooper et al., 2010b), we hypothesized that increasing NAD⁺ levels may be sufficient to increase mitochondrial activity and extend lifespan (Houtkooper and Auwerx, 2012). Here we show how supplementation of PARP inhibitors or NAD⁺ precursors led to improved mitochondrial homeostasis through the activation of the worm sirtuin homolog, sir-2.1. ” 

 Because the NAD World pathways are evolutionarily conserved in mammals and humans, there is reason to believe these results apply to us humans as well.

The 2013 publication The importance of NAMPT/NAD/SIRT1 in the systemic regulation of metabolism and ageing reports: “Ageing is associated with a variety of pathophysiological changes, including development of insulin resistance, progressive decline in β-cell function and chronic inflammation, all of which affect metabolic homeostasis in response to nutritional and environmental stimuli. SIRT1, the mammalian nicotinamide adenine dinucleotide (NAD)-dependent protein deacetylase, and nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting NAD biosynthetic enzyme, together comprise a novel systemic regulatory network, named the ‘NAD World’, that orchestrates physiological responses to internal and external perturbations and maintains the robustness of the physiological system in mammals. In the past decade, an accumulating body of evidence has demonstrated that SIRT1 and NAMPT, two essential components in the NAD World, play a critical role in regulating insulin sensitivity and insulin secretion throughout the body. In this article, we will summarize the physiological significance of SIRT1 and NAMPT-mediated NAD biosynthesis in metabolic regulation and discuss the ideas of functional hierarchy and frailty in determining the robustness of the system. We will also discuss the potential of key NAD intermediates as effective nutriceuticals for the prevention and the treatment of age-associated metabolic complications, such as type 2 diabetes.”

As mentioned above, sirtuins depend on the availability of NAD+ as a substrate and some of the negative consequences of insufficient NAD+ are associated with surtuin insufficiency.  Likewise, there is evidence that high expression of sirtuins associated with high levels of NAD+, SIRT1 and the others as well, convey a variety of longevity benefits.

One way this shows up is slowing or averting stem cell senescence.  The 2014 publication SIRT1 ameliorates age-related senescence of mesenchymal stem cells via modulating telomere shelterin reports: “Mesenchymal stem cells (MSCs) senescence is an age-related process that impairs the capacity for tissue repair and compromises the clinical use of autologous MSCs for tissue regeneration. Here, we describe the effects of SIRT1, a NAD(+)-dependent deacetylase, on age-related MSCs senescence. Knockdown of SIRT1 in young MSCs induced cellular senescence and inhibited cell proliferation whereas overexpression of SIRT1 in aged MSCs reversed the senescence phenotype and stimulated cell proliferation. These results suggest that SIRT1 plays a key role in modulating age-induced MSCs senescence. Aging-related proteins, P16 and P21 may be downstream effectors of the SIRT1-mediated anti-aging effects. SIRT1 protected MSCs from age-related DNA damage, induced telomerase reverse transcriptase (TERT) expression and enhanced telomerase activity but did not affect telomere length. SIRT1 positively regulated the expression of tripeptidyl peptidase 1 (TPP1), a component of the shelterin pathway that protects chromosome ends from DNA damage. Together, the results demonstrate that SIRT1 quenches age-related MSCs senescence by mechanisms that include enhanced TPP1 expression, increased telomerase activity and reduced DNA damage.”

The 2011 publication Dissecting systemic control of metabolism and aging in the NAD World relates “Accumulating bodies of evidence have suggested that SIRT1 plays an important role in retarding age-associated pathophysiological changes and preventing from diseases of aging, such as type 2 diabetes, Alzheimer’s disease, and cancer. Whole-body SIRT1-overexpressing transgenic mice show significant protection from the adverse effects of high-fat diet or aging on glucose metabolism [9] and [10]. SIRT1-activating compounds are also able to improve glucose homeostasis and insulin sensitivity in diet-induced and genetic type 2 diabetes animal models [9] and [10]. ”

From the same publication:  “It has recently been demonstrated that SIRT1 prevents two critical pathological aspects of Alzheimer’s disease: Aβ amyloid deposition and tauopathy. SIRT1 decreases the production of Aβ amyloid by deacetylating the retinoic acid receptor β and thereby up-regulating ADAM10, a major component of α-secretase [19]. SIRT1 also promotes degradation of phosphorylated tau by deacetylating it and prevents tau-mediated neurodegeneration [20]. Furthermore, SIRT1 regulates memory and synaptic plasticity, providing insight into potential intervention against age-associated cognitive disorders [21] and [22]. It has also been reported that SIRT1 transgenic mice show a lower incidence of spontaneous carcinomas and sarcomas and a reduced susceptibility to high-fat diet/carcinogen-induced liver tumors, compared to wild-type control mice [23]. These findings provide strong support for the importance of SIRT1 in the prevention of major age-associated diseases.”

The 2011 publication  NAD+ treatment decreases tumor cell survival by inducing oxidative stress reports: “NAD+ plays important roles in various biological processes. It has been shown that NAD+ treatment can decrease genotoxic agent-induced death of primary neuronal and astrocyte cultures, and NAD+ administration can reduce ischemic brain damage. However, the effects of NAD+ treatment on tumor cell survival are unknown. In this study we found that treatment of NAD+ at concentrations from 10 micromolar to 1 mM can significantly decrease the survival of various types of tumor cells such as C6 glioma cells. In contrast, NAD+ treatment did not impair the survival of primary astrocyte cultures. Our study has also indicated that oxidative stress mediates the effects of NAD+ on the survival of tumor cells, and P2X7 receptors and altered calcium homeostasis are involved in the effects of NAD+ on the cell survival. Collectively, our study has provided the first evidence that NAD+ treatment can decrease the survival of tumor cells by such mechanisms as inducing oxidative stress. Because NAD+ treatment can selectively decrease the survival of tumor cells, NAD+ may become a novel agent for treating cancer.”

The 2012 publication Age-associated changes in oxidative stress and NAD+ metabolism in human tissue reports: “Nicotinamide adenine dinucleotide (NAD(+)) is an essential electron transporter in mitochondrial respiration and oxidative phosphorylation. In genomic DNA, NAD(+) also represents the sole substrate for the nuclear repair enzyme, poly(ADP-ribose) polymerase (PARP) and the sirtuin family of NAD-dependent histone deacetylases. Age associated increases in oxidative nuclear damage have been associated with PARP-mediated NAD(+) depletion and loss of SIRT1 activity in rodents. In this study, we further investigated whether these same associations were present in aging human tissue. Human pelvic skin samples were obtained from consenting patients aged between 15-77 and newborn babies (0-1 year old) (n = 49) previously scheduled for an unrelated surgical procedure. DNA damage correlated strongly with age in both males (p = 0.029; r = 0.490) and females (p = 0.003; r = 0.600) whereas lipid oxidation (MDA) levels increased with age in males (p = 0.004; r = 0.623) but not females (p = 0.3734; r = 0.200). PARP activity significantly increased with age in males (p<0.0001; r = 0.768) and inversely correlated with tissue NAD(+) levels (p = 0.0003; r = -0.639). These associations were less evident in females. A strong negative correlation was observed between NAD(+) levels and age in both males (p = 0.001; r = -0.706) and females (p = 0.01; r = -0.537). SIRT1 activity also negatively correlated with age in males (p = 0.007; r = -0.612) but not in females. Strong positive correlations were also observed between lipid peroxidation and DNA damage (p<0.0001; r = 0.4962), and PARP activity and NAD(+) levels (p = 0.0213; r = 0.5241) in post pubescent males. This study provides quantitative evidence in support of the hypothesis that hyperactivation of PARP due to an accumulation of oxidative damage to DNA during aging may be responsible for increased NAD(+) catabolism in human tissue. The resulting NAD(+) depletion may play a major role in the aging process, by limiting energy production, DNA repair and genomic signalling.”

What eats up and depletes NAD?

 In the normal operation of the NAD salvage cycle, NAD is conserved, shuttled back and forth between its two forms NAD+ and NADH.  In this process, NAD acts as a cofactor in a major metabolic cycle.  In other vital processes, however, NAD acts as a substrate and is consumed. It is quite possibly the case that age-related decline in levels of NAD are mainly associated with increased demand by those substrate processes with aging. Here I discuss consumption of NAD by 1. PARPS, 2. Production of sirtuins, and 3. CD38  I also mention 4. CCAR2, a negative regulator of SIRT1 which can nullify health benefits of the availability of NAD+.

1.  PARPS are major consumers of NAD+, using it for DNA repair.

 Probably they are the largest consumer of NAD+, especially in older people where the need for DNA repair accelerates with age.  As a matter of fact, one strategy for increasing levels of NAD in mice is to inhibit PARPs,  Although this strategy seems to increase lifespans of mice by making more NAD available to mitochondria, I suspect it is a shortsighted approach when it comes to human health and longevity,  ” Nuclear poly(ADP ribose) polymerase 1 (1) generates polyADP ribose and probably accounts for the majority of nuclear NAD⁺ degradation. Direct interaction with NMNAT1 facilitates NAD⁺ supply to activated PARP1.(ref)”

The 2011 publication PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation reports: “SIRT1 regulates energy homeostasis by controlling the acetylation status and activity of a number of enzymes and transcriptional regulators. The fact that NAD(+) levels control SIRT1 activity confers a hypothetical basis for the design of new strategies to activate SIRT1 by increasing NAD(+) availability. Here we show that the deletion of the poly(ADP-ribose) polymerase-1 (PARP-1) gene, encoding a major NAD(+)-consuming enzyme, increases NAD(+) content and SIRT1 activity in brown adipose tissue and muscle. PARP-1(-/-) mice phenocopied many aspects of SIRT1 activation, such as a higher mitochondrial content, increased energy expenditure, and protection against metabolic disease. Also, the pharmacologic inhibition of PARP in vitro and in vivo increased NAD(+) content and SIRT1 activity and enhanced oxidative metabolism. These data show how PARP-1 inhibition has strong metabolic implications through the modulation of SIRT1 activity, a property that could be useful in the management not only of metabolic diseases, but also of cancer.”

2.  Sirtuins also draw down on NAD+ as a substrate, although the exact mechanisms of sirtuin activation are not clear to me.

 From the 2011 publication Mammalian Sirtuins and Energy Metabolism “Despite diverse subcellular localizations and a broad range of substrate specificities, the activity of all sirtuins is directly controlled by cellular NAD⁺ levels, which is an indicator of cellular metabolic status. The activity of these enzymes is also inhibited by their common enzymatic product, nicotinamide (19), and possibly by NADH (20). It is therefore not surprising that the activity of sirtuins changes in response to environmental cues that impact cellular metabolic state.”

The 2011 publication Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats reports: “The cofactor nicotinamide adenine dinucleotide (NAD+) has emerged as a key regulator of metabolism, stress resistance and longevity. Apart from its role as an important redox carrier, NAD+ also serves as the sole substrate for NAD-dependent enzymes, including poly(ADP-ribose) polymerase (PARP), an important DNA nick sensor, and NAD-dependent histone deacetylases, Sirtuins which play an important role in a wide variety of processes, including senescence, apoptosis, differentiation, and aging. We examined the effect of aging on intracellular NAD+ metabolism in the whole heart, lung, liver and kidney of female wistar rats.  Our results are the first to show a significant decline in intracellular NAD+ levels and NAD:NADH ratio in all organs by middle age (i.e.12 months) compared to young (i.e. 3 month old) rats. These changes in [NAD(H)] occurred in parallel with an increase in lipid peroxidation and protein carbonyls (o- and m- tyrosine) formation and decline in total antioxidant capacity in these organs.  An age dependent increase in DNA damage (phosphorylated H2AX) was also observed in these same organs. Decreased Sirt1 activity and increased acetylated p53 were observed in organ tissues in parallel with the drop in NAD+ and moderate over-expression of Sirt1 protein.  Reduced mitochondrial activity of complex I-IV was also observed in aging animals, impacting both redox status and ATP production.  The strong positive correlation observed between DNA damage associated NAD+ depletion and Sirt1 activity suggests that adequate NAD+ concentrations may be an important longevity assurance factor.“

 The 2012 publication The NAD⁺-dependent protein deacetylase activity of SIRT1 is regulated by its oligomeric status reports “SIRT1, a NAD⁺-dependent protein deacetylase, is an important regulator in cellular stress response and energy metabolism. While the list of SIRT1 substrates is growing, how the activity of SIRT1 is regulated remains unclear. We have previously reported that SIRT1 is activated by phosphorylation at a conserved Thr522 residue in response to environmental stress. Here we demonstrate that phosphorylation of Thr522 activates SIRT1 through modulation of its oligomeric status. We provide evidence that nonphosphorylated SIRT1 protein is aggregation-prone in vitro and in cultured cells. Conversely, phosphorylated SIRT1 protein is largely in the monomeric state and more active. Our findings reveal a novel mechanism for environmental regulation of SIRT1 activity, which may have important implications in understanding the molecular mechanism of stress response, cell survival, and aging.”

3.  CD38 is a key enzyme sitting on the outside of the cell surface that is a major consumer of NAD that plays an important role in cell calcium homeostasis

 From Mouse embryonic fibroblasts from CD38 knockout mice are resistant to oxidative stresses through inhibition of reactive oxygen species production and Ca(2+) overload: “CD38 is a multifunctional enzyme that has both ADP-ribosyl cyclase and cADPR hydrolase activities, being capable of cleaving NAD(+) to cyclic ADP ribose (cADPR) and hydrolyzing cADPR to ADPR. It has been reported that there is markedly a reduction of cADPR and elevation of NAD in many tissues from CD38 knockout (CD38(-/-)) mice. Cyclic ADPR is a potent second messenger for intracellular Ca(2+) mobilization, and NAD is a key cellular metabolite for cellular energetic and a crucial regulator for multiple signaling pathways in cells. We hypothesize that CD38 knockout may have a protective effect in oxidative stresses through elevating NAD and decreasing cADPR. In the present study, we observed that the mouse embryonic fibroblasts (MEFs) from CD38(-/-) mice were significantly resistant to oxidative stress such as H(2)O(2) injury and hypoxia/reoxygenation compared with wild type MEFs (WT MEFs). We further found that production of reactive oxygen species (ROS) and concentrations of intracellular Ca(2+) ([Ca(2+)](i)) in CD38(-/-) MEFs were markedly reduced compared with WT MEFs during hypoxia/reoxygenation. Coincidence with these results, a remarkably lower mRNA level of Nox1, one of the enzymes responsible for ROS generation, was observed in CD38(-/-) MEFs. Furthermore, we found that transcription of Nox1 mRNA in WT MEFs could be elevated by calcium ionophore ionomycin in a dose-dependent manner, indicating that the expression of Nox1 mRNA can be regulated by elevation of intracellular [Ca(2+)]. Therefore we concluded that CD38(-/-) MEFs are resistant to oxidative stresses through inhibiting intracellular Ca(2+) overload and ROS production which may be regulated by Ca(2+)-mediated inhibition of Nox1 expression. Our data should provide an insight for elucidating the roles of CD38 in oxidative stresses and a novel perspective of dealing with the ischemia/reperfusion-related diseases.”

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Image and legend source    “CD38, a transmembrane glycoprotein with ADP-ribosyl cyclase activity, catalyses the formation of Ca²⁺ signalling molecules and triggers proliferation and immune responses in lymphocytes —  These results indicate that CD38 has a key role in neuropeptide release, thereby critically regulating maternal and social behaviours, and may be an element in neurodevelopmental disorders.”

NAD+ can be pumped out of cells to make CD38

The 2011 publication Connexin-43 hemichannels mediate cyclic ADP-ribose generation and its Ca2+-mobilizing activity by NAD+/cyclic ADP-ribose transport reports:  “The ADP-ribosyl cyclase CD38 whose catalytic domain resides in outside of the cell surface produces the second messenger cyclic ADP-ribose (cADPR) from NAD(+). cADPR increases intracellular Ca(2+) through the intracellular ryanodine receptor/Ca(2+) release channel (RyR). It has been known that intracellular NAD(+) approaches ecto-CD38 via its export by connexin (Cx43) hemichannels, a component of gap junctions. —  Our data suggest that Cx43 has a dual function exporting NAD(+) and importing cADPR into the cell to activate intracellular calcium mobilization.”

CD38 and its NAD+ and SIRT1 reducing effects can be inhibited by the phytosubstances quercetin and apigenin

The 2013 pubication Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome reports: “Metabolic syndrome is a growing health problem worldwide. It is therefore imperative to develop  new strategies to treat this pathology. In the past years, the manipulation of NAD(+) metabolism has emerged as a plausible strategy to ameliorate metabolic syndrome. In particular, an increase in cellular NAD(+) levels has beneficial effects, likely because of the activation of sirtuins. Previously, we reported that CD38 is the primary NAD(+)ase in mammals. Moreover, CD38 knockout mice have higher NAD(+) levels and are protected against obesity and metabolic syndrome. Here, we show that CD38 regulates global protein acetylation through changes in NAD(+) levels and sirtuin activity.  In addition, we characterize two CD38 inhibitors: quercetin and apigenin. We show that pharmacological inhibition of CD38 results in higher intracellular NAD(+) levels and that treatment of cell cultures with apigenin decreases global acetylation as well as the acetylation of p53 and RelA-p65. Finally, apigenin administration to obese mice increases NAD(+) levels, decreases global protein acetylation, and improves several aspects of glucose and lipid homeostasis. Our results show that CD38 is a novel pharmacological target to treat metabolic diseases via NAD(+)-dependent pathways.”

 4.  CCAR2  can be an important negative regulator of SIRT1

CCAR2 was until very recently called DBC1 after what was originally thought to be its main property (deleted in breast cabcer), so most of the research literature relating to it talks only about DBC1.

The 2012 publication Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase  reports: “The NAD(+)-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that isDBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD(+). We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex.”

The 2008 publication DBC1 is a negative regulator of SIRT1  reports: “The NAD-dependent protein deacetylase Sir2 (silent information regulator 2) regulates lifespan in several organisms1, 2, 3. SIRT1, the mammalian orthologue of yeast Sir2, participates in various cellular functions4, 5, 6, 7 and possibly tumorigenesis8. Whereas the cellular functions of SIRT1 have been extensively investigated, less is known about the regulation of SIRT1 activity. Here we show that Deleted in Breast Cancer-1 (DBC1), initially cloned from a region (8p21) homozygously deleted in breast cancers9, forms a stable complex with SIRT1. DBC1 directly interacts with SIRT1 and inhibits SIRT1 activity in vitro andin vivo. Downregulation of DBC1 expression potentiates SIRT1-dependent inhibition of apoptosis induced by genotoxic stress. Our results shed new light on the regulation of SIRT1 and have important implications in understanding the molecular mechanism of ageing and cancer.”

The DBC1–SIRT1 interaction increases following DNA damage and oxidative stress

The 2012 publication Regulation of SIRT1 activity by genotoxic stress reports: “SIRT1 regulates a variety of cellular functions, including cellular stress responses and energy metabolism. SIRT1 activity is negatively regulated by DBC1 (Deleted in Breast Cancer 1) through direct binding. However, how the DBC1–SIRT1 interaction is regulated remains unclear. We found that the DBC1–SIRT1 interaction increases following DNA damage and oxidative stress. The stress-induced DBC1–SIRT1 interaction requires the ATM-dependent phosphorylation of DBC1 at Thr 454, which creates a second binding site for SIRT1. Finally, we showed that the stress-induced DBC1–SIRT1 interaction is important for cell fate determination following genotoxic stress. These results revealed a novel mechanism of SIRT1 regulation during genotoxic stress.”  So, there is another unvirtuous loop:  if there is insufficient NAD+, the PARP DNA repair machinery will not work well creating greater oxidative stress which down-regulates SIRT1 further impairing the NAD salvage cycle.

 Interestingly, it apears that resveratrol can block the SIRT1 down-regulating effect of DBC1/CCAR2.

The 2014 publication Resveratrol delays Wallerian degeneration in a NAD(+) and DBC1 dependent manner  reports: “Axonal degeneration is a central process in the pathogenesis of several neurodegenerative diseases. Understanding the molecular mechanisms that are involved in axonal degeneration is crucial to developing new therapies against diseases involving neuronal damage. Resveratrol is a putative SIRT1 activator that has been shown to delay neurodegenerative diseases, including Amyotrophic Lateral Sclerosis, Alzheimer, and Huntington’s disease. However, the effect of resveratrol on axonal degeneration is still controversial. Using an in vitro model of Wallerian degeneration based on cultures of explants of the dorsal root ganglia (DRG), we showed that resveratrol produces a delay in axonal degeneration. Furthermore, the effect of resveratrol on Wallerian degeneration was lost when SIRT1 was pharmacologically inhibited. Interestingly, we found that knocking out Deleted in Breast Cancer-1 (DBC1), an endogenous SIRT1 inhibitor, restores the neuroprotective effect of resveratrol. However, resveratrol did not have an additive protective effect in DBC1 knockout-derived DRGs, suggesting that resveratrol and DBC1 are working through the same signaling pathway. We found biochemical evidence suggesting that resveratrol protects against Wallerian degeneration by promoting the dissociation of SIRT1 and DBC1 in cultured ganglia. Finally, we demonstrated that resveratrol can delay degeneration of crushed nerves in vivo. We propose that resveratrol protects against Wallerian degeneration by activating SIRT1 through dissociation from its inhibitor DBC1.”

Other relevant publications on DBC1/CCAR2 include:

• Deleted in breast cancer 1 (DBC1) protein regulates hepatic gluconeogenesis. (2014)
• DBC1 (Deleted in Breast Cancer 1) modulates the stability and function of the nuclear receptor Rev-erbα. (2013)
• Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase. (2012)
• Deleted in breast cancer-1 regulates SIRT1 activity and contributes to high-fat diet-induced liver steatosis in mice. (2010)

Age-related decline in NAD+

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Image and legend source  Correlation between NAD+ levels and Age in (A) Males (B) Females.. From: Age-Associated Changes In Oxidative Stress and NAD+ Metabolism In Human Tissue .
(A) NAD+ concentrations decline with age in males. NAD+ levels decreased significantly in males aged between 0–77 years (line a; p = 0.0007; n = 27). Pearson’s correlation coefficient for a normally distributed population, r = −0.769. The post-pubescent data for male subjects also showed a decline in NAD+ levels with age (line b; r = −0.706; p = 0.0001; n = 19). An exponential (first-order) least squares fit was used to generate the nonlinear trend lines (line a and b). (B)NAD+ concentrations decreased significantly with age (36–76) in post-pubescent females (p = 0.01; n = 22).Pearson’s correlation coefficient for a normally distributed population,r = −0.537. An exponential (first-order) least squares fit was used to generate the nonlinear trend line.”

NAD SYNTHESIS

in general, there are two major pathways via which NAD can be synthesized in the body: the de-novo pathway for synthesizing NAD from raw materials, and the salvage pathway which involves recycling NAD between its two states NAD+ and NADH.

The de-novo pathway.

Again from Dissecting Systemic Control of Metabolism and Aging in the NAD World: The Importance of SIRT1 and NAMPT-mediated NAD Biosynthesis:   “NAD is synthesized from three major precursors—tryptophan, nicotinic acid, and nicotinamide [38] and [39]. Lower eukaryotes and invertebrates, such as yeast, worms, and flies, use nicotinic acid, a form of vitamin B3, as a major NAD precursor, whereas mammals predominantly use nicotinamide, another form of vitamin B3, for NAD biosynthesis. In mammals, NAMPT initiates the major NAD biosynthesis pathway by converting nicotinamide and 5′-phosphoribosyl-1-pyrophosphate (5′-PRPP) to nicotinamide mononucleotide (NMN), which is the rate-limiting step in this NAD biosynthesis [11] and [12]. The second enzyme, nicotinamide/nicotinic acid mononucleotide adenylyltransferase (NMNAT), completes NAD biosynthesis by transferring adenine from ATP to NMN. There are three distinct isoforms for NMNAT, NMNAT1-3, which are localized in nucleus, cytoplasm, and mitochondria, respectively, suggesting that NAD biosynthesis mediated by NAMPT and NMNAT might be compartmentalized in each subcellular compartment [40] and [41].”  Later. we discuss how one possible strategy for enhancing body level of NAD is supplementation with NMN.  This has worked remarkably well to produce health benefits in small animals, but the substance is now extremely expensive.

PROPERTIES OF THE NAD SALVAGE CYCLE

A diagram for the salvage pathway has already been offered above.  the following diagram shows both the de-novo and the salvage pathway.

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Image source

Important aspects of the salvage cycle are 1.  The cycle is under circadian clock and SIRT1 control, 2.  salvage cycle activities regulate expression of several genes via SIRT1 promoter activities, 3.  ROS stress activating p53 is a regulator of the cycle, and 4.  The NAD World includes extra-cellular actions as well as ones in different cell compartments. We expand on each of these points.

1.  Circadian clock regulation of the NAD salvage cycle

The 2009 publication Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1  reports: “Many metabolic and physiological processes display circadian oscillations. We have shown that the core circadian regulator, CLOCK, is a histone acetyltransferase whose activity is counterbalanced by the nicotinamide adenine dinucleotide (NAD+)-dependent histone deacetylase SIRT1. Here we show that intracellular NAD+ levels cycle with a 24-hour rhythm, an oscillation driven by the circadian clock. CLOCK:BMAL1 regulates the circadian expression of NAMPT (nicotinamide phosphoribosyltransferase), an enzyme that provides a rate-limiting step in the NAD+ salvage pathway. SIRT1 is recruited to the Nampt promoter and contributes to the circadian synthesis of its own coenzyme. Using the specific inhibitor FK866, we demonstrated that NAMPT is required to modulate circadian gene expression. Our findings in mouse embryo fibroblasts reveal an interlocked transcriptional-enzymatic feedback loop that governs the molecular interplay between cellular metabolism and circadian rhythms.”

The 2010 publication Clocks  in the NAD World: NAD as a metabolic oscillator for the regulation of metabolism and aging relates: ” Most recently, it has been demonstrated that SIRT1 regulates the amplitude and the duration of circadian gene expression through the interaction and the deacetylation of key circadian clock regulators, such as BMAL1 and PER2. More strikingly, we and others have discovered a novel circadian clock feedback loop in which both the rate-limiting enzyme in mammalian NAD biosynthesis, nicotinamide phosphoribosyltransferase (NAMPT), and NAD levels display circadian oscillations and modulate CLOCK:BMAL1-mediated circadian transcriptional regulation through SIRT1, demonstrating a new function of NAD as a “metabolic oscillator.” These findings reveal a novel system dynamics of a recently proposed systemic regulatory network regulated by NAMPT-mediated NAD biosynthesis and SIRT1, namely, the NAD World. In the light of this concept, a new connection between physiological rhythmicity, metabolism, and aging will be discussed.”

Here is a diagram of the situation:

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Image source ” The circadian clock controls the expression of nicotinamide phosphoribosyltransferase (NAMPT), which encodes the rate-limiting enzyme in mammalian NAD⁺ biosynthesis from nicotinamide. NAMPT catalyses the transfer of a phosphoribosyl residue from 5-phosphoribosyl-1-pyrophosphate (PRPP) to nicotinamide to produce nicotinamide mononucleotide (NMN), which is then converted to NAD⁺ by nicotinamide mononucleotide adenylyltransferases (NMNATs; there are three NMNAT genes). Oscillations in NAMPT levels result in circadian variations in NAD⁺ levels, which determines the activity of sirtuin 1 (SIRT1) and poly(ADP-ribose) polymerases (PARPs). Therefore, SIRT1 determines the oscillatory levels of its own coenzyme, NAD⁺ (Ref. 23). SIRT1 can also deacetylate and regulate proteins involved in metabolism and cell proliferation. Orange indicates circadian oscillation. FOXO1, forkhead box O1; LXR, liver X receptor; PPARGC1α, PPARγ co-activator 1α; PPi, pyrophosphate.”

Note that SIRT1 is intrinsic to the operation of the cycle, both regulating the cycle and being regulated by it.

Here is another Depiction of clock regulation of the salvage cycle via BMAL1 and CLOCK:

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Image and legend source  source   ” A circadian oscillatory feedback loop regulated by NAMPT, SIRT1, and CLOCK/BMAL1 and a possible functional interplay between adipose tissue and two frailty tissues in the NAD World, pancreatic β cells and neurons. NAMPT and NAD levels display circadian oscillations that are regulated by CLOCK/BMAL1 in peripheral tissues, such as the liver and WAT. This NAD oscillation periodically activates SIRT1, which represses CLOCK/BMAL1-mediated transcription of clock target genes, including Nampt itself, completing an interlocked transcriptional-enzymatic feedback loop involving NAMPT-NAD and SIRT1-CLOCK/BMAL1. In pancreatic β cells and central neurons, intracellular NAMPT (iNAMPT) levels are so low [45] that they may not be able to drive the NAMPT–NAD–SIRT1-dependent circadian feedback loop. However, their NAD oscillation might be generated by incorporating NMN that is likely synthesized from nicotinamide (Nic) by extracellular NAMPT (eNAMPT) that could be periodically secreted from adipose tissue.”

2.  The salvage pathway and SIRT1 regulation of gene expression. 

NAD+ regulates gene expression via histone deacetylase activity of SIRT1 at gene promoter sites.

The 2009 publication Enzymes in the NAD+ salvage pathway regulate SIRT1 activity at target gene promoters reports: “In mammals, nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide mononucleotide adenylyltransferase 1 (NMNAT-1) constitute a nuclear NAD(+) salvage pathway which regulates the functions of NAD(+)-dependent enzymes such as the protein deacetylase SIRT1. One of the major functions of SIRT1 is to regulate target gene transcription through modification of chromatin-associated proteins. However, little is known about the molecular mechanisms by which NAD(+) biosynthetic enzymes regulate SIRT1 activity to control gene transcription in the nucleus. In this study we show that stable short hairpin RNA-mediated knockdown of NAMPT or NMNAT-1 in MCF-7 breast cancer cells reduces total cellular NAD(+) levels and alters global patterns of gene expression. Furthermore, we show that SIRT1 plays a key role in mediating the gene regulatory effects of NAMPT and NMNAT-1. Specifically, we found that SIRT1 binds to the promoters of genes commonly regulated by NAMPT, NMNAT-1, and SIRT1 and that SIRT1 histone deacetylase activity is regulated by NAMPT and NMNAT-1 at these promoters. Most significantly, NMNAT-1 interacts with, and is recruited to target gene promoters by SIRT1. Collectively, our results reveal a mechanism for the direct control of SIRT1 deacetylase activity at a set of target gene promoters by NMNAT-1. This mechanism, in collaboration with NAMPT-dependent regulation of nuclear NAD(+) production, establishes an important pathway for transcription regulation by NAD(+).”

3.  NAD+ synthesis in the salvage cycle is in part mediated by ROS stress and p53 activation.

The 2014 publication The NAD+ synthesizing enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2) is a p53 downstream target  reports: “NAD(+) metabolism plays key roles not only in energy production but also in diverse cellular physiology. Aberrant NAD(+) metabolism is considered a hallmark of cancer. Recently, the tumor suppressor p53, a major player in cancer signaling pathways, has been implicated as an important regulator of cellular metabolism. This notion led us to examine whether p53 can regulate NAD(+) biosynthesis in the cell. Our search resulted in the identification of nicotinamide mononucleotide adenylyltransferase 2 (NMNAT-2), a NAD(+) synthetase, as a novel downstream target gene of p53. We show that NMNAT-2 expression is induced upon DNA damage in a p53-dependent manner. Two putative p53 binding sites were identified within the human NMNAT-2 gene, and both were found to be functional in a p53-dependent manner. Furthermore, knockdown of NMNAT-2 significantly reduces cellular NAD(+) levels and protects cells from p53-dependent cell death upon DNA damage, suggesting an important functional role of NMNAT-2 in p53-mediated signaling. Our demonstration that p53 modulates cellular NAD(+) synthesis is congruent with p53’s emerging role as a key regulator of metabolism and related cell fate.

4.  Action in the NAD World takes place in multiple cell compartments: the cytoplasm, the mitochondria and in the nucleus – and also in the plasma.

This point is illustrated in this diagram:

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Image and legend source   “The subcellular distribution of NAD+ biosynthetic enzymes in mammalian cells is shown on the left-hand side of the figure. All known biosynthetic reactions can take place in the cytosol, although conversion of nicotinamide (Nam) to NAD+ also takes place in the nucleus. However, NAD+ and its biosynthetic intermediates are likely to be freely exchangeable between the cytosol and the nucleus. Mitochondria contain nicotinamide mononucleotide (NMN) adenylyltransferase (NMNAT) activity and can therefore generate NAD+ from NMN. An extracellular form of Nam phosphoribosyltransferase (NamPRT), known as eNamPRT (also known as visfatin), is secreted from adipose tissue. Pyridine bases (Nam and nicotinic acid (NA)) and nucleosides (such as Nam riboside (NR)) enter cells by different transport mechanisms. The compartments of predominant NAD+ generation, the nucleus and mitochondria, also harbour the majority of intracellular NAD+-dependent signalling processes (right-hand side of the figure). Nuclear poly(ADP ribose) polymerase 1 (PARP1) generates polyADP ribose and probably accounts for the majority of nuclear NAD+ degradation. Direct interaction with NMNAT1 facilitates NAD+ supply to activated PARP1. Members of the sirtuin family are found in the nucleus, the cytosol and the mitochondria. SIRT1 is a key regulator of gene expression by deacetylating histone and non-histone targets such as p53. SIRT6 is also a nuclear protein. Among others, it activates PARP1, thereby facilitating DNA repair. SIRT7 is mostly localized to the nucleolus. SIRT2 localizes to the cytosol. SIRT3, SIRT4 and SIRT5 are located in mitochondria. SIRT3 is the major deacetylase in these organelles and a key regulator of metabolic pathways, including β-oxidation of fatty acids and the tricarboxylic acid cycle (TCA). Moreover, it deacetylates and activates superoxide dismutase 2 (SOD2), thereby suppressing reactive oxygen species (ROS) production. CD38, the main mammalian NAD+ glycohydrolase, and the majority of monoADP ribosyltransferases (mARTs) are found on the cell surface. All signalling reactions generate Nam as a common product, which can be recycled into the NAD+biosynthesis pathway by NamPRT.”

THE AVAILABILITY OF NAD+ IS CRITICAL FOR MITOCHONDRIAL FUNCTIONALITY

This has been known for a decade now.   The 2004 publication Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition  relates “—. In astrocytes, extracellular NAD(+) can raise intracellular NAD(+) concentrations. To determine whether NAD(+) depletion is necessary for PARP-1-induced MPT, NAD(+) was restored to near-normal levels after PARP-1 activation. Restoration of NAD(+) enabled the recovery of mitochondrial membrane potential and blocked both MPT and cell death. Furthermore, both cyclosporin A and NAD(+) blocked translocation of the apoptosis-inducing factor from mitochondria to nuclei, a step previously shown necessary for PARP-1-induced cell death. These results suggest that NAD(+) depletion and MPT are necessary intermediary steps linking PARP-1 activation to AIF translocation and cell death.”

The 2011 publication Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis  reported: “During the last several years, evidence that various enzymes hydrolyze NAD into bioactive products prompted scientists to revisit or design strategies able to increase intracellular availability of the dinucleotide. However, plasma membrane permeability to NAD and the mitochondrial origin of the dinucleotide still wait to be clearly defined. Here, we report that intracellular NAD contents increased upon exposure of cell lines or primary cultures to exogenous NAD (eNAD). NAD precursors could not reproduce the effects of eNAD, and they were not found in the incubating medium containing eNAD, thereby suggesting direct cellular eNAD uptake. We found that in mitochondria of cells exposed to eNAD, NAD and NADH as well as oxygen consumption and ATP production were increased. Conversely, DNA repair, a well known NAD-dependent process, was unaltered upon eNAD exposure. We also report that eNAD conferred significant cytoprotection from apoptosis triggered by staurosporine, C2-ceramide, or N-methyl-N’-nitro-N-nitrosoguanidine. In particular, eNAD reduced staurosporine-induced loss of mitochondrial membrane potential and ensuing caspase activation. Of importance, pharmacological inhibition or silencing of the NAD-dependent enzyme SIRT1 abrogated the ability of eNAD to provide protection from staurosporine, having no effect on eNAD-dependent protection from C2-ceramide or N-methyl-N’-nitro-N-nitrosoguanidine. Taken together, our findings, on the one hand, strengthen the hypothesis that eNAD crosses the plasma membrane intact and, on the other hand, provide evidence that increased NAD contents significantly affects mitochondrial bioenergetics and sensitivity to apoptosis.”

Declining NAD+ can lead to the collapse of mitochondrially-mediated energy metabolism,  The process appears to be reversible.

The December 2013 publication that catalyzed the current interest in NAD+ and mentioned at the begenning of this blog entry is Declining NAD⁺ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging.

“Highlights
• A specific decline in mitochondrially encoded genes occurs during aging in muscle
• Nuclear NAD+ levels regulate mitochondrial homeostasis independently of PGC-1α/β
• Declining NAD+ during aging causes pseudohypoxia, which disrupts OXPHOS function
• Raising nuclear NAD+ in old mice reverses pseudohypoxia and metabolic dysfunction

“Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1α under normoxic conditions, with parallels to Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1α/β-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.”

” In oncology, the Warburg effect is the observation that most cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation in the cytosol, rather than by a comparatively low rate of glycolysis followed by oxidation of pyruvate in mitochondria as in most normal cells.[4][5][6] The latter process is aerobic (uses oxygen). Malignant, rapidly growing tumor cells typically have glycolytic rates up to 200 times higher than those of their normal tissues of origin; this occurs even if oxygen is plentiful(ref).”

The following diagrams illustrate aspects of the NAD+ – mitochondrial connection:

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Image and legend source ” We show that, during aging, there is a specific loss of mitochondrial, but not nuclear, encoded oxphos subunits. We trace the cause to an alternate pgc-1a/b-independent pathway of nuclear-mitochondrial communication that is induced by a decline in nuclear NAD+ and the accumulation of hif-1a under normoxic conditions, with parallels to warburg reprogramming. Deleting sirt1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function to that of a young mouse in a sirt1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1a/b-independent nuclear-mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.”

The following two diagrams illustrates matters discussed above that go on in key cell compartments and outside the cell:

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Images and legend source ” Maintenance of the mitochondrial NAD pool. Although separate, the mitochondrial and nuclear/cytoplasmic NAD pools are intricately connected through the NAD/NADH-redox shuttles (most commonly the malate/aspartate and the glycerol-3-phosphate shuttles) and NAD biosynthetic pathways in each subcellular compartment. Multiple cellular processes play an important role in maintaining an optimal NAD/NADH ratio between mitochondria and the cytoplasm, including glycolysis, the tricarboxylic acid (TCA) cycle, and oxidative phosphorylation by the electron transport chain (ETC). Mitochondrial and nuclear/cytoplasmic NAD biosynthetic pathways are balanced in response to nutritional and environmental stimuli.”

Why this is all important for longevity is illustrated by these diagrams for mice:

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“Activity of brain mitochondrial enzymes that are markers of aging in relation to mice survival. Complex I, complex IV, and mitochondrial nitric oxide (NO) synthase (mtNOS) activities in male (A) and female (B) mice.”

More to come

This blog entry has covered some important basics related to the NAD world, but there is a great deal more to say about the topic. There will be at least one more blog entry in this series where we will go into further detail about the relationship of NAD+ to certain disease processes, discuss possible practical interventions for increasing pools of NAD+ especially in diseased or aging individuals, and discuss the key issue of how the efficacy of such interventions can be measured and dynamically tracked.

By James P Watson with contributions and assistance by Vince Giuliano

This is Part 2 of what will likely be a three or four-part series of blog entries related to the metabolic cofactor NAD+. The  Part 1 blog entry provided an overview treatment of the NAD World and its nuances: identified the major molecular entities involved, their roles, health and longevity ramifications, the reasons for the current excitement, and begin to clarify what is actually known and what the remaining uncertainties are.  We also discussed a few important facets of the general topic which are ignored in many of the current publications, such as the high degree of circadian regulation of NAD+ .and certain proteins that regulate NAD+ and its cousins such as CD38 and CCAR2,

Introduction to part two by Vince Giuliano.

Personally, I am finding that developing a thorough understanding of the World of NAD+, including what is relevant about all of the Sirtuin’s, cofactors and pathways, is one of the more-difficult intellectual challenges I have ever faced.  I am reminded of the process of  understanding quantum physics I went through as a university student.  And the difficulty of getting my mind around a complex of strange and completely non-intuitive concepts, dreaming strange dreams at night  until I was finally willing to live with what I could not completely fathom. I am reminded of the wisdom of the often-quoted Winston Churchill graduation speech to his high school where he virtually flunked out years earlier “never give up. Never give up. Never give up!  Never.  Never.  Never, (ref)” For personally I think the NAD World is extremely important pathway and I want to understand it to the limit of my capability and what is actually known. In that light, I acknowledge that this present blog entry covers much of the same territory as that already covered in the Part 1 blog entry.  However, the coverage is from a different set of perspectives and includes much new material.  While there is significant redundancy between what is discussed here and in the Part 1 blog entry, and additional redundancy within this blog entry itself, I believe this can contribute to understanding.

This blog entry concentrates on the reasons for focusing on NAD+, particularly with respect to interventions that are seriously likely to lead to longer healthier lives.  It discusses molecular processes in the NAD salvage cycle that are responsible for the health-inducing and life-extending properties of calorie restriction and further discuss the key roles of Sirtuins, SIRT1, SIRT6 and SIRT7 in particular. It goes into more detail in several areas covered less- deeply in the Part 1 blog entry. It points to some difficult-to-integrate contrarian research and basic uncertainties. Also, Jim amfd I are centrally concerned with developing a set of biomarkers that can tell us what the various approaches to enhancing body levels of NAD+ actually accomplish or fail to accomplish.  So many of our comments are addressed to this point.

We expect Part 3 will go into additional detail more explicitly describing a number of strategies currently being researched or proposed for maintaining high constituent levels of NAD+.  We expect to further explain reasons why simply enhancing NAD+ levels might not work to increase health or longevity and speculates on what else might have to be done in addition.  Finally, we expect further to discusses a variety of practical biomarkers to enable comparative evaluation of the NAD+ enhancement strategies, including information that might be provided by a new generation of health and fitness smart watches, smart bands, and other consumer measuring devices just now coming on the market.

Like many of our blog entries, this one is crammed full of detail. So, I would like to telegraph a few of the central take-home messages as I see them:

Our perception is that a big new area of longevity science may be opening up connected wih NAD, metabolisn and Sirtuins, one that may well offer new practical interventions that could lead to longer healthier lives. There are strong theoretical as well as experimental reasons for believing that enhancing body levels of NAD+ may have a chance of enhancing health and longevity while more conventional approaches do a limited job or fail.  We identify those reasons here.

1. There are several alternative approaches now being investigated to achieve such enhancement, including supplementation with NMN, or with nicotinamide riboside (NR) and direct IV infusion of NAD. We don’t know which one will be best. or even the extent to which any one really works in humans to enhance health and longevity.   Nonetheless, we probably are seeing a commercial rush to bring NMN supplements to the market, and NR is already being sold as a supplement.
2. There are additional theoretical reasons for suspecting that such benefits may not emerge or that they may require additional interventions for their realization. One approach for enhancing NAD+ levels may be far superior to the others.  For example many of the benefits of NAD plus supplementation can be attributed to higher activity of the Surtuin SIRT1.  However, what actually happens with Sirtuin expression is the result of a complex network of feedback-inhibition loops and many known factors can work to limit or eliminate Sirtuin levels or activity.  We have very poor understanding of how these feedback inhibition loops ultimately net out in live mice under various real-life conditions, let alone in us.
3. We offer many predictions below about probable health benefits of NAD+ supplementation. We need to acknowledge that some or all of these could be wrong.
4. For this last reasons, it is very important to develop a panel of practically measurable bottom-line biomarkers that tell us what a form of NAD+ enhancement is actually doing in our body to enhance health and possible longevity. And, to tell us other things we don’t understand well, such as how best to synchronize NAD+ enhancement with our circadian rhythms and additional interventions such as fasting and exercise. Otherwise, we will be flying blind with respect to the efficacy of NAD+ enhancement, much as we have been traditionally flying blind with respect to the efficacy of consuming dietary health supplements. We identify a number of such possible biomarkers in the course of this blog entry, although we leave discussion of practical means to measure these biomarkers to a subsequent blog entry in this series.

The Discovery of NAD+ and NAD+ Metabolism

 How NAD+ was discovered – Yeast fermentation of sugar

Four Nobel prize winners contributed to the discovery of NAD+^, starting with Sir Arthur Harden who studied yeast fermentation of sugar back in 1904 and isolated a low molecular weight fraction and a high molecular weight fraction, both of which were necessary for the fermentation to occur.  He did not know that the low molecular weight fraction was NAD+, but called this low molecular weight fraction “cozymaze”.  Later on Hans von Euhler-Chelpin  showed that cozymaze was made two mononucleotides, AMP and NMN. Then in 1936 Otto Warburg, showed that cozymaze was involved in hydrogen transfer (now called redox reactions).  In 1948, Arthur Kornberg showed that NAD+ was synthesized enzymatically within cells by an enzyme that consumed ATP and linked NMN with AMP to form NAD+. All of these scientists won Nobel prizes during their career. However, it would be another 55 years before the primary structure of the NAD biosynthetic enzyme (NMNAT) was determined. Thus the entire discovery process of NAD+ and NAD+ biosynthesis took over 100 years to get to where we are today.

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Popular but questionably effective downstream attempts to increase longevity and calorie restriction that actually does this

Next, again as background we review a) why antioxidants, vitamins, and hormones fail to reverse aging, b) the molecular pathways of calorie restriction (CR) which is the best known intervention for life extension, and c) how Sirtuins are the key molecular actors for health and longevity resulting from both calorie restriction and NAD+ enhancement.

Why Antioxidants, Vitamins, and Hormones failed to reverse aging – They treat the symptoms and not the causes

Although many drivers of human aging can be slowed or delayed, many of these factors are thought to be non-reversible reversible. (Ex: DNA gene mutations are not reversible). Most “anti-aging” supplements like anti-oxidants cannot reverse such aspects of aging.  Instead, they merely “control damage,” and many have a mixed track record of efficacy in clinical trials.  Likewise, hormone replacement therapy (HRT) does not reverse aging, despite the claims of some “anti-aging” clinics. In fact, emerging scientific studies have shown that exogenous anti-oxidant supplements and HRTs have a paradoxical effect. For instance, exercise has been shown to increase the expression of anti-oxidant genes and increase the expression of endogenous hormones (hGH, etc.). However, when exogenous anti-oxidants are used, this reduces the expression of anti-oxidant genes induced by the exercise producing a negative effect. Likewise, exogenous HRT use suppresses endogenous hormone production and thereby accelerates the decline in hormone gene expression that occurs with aging (via an epigenetic feedback-this is so good inhibition mechanism). This is why many testosterone users have testicular atrophy. Thus antioxidants and HRTs treat the symptoms (i.e. “downstream effects) of aging, rather than the cause (“upstream events”) of aging.

Current initiatives to restore NAD+ levels in individuals are attempts to affect what we believe are “upstream events” in aging to produce positive downstream events, such as already has been shown to be possible in various studies – like reversal of muscle aging in rodents.

Because nuclear NAD+ deficiency has so many impacts that mimic those of caloric restriction (CR), we look next at how it affects upstream events in molecular aging.  We are also looking at CR for ideas for the best biomarkers for objectively evaluating the “upstream effects” of restoring NAD+ levels in the nucleus.

Why Calorie Restriction (CR) increases life span  – CR affects the “upstream events” in molecular aging

Caloric restriction (CR) is the most scientifically-validated method of increasing life span and health span.  This involves reducing caloric intake by 10-40% (below RDA) without inducing protein-calorie malnutrition. The molecular mechanisms of CR are complex, but simply put, they impede and slow “pro-aging pathways” and amplify “longevity pathways”.  The two major “pro-aging pathways” are the Insulin/IGF-1 pathway and the mTOR pathway. CR inhibits both of these.  The “longevity pathways” involve AMPK,  Sirtuins, Glucagon, and Adiponectin. CR activates all of these. The diagram below shows the molecular mechanism of each of these factors in CR.

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The Role of Sirtuins in Caloric Restriction (CR) – Sirtuins are integrally involved with all of the CR pathways and are the main actors for both calorie restriction and NAD+ health and longevity interventions.

In the diagram above, there are three red ovals that comprise a major molecular pathway of CR involving the family of enzymes called Sirtuins. One red ovals contain the symbols for the one of the 7 Sirtuin enzymes called Sirtuin 1 (SIRT1).  Another red oval contains symbol for Nicotinamide adenine dinucleotide (NAD+), which is the substrate that activates all of the Sirtuin enzymes. Sirtuin enzymes consume NAD+, producing a by product called Nicotinamide (Nam), which inhibits SIRT enzymes.  The last red oval represents the enzyme called Nampt, which is the rate-limiting enzyme in the pathway that converts Nam back into NAD+, thereby renewing the supply of NAD+ to activate SIRT enzymes.  If you have read the Part 1 blog entry, you will recognize this pathway of regenerating NAD+ from Nam which is called the “NAD+ salvage pathway.” Activating these “red ovals” and restoring normal Sirtuin function is the goal of interventions intended to elevate or restore nulcear levels of NAD+.

The diagram above only shows one Sirtuin enzyme, SIRT1.  In reality, there are 7 NAD+-dependent Sirtuins (SIRT1-7) which all play a role in CR molecular mechanisms. Sirtuins affect almost every oval in the diagram below because they activate or inhibit other CR proteins by removing acetyl groups from these CR proteins.  In the diagram above, the small “Ac” stands for an “acetyl” group. SIRT1 directly affects all of the CR proteins that have an “Ac” on the diagram. It deacetylates and therefore tends to activate them.

For discussion of many of the benefits of Sirtuins, you can review the blogs on the series Slaying Two Dragons With One Stone (the Sound of Silence), Part 1, Part 2 and Part 3.

In addition to the major molecular pathway of CR on aging shown above, there are more important but lesser- known factors that change with CR, such as Klotho, p66shc, and DNA repair pathways. Although none of these pathways alone can account for the beneficial effects of CR, all of them probably work together to increase healthspan in all organisms studied to date.  It is the involvement of NAD+ as a required substrate for the Sirtuin enzymes that is the focus of initiatives to restore the nuclear levels of NAD+. 

Although the diagram does not illustrate this, NAD+ drives the Sirtuin-dependent deacetylation of many of the enzymes shown above, including eNOS, NF-kB, PGC-1a, Foxos, LKB1, and IRS1/IRS2. By NAD+-dependent deacetylation of these enzymes, Sirtuins thereby affect every major molecular pathway that involves aging. This is why many researchers believe restoring NAD+ levels in the cell is so important for affecting the “cause” of aging, rather than the “effects” of aging.

A 2012 review article, Cantó et. al.  Targeting SIRT1 to improve metabolism: all you need is NAD+? anallyzes ” the pros and cons of the current strategies used to activate SIRT1 and explore the emerging evidence indicating that modulation of NAD+ levels could provide an effective way to achieve such goals.”

The Two Roles of NAD+ – Contrasting the Cofactor vs Signaling role of NAD+

(Note that the Part 1 blog entry introduces the main actors in the cast of NAD World, molecular entities mentioned below, like NAD+, NADH, NADPH. ATP. etc.)

NAD+ does two things: a) it serves as a cofactor in redox and important metabolic reactions in which case it is shuttled back and forth between the NAD+ and NADH forms but is not consumed, and b) it serves as a signaling molecule in which case it is consumed as a substrate.

When NAD+ and NADP were discovered, they were thought to function only as a cofactor in redox reactions. Here they functioned as an “energy currency”, transferring high energy electrons from fuel oxidation (carbohydrates, fats, protein or alcohol) to the mitochondria (NADH) or the plasma membrane (NADPH) to generate ATP.  In these reactions, NAD(P)  “accepted” hydride groups (2 electrons and one proton) and NAD(P)H “donated” hydride groups, transferring the high energy electrons to another substrate.  Thus NAD/NADH was the recycled cofactor pair in the catabolic oxidation of carbohydrates, fatty acids, proteins, and alcohol.  On the other hand, NADP/NADPH was the recycled cofactor pair in the anabolic synthesis of fatty acids and cholesterol.  In these redox reactions, no NAD or NADP molecules are consumed.  Over 200 cellular enzymes utilize either NAD+/NADH or NADP/NADPH as a cofactor this way.

It was not until recently, however, that a second role for NAD+ as a signaling molecule was discovered. In these reactions, NAD+ does not accept hydride groups and the molecule is not recycled.  Instead, the NAD+ molecule is consumed as a substrate and the glycosidic bond between the ADP-ribose moiety and the nicotinamide moiety (NAM) of the molecule is broken, releasing NADM and ADP-ribose as byproducts of the reaction. Sirtuins are just one of many enzymes that “consume” NAD+ as a substrate.  The two contrasting roles for NAD+ as a cofactor vs a substrate are illustrated below:

The Redox/Co-Factor Role of NAD+ and NADPH  and The Signaling Compound/Substrate Role of NAD+

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What makes and what eats-up NAD+?  Causes of NAD+ deficiency with aging.

Although NAD+ can be made from the amino acid tryptophan by an 8-step pathway called the de novo pathway,  this pathway does not work very well in most cells.  For this reason, most of the consumed NAD+ is converted to nicotinamide and then “salvaged” by a two step process that converts it back into NAD+.  This pathway is called the salvage pathway and is rate-limited by the first enzyme in the two-step pathway called NAMPT.  NAMPT gene expression can be activated by caloric restriction (CR) or exercise and this may be a major reason why both CR and exercise have long term health benefits.  However even with exercise and caloric restriction, nuclear NAD+ levels decline with aging.

It is unclear now if the cause of age-related nuclear NAD+ deficiency is due to Sirtuins, PARPs, CD38, or a decline in the expression of NAMPT.  Most studies suggest that it is a combination of all four factors and that “genotoxic stress” is a major driving force for nuclear NAD+ deficiency.  The synergistic effects of genotoxic stress inducing single stranded DNA breaks and double-stranded DNA breaks and the combined effect of Sirtuins and PARPs is illustrated below:

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Also, NAD+ can be provided by an exogenous source such as an IV, and its level can possibly enhanced such as by NMN or NR supplementation as discussed below. And also as outlined below numerous factors can affect SIRT activation levels.

NAD+ and aging

Nuclear NAD+ decline and Aging – One cause of aging is thought to be the effect of NAD+-consuming enzymes in the cell nucleus

One of the “upstream events” in aging appears to be the decline in NAD+ within the nucleus of the cell. This appears to occur within all 37 trillion cells within the human body.  The decline is due to several classes of NAD+-consuming enzymes called Sirtuins, PARPs, and ADP-ribose glycohydrolases.  These enzymes perform absolutely essential functions required for cell health and each consumes NAD+ as a substrate.  Aging appears to be associated with increasing insufficiency of enough NAD+ to support the growing demands of these.  Sirtuins are a class of NAD+-consuming enzymes needed for double stranded DNA repair, for epigenetic gene silencing, and for chromatin remodeling.  Poly-ADP-ribose Polymerases (PARPs) is another class of NAD+-consuming enzymes involved with repair of single stranded break DNA damage.  PARPs also initiate cell death (apoptosis) in cancer cells and cells whose DNA is beyond repair.  More recently, a third type of NAD+-consuming enzyme has been found in the nucleus called CD38, once thought to only exist outside the cell.  CD38 is a part of the family of enzymes called ADP-ribose glycohydrolases.  Thus there are at least three families of enzymes all competing for the same pool of NAD+ within the nucleus of the cell.

Aging and NAD+ – Aging in the NAD world-view is due  to a “nuclear NAD+ deficiency” incurred from NAD+-consuming enzymes

For quite some time, caloric restriction (CR) has been regarded as the most scientifically-validated method to retard aging. CR research has lead to the discovery of several major molecular pathways that accelerate or retard aging, including the Insulin/IGF-1 pathway (age accelerator), the mTOR pathway (age accelerator), the AMPK pathway (aging brake), and Sirtuins (aging brake).  It is the role of NAD+ as a required substrate for Sirtuins that lead to the recent discovery that there is a nuclear NAD+ deficiency that affects not only Sirtuins, but another set of important NAD+ consuming enzymes called Poly-ADP-ribose Polymerases (PARPs).  Both the Sirtuins and the PARPs are involved with the DNA damage response to genotoxic stress.  Sirtuins play a major role in double-stranded DNA breaks whereas PARPs play a major role in single-stranded DNA breaks.  Here is a simplified diagram showing how both SIRTs and PARPs use up NAD+ in the nucleus of the cell:

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Restoring nuclear NAD+ levels may reverse aging

Recent work has shown that restoring nuclear NAD+ levels in aging mice actually reversed a common phenotypes of aging – that of mitochondrial dysfunction.  Mitochondrial dysfunction manifests itself as a decline in ATP production, a decline in heat production, excessive cellular free radical production, and increased DNA damage.

Although there are many proposed strategies for restoring nuclear NAD+ levels to normal, the only one that has been successfully accomplished this experimentally so far (with mice) is nicotinamide mononucleotide (NMN).  However, many experts believe that supplementation with nicotinamide riboside (NR) or directly with NAD will also restore nuclear NAD+ levels.  See the 2012 publication Canto et. al.  The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet induced obesity.  “As NAD⁺ is a rate-limiting cosubstrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38—both NAD⁺ consumers—increases NAD⁺ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD⁺ precursor with the ability to increase NAD⁺ levels, Sir2-dependent gene silencing, and replicative life span in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD⁺ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high-fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin NR could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.”

Others have suggested alternative strategies such as PARP inhibition, CD38 inhibition,  or increasing NAMPT activity.  We are interested in all of the above.  We are not interested in limiting our approach to one particular strategy.  Likewise, we do not believe that any particular NAD precursor, NAD metabolite, or enzyme inhibitor is a replacement for sleep, exercise, and caloric restriction, which have already been scientifically proven to help ameliorate the nuclear NAD+ problem.  Rather, we are interested in doing whatever it takes to restore nuclear NAD+ levels, using sleep, exercise, and CR as fundamental strategies and various NAD precursors, PARP inhibitors, and CD38 inhibitors as adjuncts to a healthy lifestyle.  Further, we strongly suspect that determination of what works best and under what conditions cannot be based on theory and will require monitoring a panel of biomarkers.  My personal  goal is not to increase lifespan but to increase healthspan, although increasing healthspan could increase lifespan.

This diagram projects an increase of lifespan possibly associated with NMN

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How NAD+ activates SIRTs and the competing “NAD+ consumers”

Based on the evidence from CR studies, there is strong scientific evidence that activating Sirtuins has major positive health effects and longevity benefits in most all animal models.  Although there are 7 different Sirtuin enzymes (SIRT1-7), SIRT1 is the most well understood member of this family of enzymes. All 7 Sirtuins require NAD+ as a substrate for the enzymes to work. Sirtuin enzymes consume NAD+, converting it into nicotinamide (NAM). NAM itself inhibits SIRT1 activity, but NAM can be “salvaged” and converted back into NAD+ (see diagram below). This conversion of NAM back into NAD+ is a two-step enzymatic process called the “NAD+ salvage pathway” described in our Part 1 blog entry and  covered more in additional detail here.

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Image source  NAD⁺ is consumed by enzymatic activity of all 7 of the Sirtuin isoforms (SIRT1-7). The consumption of NAD+ produces the byproduct, Nicotinamide (NAM) which can be “recycled” to NAD⁺  in the 2-step process shown above that is called the salvage cycle, which requires PRPP & ATP to run the cycle.

As discussed in the Part 1 blog entry, there are other enzymes that also require NAD+ as a substrate, such as the PARP family of enzymes as well as CD38 (see diagram below).  These other NAD+-consuming enzymes “compete” with SIRT enzymes in the nucleus and this competition may be the primary reason why nuclear NAD+ levels decline with aging.  Again, this is why restoring nuclear NAD+ levels is thought to be a good idea.

The above two diagrams illustrate the key “suspects” in the etiology of SIRT deficiency-induced aging.  As you can see, this “SIRT1 deficiency” is not a deficiency in the protein, but a deficiency in the activity of the protein.  It has been clearly shown that in most scenarios, there is no decline in SIRT1 protein availability with aging.  This may be because the SIRT1 protein can regulate its own gene expression.  This auto-regulation may be why the expression of SIRT1 protein does not decline with aging.

What is also a mystery is that aging also is associated with a decline in the expression of SIRT3.  It appears that the reason for this decline in SIRT3 expression may be due to the fact that SIRT1 regulates the gene expression of SIRT3. This is why many experts believe that the decline in SIRT1 activity is an “upstream event”, whereas the decline in SIRT3 is a “down stream events”.  The following possible causes for the decline in SIRT1 activity are illustrated in the diagrams above and listed in the table below:

Possible Causes of the Decline in SIRT1 activity with Aging1.

Decline in iNAMPT activity – The NAD+ salvage pathway has a rate-limiting enzyme called Nicotinamide phosphoribosyltransferase (NAMPT). Because there is both an intracellular and an extracellular localization of NAMPT, it is sometimes abbreviated as iNAMPT (intracellular NAMPT) and eNAMPT (extracellular form, which has also been called eNAMPT, PBEF, or Visfatin). eNAMP circulates in the blood and will be discussed elsewhere.  With iNAMPT, several independent studies have shown that iNAMPT gene expression and iNAMPT protein activity declines with aging and that this decline in iNAMPT activity precedes replicative senescence of cells, such as human vascular smooth muscle cells (VSMCs) (Eric van der Veer, Extension of Human Cell Lifespan by Nicotinamide Phosphoribosyltransferase 2007).  Even more interestingly, before the VSMCs became senescent, the decrease in NAMPT enzymatic activity was even greater than the decline in NAMPT gene expression with NAMPT enzyme activity dropping to 14% of baseline prior to VSMC senescence.   This is a very powerful argument that NAMPT is a “longevity protein” that extends the lifespan of VSMCs by activating SIRT1 and restraining the accumulation of p53 (SIRT1 mediates p53 degradation).

The “good news” is that there are 4 ways to increase NAMPT activity: caloric restriction (CR), fasting, exercise, and ATR1 blockade. The mechanism by which CR and fasting increase NAMPT is probably due to the fact that the SIRT1 protein regulates the NAMPT gene (Zhang, Enzymes in the NAD+ Salvage Pathway Regulate SIRT1 Activity at Target Gene Promoters  2009). The mechanism by which exercise induces an increase in NAMPT expression is via AMPK-mediated PGC-1α activity.  Athletes have a 2-fold higher levels of NAMPT in their skeletal muscle compared with sedentary adults (obese, non-obese, and T2DM). Three weeks of exercise training increases NAMPT protein in skeletal muscles by 127%.

AICAR, a potent “exercise mimetic”, increases skeletal muscle NAMPT mRNA by 3.4-fold. (Costford et.al., Skeletal muscle NAMPT is induced by exercise in humans, 2010). Recently, a fourth method to increase iNAMPT gene expression was found: angiotensin receptor 1 (ATR1) blockade. ATR1 blockers such as candesartan and     telmisartan increase expression of NAMPT and SIRT3 genes, increased NAD+ levels, increased mitochondrial biogenesis, and reduced atherosclersosis in in vivo studies.  Even more surprising was that in ATR1 gene knock-out mice, there was a 26% longer lifespan. (Benigni et.al., Disruption of the Ang II type 1 receptor promotes longevity in mice 2009).  This effect was not mediated by the usual CR  pathways, but appeared to be a CR independent mechanism, mediated by an increase in iNAMPT and SIRT3.

Conclusion:  CR, fasting, and regular exercise decelerates the deleterious effects of aging via SIRT1-dependent pathways by stimulating of NAD+ biosynthesis via increase in NAMPT gene expression.  ATR1 blockade increases NAMPT gene expression via an unknown, CR-independent mechanism.

2. Increase in PARP activity – There is recent evidence that a decline in NAD+ levels occurs in the nucleus of the cell with aging. As we have already indicted, the exact cause of this “nuclear NAD+ decline” is unknown and is the focus of research initiatives for restoration of nuclear NAD+. We have already discusssed the role of the PARPs as NAD+ consumers above and in the Part 1 blog entry.

SIRT1 – The Most Important Sirtuin in Aging and Age-related Diseases

Here, we review some of the most compelling reasons why SIRT1, the queen of the Sirtuins, is so important for health and longevity.

Depletion of SIRT1 by siRNA knockdown significantly alters the expression of about 200 genes (Zhang, Enzymes in the NAD+ Salvage Pathway Regulate SIRT1 Activity at Target Gene Promoters  2009). Here are some of the effects of SIRT1 on various organs in the body that have been scientifically proven in animal models. We believe that restoring nuclear NAD+ levels to normal will probably have all of these effects on the body, as well as many other positive effects, since NAD+ activates all 7 of the Sirtuins, not just the SIRT1 isoform.

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Image source: Sirtuins — novel therapeutic targets to treat age-associated diseases

As you can see from the diagram above, there are beneficial effects of SIRT1 activation on almost every organ system in the body. Many of these have to do with ameliorating the signs or symptoms of metabolic syndrome and type II diabetes mellitus. For those that are not familiar with Sirtuin proteins, it is important to understand that SIRT1 does not circulate as a hormone, but instead, is a protein largely confined to the nucleus of the individual cell.  In the cell nucleus, SIRT1 deacetylates many proteins that activate or inhibit gene expression.  These effects are thought to be largely but not exclusively  due to epigenetic mechanisms involving the deacetylation of histone proteins.  SIRT1 deacetylates many other proteins found in the nucleus other than histone proteins. The following sections will cover both the histone and the “non-histone” effects of SRIT1 in the nucleus of the cell. All of these benefits of SIRT1 are easier to understand if one familiarizes themselves with the fundamental aspects of epigenetics. This article does not go into detail about epigenetics, but it would be wise for the unfamiliar reader to delve into this topic first before proceeding to read the following sections.  Past blog entries related to epigenetics are listed here.  And the many prevous blog entries related to Sirtuins are listed here.  For now, we will focus on the specific effects of SIRT1 within the nucleus of a cell, as described below.  Then we’ll cover the other nuclear-localized members of the Sirtuin family, SIRT6 and SIRT7.

Major SIRT1 Functions #1: Preventing cellular senescence  –SIRT1 prevents cell cycle arrest via six mechanisms.  We first list these mechanisms and then review what cellular senescence is and further discuss why it is important, and what these SIRT1 impacts are.

1) SIRT1 helps repair double stranded DNA breaks, which is a major “upstream cause” of cellular senescence

2) SIRT1 epigenetically prevents expression of the p16^INK4a/ARF promoter by maintaining promoter hypermethylation

3) SIRT1 epigenetically prevents gene expression of p16^INK4a/ARF decetylation of H3K9 histone proteins

4) SIRT1 genetically activates Akt/p70S6K1 signaling, thereby inducing p16INK^4a/ARF repression

5) SIRT1 prevents p53-induced cell cycle arrest by deacetylating p53

6) SIRT1 prevents the formation of heterochromatin by deacetylating histone linker proteins (H1K24)

About cellular senescence

Cellular senescence is defined as “cell cycle arrest” and is a major hallmark of aging in all species. In old age, approximately 15-20% of cells in non-human primates show markers for cellular senescence (Herbig, Cellular Senescence in Aging Primates 2006). In human bone marrow stromal cells cultured in vitro, the percentage of senescent cells increases 4% per population doubling in old bone marrow cells vs only 0.4% per population doubling in young bone marrow cells (Stenderup, Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells  2003).

Some past blog entries discussing cell senescence and the roles of p16^INK4a/ARF are listed here.

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Image and legend source: Cellular Repair and Reversal of Aging: the Role of NAD  “Schematic illustration of NAD+-mediated sirtuins actions on NF-κB. During inflammation reduced levels of NAD+ do not impaired the hyperacetylation of NF-κB which in turn through promoter region of target genes activate pro-inflammatory pathway and senescence. Conversely, increased cellular levels of NAD+ activate SIRT1, 2 and 6 which deacetylate NF-κB inhibiting its transcriptional role and then inflammation.”

Although cell senescence has many beneficial, vital roles in normal life (embryogenesis, wound healing, cancer prevention, red blood cell aging, etc.) senescent cells are rare in young organisms.  In old age, all species start to accumulate old cells that will not divide (i.e. are senescent). Normally old cells undergo apoptosis, but these senescent cells are resistant to apoptosis, which means they hang around for longer than cells should. Moreover, they secrete a toxic mixture of secretions that are pro-inflammatory and actually help cancers grow (called the senescence associated secretory phenotype, aka SASP).  Other than genetic modifications of cells, no “cure” for the accumulation of senescent cells has been found to date.  For this reason, many researchers have believed the best way to prevent aging would be to prevent cellular senescence without increasing the risk of cancer.  Caloric restriction (CR) is the most effective method to date that has been discovered for preventing cellular senescence.  SIRT1 appears to be an important reason why CR prevents cellular senescence.

To understand cellular senescence on a molecular level, it is important to understand the major causes of cellular senescence, which includes DNA damage and telomere shortening. When either of these events occur, three key proteins trigger cell cycle arrest. They are p16INK^4a protein, the ARF protein, and the p53 protein.  Since p16INK4a and ARF are both transcribed from the same gene, I will discuss them together here. However, I will briefly discuss DNA damage and telomere shortening first.

The initial discovery of the phenomena of cellular senescence was by Leonard Hayflick when he noted that diploid fibroblasts in culture would only keep dividing for a finite number of “population doublings”, and then would stop dividing. The number of cell divisions that would trigger this phenomena was referred to as the “Hayflick limit”, but the cause was unknown until researchers discovered that telomeres shortened with each cell division due to the DNA “end replication problem”. Later on, an enzyme called telomerease was discovered that could counteract the telomere shortening that occurred with cell divisions.  The researchers who discovered telomerase won the Nobel prize in 2009, but since then, many other factors have been discovered that regulate telomere length (Ex: oxidative stress, the lncRNA called TERRA, Shelterin protein caps on the telomeres that protect the ends, Rap1, Tankyrases, SIRT6, histone protein silencing of telomeric DNA, etc.).

Ten years ago, many of us concerned with aging thought that since telomeres usually get shorter with age and shorter telomeres can lead to cell senescence, health and longevity could be enhanced by interventions that make telomeres longer.  That was a very exciting idea, but it turned out to be wrong.  We now know that telomere lengths are mostly downstream responses to upstream events, just like grey hair and wrinkles are downstream consequences of aging.   See the blog entries Nuclear Aging: The View from the Telomere end of the Chromosome –  Part 1 – context, history, and about telomere lengths and Part 2 – Telomere Molecular Biology and many other blog entries we have writtem about telomeres and telomerase for details.  There is just a possibility that we could be equally wrong about our new exciting idea – that of enhancing NAD+ as a health and longevity intervention.  Time will tell.

When telomeres shorten to a critical point, they trigger the DNA damage response (DDR), which activates p53 and can induce cellular senescence.  Later on, it was discovered that even non-dividing cells and cells with longer telomeres could undergo cellular senescence.  For instance, X-ray or gamma ray radiation can induce cellular senescence in cells with longer telomeres.  Likewise, oxidative stress from intrinsically-produced ROS or exposure to oxidative stress by external ROS (Ex: exogenous H₂O₂) can also trigger cellular senescence.   In these cases, the cause for the cellular senescence appears to be DNA damage.  Double stranded DNA breaks (DSBs) appear to be the most closely associated with cellular senescence, regardless of whether the DSBs were induced by ionizing radiation (X-rays, UV light, etc.), DNA damaging drugs (chemotherapy), or oxidative stress (endogenously generated or exogenously applied).

A key factor in cellular senescence is the increased expression of two proteins that trigger cellular senescence, p16INK4a and ARF. These two proteins are actually part of the same gene (i.e. they share exons) but because they are encoded in different reading frames, they have different amino acid sequences and different functions. As a result of this “shared gene”,  they are often referred to as the “INK4a/ARF locus”. p16INK4a is an inhibitor of the cyclin-dependent kinases CDK4 and CDK6.  ARF regulates p53 stability through inactivation of the p53-degrading ubiquitin ligase, MDM2.  In young cells and young organisms, the INK4a/ARF locus is epigenetically repressed by Polycomb proteins, but in old age, the INK4a/ARF locus is “derepressed”, the gene is transcribed, and production of these two proteins increases by as much as 42-fold in different organs in older mice (Krishnamurthy, Ink4a/Arf expression is a biomarker of aging 2004). In this context, the activities of SIRT1 listed above  (Numbers 2, 3, and 4) mitigate against cell senescence by repressing p16^INK4a/ARF or its effects. 

Interestingly, calorie restriction retards the increase in INK4a/ARF protein accumulation in all organs, reducing the age-associated increase in INK4a/ARF proteins by 2 and 16-fold. (Also Krishnamurthy, Ink4a/Arf expression is a biomarker of aging 2004).   Although the loss of Polycomb protein repression is a major reason for the increase in INK4a/ARF proteins, decline of SIRT1 expression plays a major role here as well.   For instance, glucose restriction (GR) has been found to increase SIRT1 mRNA levels and SIRT1 protein levels within cells and to inhibit p16INK^4a/ARF expression. As a result, cellular lifespan was found to be dramatically increased (by 4 weeks) in normal cells with GR, whereas the same glucose restriction increased apoptosis (18%) in immortalized cells causing them to die (Li, Tollefsbol, Glucose restriction can extend normal cell lifespan and impair precancerous cell growth through epigenetic control ofhTERT and p16 expression 2010)(Li, Tollefsbol, p16INK4a Suppression by Glucose Restriction Contributes to Human Cellular Lifespan Extension through SIRT1-Mediated Epigenetic and Genetic Mechanisms 2011).  When the molecular mechanisms were studied on the role of GR in repressing p16INK^4a/ARF expression, several mechanisms were found to play a role as follows:

1. Promoter site DNA (CpG) methylation

Glucose restriction induced hypermethylation of the p16INK4a/ARF promoter, which then prevents binding of the E2F-1 transcription factor.  This results in down-regulation of the p16INK4a/ARF locus, preventing cell apoptosis and cellular senescence, which leads to increased cell lifespan. (Li, et.al. as above, 2010).  Recently, other researchers have shown that SIRT1 can deacetylate DNA methyltransferase 1(DNMT1) and that compared to other HDACs, SIRT1 is the most robust deacetylator of DNMT1 (Peng, SIRT1 Deacetylates the DNA Methyltransferase 1 (DNMT1) Protein and Alters Its Activities  2011). “In summary, we show that DNMT1 is acetylated at multiple lysines and that SIRT1 deacetylates DNMT1 in vitro and in vivo. Deacetylation of DNMT1 at specific lysines enhanced its methyltransferase activity, changed its transcription repression activity and cell cycle regulatory function, and impaired its capacity to silence TSGs. In contrast to class I HDACs, which boost the silencing effect of DNMT1 by chromatin modification or stabilization of DNMT1 (59, 81), SIRT1 directly modifies DNMT1 activity.”

The same authors showed that nicotinamide, an inhibitor of SIRT1, reduced DNA promotor methylation by DNMT1. Since the main role of DNMT1 is to maintain CpG methylation (as opposed to de novo methylation),  Thus it is likely that SIRT1 plays a role in maintaining DNA promoter site (CpG) methylation of the p16 promoter, although this mechanism not yet been specifically proven for the INK4a/ARF locus.  Again, the key point of interest here is that supporting methylation and therefore inactivation of ther p16 promoter site is a likely action of SIRT1 for countering age-related cell senescence.

2. Promoter site Histone deacetylation

The best way to understand the role of SIRT1 in cellular senescence is to understand the molecular mechanisms of how  Sirtuin enzymes work. Although Sirtuins do have other molecular functions, their primary job is to remove acetyl groups from lysine amino acids on proteins. In the nucleus, SIRT1 removes acetyl groups (i.e. deacetylation) from many proteins, but the major target of SIRT1 are histone proteins (H1, H3, and H4). Histone proteins make up the “spools” (called nucleosomes) which DNA is wound around.  When the spools are tightly compacted together, genes cannot be transcribed from the DNA wound around the nucleosomes. This chromatin compacted state is called “heterochromatin”. When the nucleosomes are spread out and not compacted, genes can be transcribed from the DNA and this chromatin state is called “euchromatin”. SIRT1 plays a major role in determining if chromatin is in the heterochromatin vs the euchromatin state. Specifically, SIRT1 rmoves an acetyl group from histone subunit 1 (abbreviated as H1K24). When this occurs, the chromatin remains in the euchromatin state and can be transcribed.  When there is a nuclear deficiency of NAD+, H1K24 becomes acetylated and chromatin compaction (heterochromatin) occurs.  The formation of heterochromatin is a hallmark of cellular senescence and aging. This is illustrated in the diagram below.  On a molecular level, there is no clearer “upstream explanation for aging” than this diagramt, illustrating what happens in young cells when NAD+ levels are high (SIRT1 activation) and what happens in old age when nicotinamide levels are high (SIRT1 inhibition). The 4 main SIRT1 anti-aging effects involve protein deacetylation in the nucleus:

1) deacetylation of the histone linker protein, H1;

2) deacetylation of the  tumor suppressor, p53;

3) deacetylation of histone H4 at lysine 16 and

4) deacetylation of histone H3 at lysine 9.

When H1 is deacetylated, chromatin is relaxed and gene expression occurs (euchromatin). If p53 is deacetylated, it cell survival occurs and cell cycle arrest does not occur (i.e. senescence).  When H4K16 is deacetylated, genes can be silenced.  With low levels of nuclear NAD+,

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Image and legend source: Sirtuins: critical regulators at the crossroads between cancer and aging “SIRT1 expression and activity decrease during aging. High levels of SIRT1 protein in young cells in combination with high levels of nicotinamide adenine dinucleotide lead to deacetylation of p53 and histone proteins to promote longevity (left panel). SIRT1 levels are lower in aged cells, and higher levels of nicotinamide inhibit SIRT1 activity (right panel). The resulting hyperacetylation of p53 can induce replicative senescence. Deacetylation of histone H1 leads to its degradation that promotes formation of senescence-associated heterochromatic foci (SAHFs). Failure to downregulate SIRT1 during aging may promote cell survival after oxidative damage, leading to the accumulation of mutations, and an increased risk of cancer development.”

When there is inadequate SIRT1, H1K24 gets acetylated and the chromatin becomes compacted, forming “senescence associated heterochromatin” (SAH). SAH is a major cellular marker of aging. Thus the loss of SIRT1 H1K24 deacetylation directly causes this aspect of aging. However, if SIRT1 H2K24 deacetylation activity is maintained in old age, then SIRT1 becomes a “longevity protein”.  Unfortunately, with aging, there is a nuclear deficiency of NAD+.  As a result, SIRT1 cannot deacetylate H1K24 and senescent cells accumulate.

Note a point of clarification here on the different contexts for and meanings of deacetylation. The specific histone H1 relates to links between spindles of DNA,  Keeping it deacetylated via SIRT1 results in uncompacted chromatin which facilitates ready gene expression.  When it comes to histones H3 and H4, acetylation has the opposite effect; it locally uncompacts the chromatin and allows ready gene expression.  And deacetylation inhibits gene expression in these cases,  So, H3 and H4 deacetylation is very useful for silencing pro-aging genes such as senescence-associated ones.

Major SIRT1 Functions #2: Gene silencing  – H3K9 and H4K16 deacetylation in the nucleus

Two histone proteins that are deacetylated by SIRT1 are histone subunit 3 (H3) and subunit 4 (H4) . SIRT1 removes an acetyl group from a specific lysine (K9) on H3 and a specific lysine (K16) on H4.  H3K9 is a “site-specific” deacetylase target of SIRT1 and SIRT6, whereas  H4K16 is a “site-specific” deacetylase target of only SIRT1.  Both H3K9 and H4K16 deacetylation of histones have the effect of silencing genes as mentioned above.  See the discussion and the diagrams in the blog entry  Slaying Two Dragons with the Sound of Silence: – How to Keep Your Repetitive DNA Turned Off with “3 Songs”: Sirtuins, Polycomb Proteins, and DNMT3.

The major (non-histone) “longevity mechanism” is the effect of SIRT1 on p53, also shown below.

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In the diagram above, SIRT1 deacetyates the p53 protein thereby reducing its transcriptional activity.  p53 is the master “guardian of the genome”, and serves as a tumor suppressor to prevent cancer formation by shutting off the cell cycle in damaged cells. So, turning it off in cancer cells is not a good idea.  p53 also increases cell survival in non-cancerous cells and also activates apoptosis when the cells are old or too damaged to repair.

From the 2012 publication Current advances in the synthesis and antitumoral activity of SIRT1-2 inhibitors by modulation of p53 and pro-apoptotic proteins” — As sirtuins are involved in many physiological and pathological processes, their activity has been associated with different human diseases, including cancer. Especially two sirtuin members, SIRT1 and SIRT2, have been found to antagonize p53-dependent transcriptional activation and apoptosis in response to DNA damage by catalyzing p53 deacetylation. The findings that SIRT1 levels are increased in a number of tumors highlight the oncogenic role of sirtuins, in particular, in the down-modulation of p53 oncosuppressor activity. Along this lane, cancers carrying wild-type (wt) p53 protein are known to deregulate its activity by other mechanisms. Therefore, inhibition of SIRT1 and SIRT2, aimed at restoring wt-p53 transcriptional activity in tumors that retain the ability to express normal p53, might represent a valid therapeutic cancer approach specially when combined with standard therapies.”

In addition, SIRT1 deacetylates a specific site on the histone 4 subunit of the nucleosomes, which are the spools on which DNA is wound.  When SIRT1 deacetylates lysine at position 16 on the histone 4 protein (H4K16), this silences the gene on the DNA wound around this histone.  Thus SIRT1 has multiple effects all due to its ability to remove acetyl groups from proteins.  These are all dependent on the nuclear levels of NAD+ within the cell.

Image and legend source: Sirtuins: critical regulators at the crossroads between cancer and aging “ SIRT1 expression and activity decrease during aging. High levels of SIRT1 protein in young cells in combination with high levels of nicotinamide adenine dinucleotide lead to deacetylation of p53 and histone proteins to promote longevity (left panel). SIRT1 levels are lower in aged cells, and higher levels of nicotinamide inhibit SIRT1 activity (right panel). The resulting hyperacetylation of p53 can induce replicative senescence. Deacetylation of histone H1 leads to its degradation that promotes formation of senescence-associated heterochromatic foci (SAHFs). Failure to downregulate SIRT1 during aging may promote cell survival after oxidative damage, leading to the accumulation of mutations, and an increased risk of cancer development.”

Major SIRT1 Functions – #3: Circadian Clock Control – keeping the clocks in every cell regulated

We discussed the role of SIRT1 in controlling circadian clocks in the Part 1 blog entry,  Also, you can see the blog entries Circadian Regulation,NMN, Preventing Diabetes, and Longevity  and Shedding new light on circadian rhythms.   Pathways involved are illustrated in this diagram:

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Image source: Circadian integration of metabolism and energetics

Major SIRT1 Functions – #4 : Reducing Inflammation – inhibition of both glucose and fatty acid-induced inflammation.

Inflammation and aging seem to go hand-in-hand, a process that has been referred to as “inflammaging”.  Caloric restriction can reverse a significant amount of this inflammaging as a result of both the reduction in insulin/IGF-1 pathway signaling and also via a reduction in free fatty acid-induced inflammation.  Sirtuin activation associated with CR has been shown to have a major role in CR as a molecular mediator of this effect on inflammaging.  Here are some of the ways this occurs:

SIRT1 and Glucose-mediated inflammaging – Insulin resistance is the hallmark of this pathway of inflammaging.

SIRT1 improves insulin sensitivity in insulin resistant states by inhibiting protein tyrosine phosphatase 1B (PTP1B), which is a direct intracellular inhibitor of insulin action (This effect is only seen in insulin resistant states, however, and not in insulin sensitive states).  In addition, SIRT1 increases the circulating plasma hormone that is made in fat, called adiponectin, which increases insulin sensitivity.  Adiponectin also directly inhibits TNF-α and the conversion  of macrophages to foam cells.  This reduces adhesion molecules and the number of macrophages attached to endothelial cells.  These SIRT1-induced effects reduce the atherosclerotic effects of insulin, high sugar diet, and high fat diets.

Probably the greatest positive effect of SIRT1 on inflammaging is by its inhibition of NF-kβ signaling. Normally, insulin signaling increases inflammation via Akt-mediated activation of the “master inflammatory switch”, NF-kβ, a transcription factor that turns on all of the major inflammatory genes. However, when SIRT1 activity is increased, SIRT1 deacetylates NF-kβ and this inactivates the transcription factor.  As a result, all of the NF-kβ controlled inflammatory genes show less expression, including TNF-α, IL-6, C-reactive protein, and the cJun N-terminal Kinase (JNK) transcription factor. The diagram below illustrates this well:

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Image and legend source:  Sirtuins in neurodegenerative diseases: an update on potential mechanisms  ” Anti-inflammatory mechanisms of SIRT1. SIRT1 deacetylates p65 and blocks the transactivation of NF-κB-dependent gene expression. SIRT1 suppresses the activity of PARP-1, a coactivator of NF-κB-dependent transcription, by deacetylation and by inhibiting its expression. PARP-1 activation could deplete NAD+, resulting in inhibition of SIRT1 and NF-κB activation. On the epigenetic level, SIRT1 represses NF-κB-dependent inflammatory gene expression by deacetylating H4K16 and also by recruiting more components of repressor complexes. SIRT1 deacetylates and activates histone methyltransferase SUV39H1, which suppresses expression of inducible inflammatory genes. DNA methylation is associated with suppressed expression.”

In summary, SIRT1 has a net anti-inflammatory effect in glucose-mediated inflammaging and increases insulin sensitivity in insulin-resistant conditions, but not in baseline conditions of insulin-sensitive states.

SIRT1 and Fatty acid-mediated inflammaging – High fat diets have also been shown to induce inflammaging. Here the pro-inflammatory effects are independent from insulin and are mediated by intracellular free fatty acid overload within cells.  These high free fatty acid levels inhibit the forkhead transcription factor, FOXO1. As a result of FOXO1 inhibition, there is an increase in ROS, IL-6, PAI-1, and MCP-1 gene expression.  SIRT1 increases adiponectin levels which in turn, activates FOXO1 activity and increases the interaction between FOXO1 and C/EBPβ transcription factors.  In summary, SIRT1 has a net anti-inflammatory effect in fatty-acid mediated inflammaging and this effect is mediated primarily by adiponectin and the FOXO1 transcription factor.

Conclusion: Restoring NAD+ levels to normal in the nucleus of cells should increase insulin sensitivity, increase adiponectin levels, and reduce inflammation induced by both a high glucose and high fat diet.  We predict that biomarkers for insulin sensitivity (fasting blood sugar, 2-hour glucose tolerance testing, fasting insulin, 2-hour insulin tolerance testing, HOMA2, HOMA-IR, and Glycomark tests will improve with increases of nuclear NAD+, but only in insulin resistant states.  We also predict that biomarkers for inflammation will decline with such nuclear NAD+ increases, especially adiponectin (increase), TNF-α (decrease), IL-1β (decrease), IL-6 (decrease), IL-8 (decrease), andC-reactive protein (decrease).  However, if these values are normal in baseline conditions in a person undertaking to increase their nuclear NAD+, no change in these numbers will be evident.

Major SIRT1 functions: #5 – DNA repair – It is a key enzyme in repairing double stranded DNA breaks (DSBs)

Homologous recombination is the highest-fidelity mechanism for repairing DSCs.  See UBI et al.  Role of SIRT1 in homologous recombination (2010) ” — Recent reports revealed that SIRT1 also deacetylates several DNA double-strand break (DSB) repair proteins. — Using nuclear foci analysis and fluorescence-based, chromosomal DSBrepair reporter, we find that SIRT1 activity promotes homologous recombination (HR) in human cells. Importantly, this effect is unrelated to functions of poly(ADP-ribose) polymerase 1 (PARP1), another NAD(+)-catabolic protein, and does not correlate with cell cycle changes or apoptosis.”   That is, the effect is independent of the PARP-related mechanism for repairing single-stranded breaks.

Major SIRT1 functions: #6 – deacetylation nonhistone proteins – transcription factors, co-activators, co-repressors, methyl binding proteins, etc.

In addition to the effects of SIRT1 on histones, p53, and DNA repair, SIRT1 deacetylates many other proteins within the nucleus.  Most of these are transcription factors or co-activators which increase gene expression. They include the ones listed in this diagram:

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Image and legend source: Mammalian Sirtuins and Energy Metabolism  “ The diverse functions of SIRT1 in central nutrient sensing and peripheral energy metabolism. The activity of SIRT1 is regulated by the cellular metabolic status, small molecule activators, interacting proteins, as well as post-translational modifications. After activation, SIRT1 modulates a variety of metabolic activities systemically and locally through either direct protein deacetylation or indirect chromatin remodeling.”

Following is a list of the non-histone protein targets of SIRT1 shown above, along with several other CR proteins not shown in the diagram.  SIRT1 deacetylates over a dozen non-histone proteins, including p53, HSF1, eNOS, STAT3, FOXOs, PGC-1a, PPARγ, LXR, NF-kB, PER2, CLOCK, UCP2, MyoD.   Other effects of SIRT1 are paradoxic are the effects of SIRT1 on FOXA2, LKB1, and Nrf2 (E2F1).

p53^Ac – The most important non-histone target of SIRT1 is the protein called p53 already mentioned here. Both SIRT1 and SIRT2 deacetylate p53 and thereby increase cell survival.  SIRT1 deacetylates lysine 382 (K³⁸²) on the SIRT1 protein. On the other hand, inhibiting both SIRT1 and SIRT2 (but not one of these two SIRTs) induces cell death in both cancer cells and non-cancer cells. Thus p53 has often been referred to as the “master tumor suppressor” or the “guardian of the genome”.  Regardless of the terminology describing p53, it is clear that both SIRT1 and SIRT2 deacetylate p53 and promote cellular survival.  This is one of the most important functions of SIRT1/2 and is a fundamental reason why Sirtuins probably promote longevity (Peck, SIRT Inhibitors Induce Cell Death and p53 Acetylation through Targeting Both SIRT1 and SIRT2  2010).

HSF1^Ac – Heat shock factor 1 is a thermally sensitive protein that trimerizes migrates into the nucleus in response to heat stress or oxidative stress.

LXR^Ac – Caloric restriction has a positive effect on lowering cholesterol.  This is due to the fact that SIRT1 deacetylates the nuclear           receptor liver X receptor (LXR) protein

FXR^Ac - The farnesoid X receptor regulates the expression of a number of its target genes(ref).

eNOS^Ac – Reduced caloric intake decreases arterial blood pressures in healthy individuals and improves endothelium-dependent vasodilation in obese individuals.  Endothelial nitric oxide synthetase (eNOS) is an enzyme that makes nitric oxide from L-arginine, relaxes blood vessels and thereby lowers blood pressure with CR. L-arginine is a popular “anti-aging” supplement that is marketed as a “CR mimetic”, but has failed to lengthen life span or increase health span.  The likely reason for the “arginine failure” is that SIRT1 is needed to remove the “acetyl group” from lysines 496 (K⁴⁹⁶) and 506 (K⁵⁰⁶) from eNOS.   This can only be accomplished by activating SIRT1 with CR or fasting.  However, with old age, there is inadequate NAD+ to drive SIRT1 deacetylation.  It is for this reason that we predict that NAD+ or NAD+ precursors like NMN will lower blood pressure in healthy individuals and improve flow-mediated dilation (FMD) in both healthy and obese individuals.

FMD (flow-mediated dilation) can easily be measured with clinically validated testing devices such as the EndoPAT or the VENDYS systems, which use a brachial blood pressure cuff and finger sensors which measure reactive hyperemia index (EndoPAT) or finger temperature warming (VENDYS).  Both systems are clinically validated as non-invasive measurements of eNOS activity.

FOXA^Ac – The FOXA symbol in the diagram above is an upstream event that activates Glucagon signaling and inhibits Insulin signaling.  Reduced caloric intake reduces insulin levels yet increases insulin sensitivity, increases glucagon production, and improves pancreatic β-cell function via the forkhead transcription factor called FOXA2.  FOXA transcription factors play a key role in gene regulation of genes involved with glucose metabolism by binding to promoter regions on these genes with SIRT1. When nutrients are scare or when cells are starved, FOXOA2 can be acetylated in at least 7 different lysine residues. This increases FOXOA2 protein stability.  SIRT1deacetylates these lysine residues in times of CR or starvation.  NAD+ depletion disrupts this SIRT1-mediated stress response and results in excessive accumulation of FOXOA2 proteins. Restoring nuclear NAD+ levels should restore the normal interaction between FOXOA2 and SIRT1.  This could be measured with oral glucose tolerance testing (OGTT), insulin tolerance testing (ITT), fasting blood glucose and fasting insulin, as well as HbA1c and Glycomark testing.

FOXO^Ac – Caloric restriction, fasting, and reducing Insulin/IGF-1 pathway signaling all promote the nuclear localization of the Forkhead homeobox type O transcription factors (FOXOs).  FOXOs play a major role in cellular stress resistance by turning on genes that code for anti-oxidant enzymes, such as MnSOD (SOD2) and catalase.  FOXOs also control genes for apoptosis. Unfortunately, none of these benefits occur unless blood sugar levels are low and insulin/IGF-1 signaling is low.  In these conditions, the FOXOs can all migrate into the cell nucleus and turn on many genes responsible for cellular stress resistance.  Both SIRT1 and SIRT2 deacetylate FOXO transcription factors and thereby activate the FOXOs. For instance, SIRT1             deacetylates FOXO1 which then increases gluconeogenesis in the liver. SIRT2 deacetylates FOXO3a and also promotes hepatic gluconeogenesis.

We predict that restoring nuclear NAD+ levels with NAD+ precursor therapy will increase cellular stress resistance and this could be measured by stem cell survival with stem cell harvesting, stem cell transplantation, and stem cell banking (protection from freeze/thawing induced apoptosis).  This could be easily measured in the laboratory by culturing stem cells under conditions of cellular stress. 

LKB1^Ac – Liver kinase B1 (LKB1) is an enzyme that mediates much of the effect of mitochondrial biogenesis with CR or fasting. This occurs due to the fact that LKB1 is a direct activator of AMPK, the master regulator of metabolism and mitochondrial biogenesis.  Recently, it has been disclosed that LKB1 also functions as a tumor suppressor by maintaining epithelial integrity. In tumors where LKB1 is mutated, cells loose an orderly epithelial configuration and the cancers start to grow rapidly  LKB1 must be deacetylated by SIRT1 for proper tumor suppression and for AMPK activation. SIRT1 deacetylates lysine 48 (K48) on LKB1 and causes the LKB1 to leave the nucleus and go to the cytoplasm where it can associated with the adaptor protein STE20, activating itself and AMPK.

Thus, SIRT1 deacetylation of LKB1 induces LKB1 and AMPK activity in the cytoplasm. (Ruderman, SIRT1 Modulation of the Acetylation Status, Cytosolic Localization, and Activity of LKB1 2008). Both of these effects promote longevity and increase energy. For this reason, we predict that restoring nuclear NAD+ levels will have a direct effect on reducing age-induced fatigue.  This could be measured with a VO2 max measuring device and endurance testing.  We also predict that restoring nuclear NAD+ levels will re-organize skin cells that have lost their epithelial polarity, restoring better looking skin and improving skin histologic architecture on light microscopy.

MyoD^Ac – MyoD is a key transcription factor in skeletal muscle differentiation.

STAT3^Ac – Skeletal muscle insulin resistance is a key component of the underlying cause of type II diabetes. Fasting and CR both can increase whole body insulin sensitivity, even when practice for brief periods of time (4-20 days). Although there are multiple ways that CR changes Insulin/IGF-1 signaling, the primary method by which CR promotes insulin sensitivity appears to be via the (inhibition of )  STAT3.  SIRT1 deacetylates STAT3, which inactivates the transcription factor. This reduces gene expression for two subunits of the enzyme PI3K (p55a/p50a). This results in more efficient PI3K signaling with insulin stimulation.  In summary, SIRT1 inhibits STAT3 which inhibits gene expression for PI3K subunits, thereby increasing energy expenditure.

IRS1/IRS2^Ac – CR has a paradoxic effect on insulin receptor substrate 1 and 2 (IRS1/IRS2) gene expression. IRS1 and IRS2 are the first proteins in the molecular cascade of events that occurs when Insulin binds to the Insulin/IGF receptor on the cell.  With CR, however, there is an increase in protein expression of IRS1/IRS2 in muscle, which in turn increases intracellular signaling for the Insulin/IGF-1 pathway.  SIRT1 deacetylates IRS1/IRS2, thereby “turning off” this pro-aging pathway.

Nrf2^Ac (E2F1) – Sirtuins have a paradoxic effect on the transcription factor called Nuclear factor Erythroid 2-related factor 2 (Nrf2 or E2F1). Normally, cellular stress activates the nuclear localization of Nrf2 which then in turn activates gene transcription by binding to the antioxidant response element (ARE) found in the promoter sites of these genes. This can occur due to direct effect of ROS on Nrf2 binding to its binding partner, Keap-1. It can also occur by the acetylation of Nrf2 by p300/CBP (aka CREB-binding protein). Paradoxically, both SIRT1 and SIRT2 can deacetylate Nrf2, SIRT1 deacetylates at two specific  lysine residues (K⁵⁸⁸ and K⁵⁹¹) which promotes cytoplasmic localization of the Nrf2 protein and prevents nuclear localization of Nrf2. As a result, SIRT1 actually is an inhibitor of Nrf2  antioxidant gene transcription. This is one of the paradoxic effects of SIRT1 and CR on Nrf2 (Kawai, Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization 2010).

Another paradoxic effect is the effect of SIRT2 on Nrf2.  SIRT2 also deacetylates Nrf2 and therefore can decrease Nrf2 gene expression.  One of these genes is the major cellular iron exporters called FPN1.  Nrf2 activates FPN1 gene expression and SIRT2 inhibits FPN1 gene expression (Yang,  Sirtuin 2 mediated-deacetylation regulates cellular iron homeostasis

2014).  Thus SIRT2 controls regulates iron levels within the cell, decreasing iron export by FPN1, by deacetylating Nrf2.

p66^shc – The adaptor protein p66^shc is a major “pro-aging” factor within the cell.  When growth factor signaling or ROS activates p66^shc, the protein is phosphorylated on Serine (S).  This promotes mitochondrial migration of p66^shc into the mitochondrial matrix where it increases ROS production from mitochondria. SIRT1 decreases p66^shc activity by decreasing both p66^shc mRNA and p66^shc protein levels.  The molecular mechanism responsible for SIRT1 repression of p66shc gene expression is thought to be mediated by the epigenetic silencing of the p66shc gene by SIRT1-mediated histone deaetylation. Thus SIRT1 is a negative          regulator of ROS production by epigenetically repressing p66^shc gene expression (Xu, Salvianolic acid A preconditioning confers protection against concanavalin A-induced liver injury through SIRT1-mediated repression of p66shc in mice 2013) (Paneni, Molecular pathways of arterial aging 2015).

PGC-1αAc – Peroxisome proliferator activated receptor PPAR-γ co-activator-1α (PGC-1 α) is a master gene co-activator that works with over a dozen different transcription factors to turn on hundreds of genes involving peroxisome and mitochondrial biogenesis.  Thus, PGC-1α turns on all of the genes involving making and burning of fat, producing of ketones, making ATP, and generating energy.  SIRT1 also promotes gluconeogenesis in the liver by deacetylating PGC-1α (along with deacetylating FOXO1).  Earlier blog entries discussing PGC-1 α can be found in this list,

PPARγ – Caloric restriction also activates the master transcription factor for the production of peroxisomes, called peroxisome proliferator activator receptor gamma (PPARγ). PPARγ induces long chain fatty acid oxidation, which is the first step required before fats can be oxidized in the mitochondria by beta-fatty acid oxidation.  SIRT1 deacetylates PPARγ and then recruits a coactivator for PPARγ called Prdm16.  Then the PPARγ/Prdm16 complex can activate the genes required to make brown fat.  You can see the blog entry Getting skinny from brown fat,

NF-kβ – Nuclear factor kappa-β is a transcription factor that is the “master switch” for inflammation. SIRT1 deacetylates NF- kβ at the p65 subunit of NF-kB at lysine 310.  This “turns off” gene expression for all of the genes involving inflammation, such as CRP, TNF- α, IL-1β, etc.  Since arthritis is a universal feature of aging and all of these inflammatory biomarkers are elevated in joints with OA, we predict that restoring nuclear NAD+ with NAD+ precursor therapy will reduce the signs and symptoms of OA.  This could be measured with various validated clinical scoring systems, such as the K-L scoring system based on plain, X-rays with weight bearing, the WOMAC scale, the IKDC scale, etc.  All of these should improve with NMN or other NAD+ enhancing therapy.

Fine Tuning of SIRT1 Protein Activity

Endogeneous Activators and Inhibitors of Sirtuins – NAD+, NAM, AROS, lamin A, Tenovins, DBC1/CCAR2, CD38,  HIC1, Cathepsin D

Alas, in biology for any generalization there is too often one or more “An the other hands, —.”  We have discussed SIRT1expression here as if it were mainly driven by NAD+ level.   Actually, it is affected both positively and negatively by many interacting variables. SIRT1 activity is increased by increasing nuclear NAD+, by fasting, and by caloric restriction. Most of these positive effects on SIRT1 are mediated by increases in nuclear NAD+ levels. SIRT1 activity is also inhibited by nicotinamide. This may be why niacin supplementation has not been shown to have a longevity effect, since it is converted into nicotinamide within the cell.  In addition to activating SIRT1 with NAD+, there are naturally derived exogenous compounds such as reseveratrol and other plant-based compounds that bind to a different site on SIRT1, increasing it’s activity independently from NAD+. There are also several exogenous small molecule inhibitors of SIRT1, such as Tenovin-1 and Tenovin-6 which prevent the deacetylase activity of SIRT1. There are also endogenous proteins that bind directly to SIRT1 and increase or decrease the activity of SIRT1, such as AROS, lamin A, and DBC1.  AROS is a protein that binds to SIRT1 and increases its activity.  DBC1 (aka CCAR2) is a protein that binds to SIRT1 and inhibits SIRT1.  We discussed these two in the Part 1 blog entry.  HIC1 is a protein that binds to the SIRT1 promoter and increase SIRT1 gene expression. Another recently discovered protein that binds to SIRT1 is lamin A, a nuclear cytoskeletal protein that binds to SIRT1 and activates the protein.  Another endogenous inhibitor of SIRT1 is the enzyme CD38. Although initial reports labeled CD38 as an “ectoenzyme”, several reports have shown that the CD38 protein is found on the inner membrane of the nucleus of the cell. Aksoy and colleagues have  shown that CD38 is an SIRT1 inhibitor (Aksoy, Regulation of SIRT 1 mediated NAD dependent deacetylation: A novel role for the multifunctional enzyme CD38  2006).

In inflammatory joint diseases such as osteoarthritis and rheumatoid arthritis, there is another endogenous disruptor of SIRT1 – Cathepsin B.  Normally SIRT1 has a positive effect within the joint, inhibiting chondrocyte apoptosis and promoting extracellular matrix synthesis of proteins such as alpha2(I) collagen. However, when there are high levels of TNF-a within the joint, TNF-a mediates a Cathepsin-B induced cleavage of the SIRT protein at amino acid 533, creating a 75-kd SIRT1 fragment that is no longer functional.  As a result, there are low levels of SIRT1 activity within the chondrocytes and synovial cells of arthritic joints.

Some of these activators and inhibitors of SIRT1 activity are illustrated below:

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Image source: How does SIRT1 affect metabolism, senescence and cancer?

DBC1 (aka CCAR2) is one of the primary inhibitors of  SIRT1 within the nucleus and it is activated by DNA damage (genotoxic stress).  Fasting also inhibits the interaction of DBC1 and SIRT1.  This may be a NAD⁺-independent molecular mechanism for how fasting increases SIRT1 activity.  Likewise, a high fat diet activates SIRT1 binding to DBC1. This may be an NAD+-independent effect of a high-fat diet inhibiting Sirtuins.

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SIRT1 Phosphorylation – SIRT1 phosphorylation can increase or decrease SIRT1 activity and SIRT1 half life

In addition to the above factors, SIRT1 is modified  by at least 7 enzymes that phosphorylate SIRT1 at 13 “site-specific” locations on the SIRT1 protein. These enzymes phosphorylate different sites, and thereby either inactivate SIRT1 (AMPK),  activate SIRT1 (cAMP/PKA, Cyclin B/Cdk1, CK2, JNK1, DYRKs) or increase the half life of the SIRT1 protein (JNK2). For instance AMPK phosphorylates SIRT1 at Thr³⁴⁴, which inactivates the p53 deacetylase function of SIRT1.  On the other hand, DYRKs (Dual specificity tyrosine phosphorylation-regulated kinases) phosphorylate SIRT1 at Thr⁵²² in response to environmental stress. This DYRK-mediated phosphorylation prevents SIRT1 from forming oligomeric aggregates of SIRT1 proteins. As a result, the Thr⁵²² phosphorylated SIRT1 forms only monomers and thereby becomes a more active deacetylator of p53. Thus SIRT1 phosphorylation by AMPK and DYRKs have opposite effects on SIRT1 deacetylation of p53. Two phosphorylation sites on SIRT1 help regulate the cell cycle. Specifically, the CyclinB/Cdk1 complex phosphorylates SIRT1 at Thr⁵³⁰ and Ser⁵⁴⁰.  Thus CyclinB/Cdk1 phosphorylation of SIRT1 increases cell proliferation.  This is why SIRT1 is so important for cell growth and is present in high levels in mitotically active cells. Even the two cJun N-terminal kinases, JNK1 and JNK2, have different phosphorylation sites on SIRT1 and different effects. For instance, JNK1-mediated phosphorylation of the Ser⁴⁷ residue on SIRT1 increases the histone 3 (H3) deacetylase activity of SIRT1, whereas JNK2-mediated phosphorylation of the Ser²⁷ residue of SIRT1 increase the half life of the SIRT1 protein from 2 hours to 9 hours. Thus each of these different “SIRT1 phosphorylators” add a phosphate group at specific sites on SIRT1. This increase specific activities of SIRT1 and these effects are independent of NAD+ levels within the cell. Last of all, UV light and free radicals such as hydrogen peroxide (H₂O₂) inactivates SIRT1 by another protein called SENP. SENP inactivates SIRT1 by removing a sumoyl group from the SIRT1 protein.  Here are some diagrams that illustrates how “SIRT1 phosphorylators” activate SIRT1 activity and how de-sumoylation of SIRT1 inhibits SIRT1.

De-SUMOylating SIRT1

UV light or H2O2 inactivate SIRT1 via a UV/ROS sensitive protein called SENP.  SENP removes a SUMO group from SIRT1, thereby inactivating the protein.

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ROS activation of SIRT1

Free radicals such as H2O2 or superoxide can activate the SIRT1 protein via a protein kinase called c-Jun Kinase (JNK), increasing the ability of SIRT1 to deacetylate histone proteins such as H3.

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SIRT1 and Cell Survival

DYRKs are enzymes that are activated by  ROS  or (environmental stress) or DNA damage. DYRKs activate SIRT1-mediated deacetylation of p53, thus increasing cell survival in stressful times.

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SIRT1 and the Cell Cycle

Two proteins involved with cell cycle regulation form a complex with SIRT1 and phosphorylate the SIRT1 protein.  This increases cell proliferation and is a key factor in allowing mitotic cells to keep dividing.

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In summary, SIRT1 activity is dependent on NAD+ levels, nicotinamide levels, phosphorylation status, DBC-1 status, AROS status, ROS or other stressors, and the presence of exogenous SIRT1 activators (resveratrol) or inhibitors (Tenovins).

The over-all importance of Sirtuins, however, is their ability to affect gene expression in the nucleus. Changing gene expression is is considered an “upstream event” in aging.  In this aspect, SIRT1 is not the only Sirtuin in the nucleus of the cell.  There are two others that we will discuss now – SIRT6 and SIRT7.

SIRT6 – The Master DNA Repair and Genomic Stability Sirtuin

The effects of SIRT6 on Genome Stability

Another Sirtuin family member that directly affects gene expression is SIRT6.  SIRT6 is also found in the nucleus and is also an NAD+-dependent enzyme.  Like SIRT1, SIRT6 plays a vital role in both single strand DNA breaks (SSBs) and double stranded DNA breaks (DSBs). With DSBs, SIRT6 is one of the earliest factors that is recruited to the site of the DSB. This occurs within 5 seconds in a healthy cell. SIRT6 recruits the chromatin remodeler, SNF2H, to the site of the DSB and deacetylates histone 3 at lysine 56 (H3K56). This allows SNF2H to open up the chromatin at the DSB site, which then allows the DNA DSB repair proteins to be recruited (BRCA1, 53BP1, and RPA).  This is shown in the diagram below.

SIRT6 – The Early Responder to DSBs

Double stranded DNA breaks are the most lethal DNA damage type and are primarily responsible for cancer and aging.  Of the seven SIRTs, the SIRT6 isoform probably plays the greatest role in DNA repair.  In the diagram that follows, a double stranded DNA break occurs and within 5 sec, SIRT6 shows up to deacetylate H3K56. This recruits the chromatin remodeler, SNF2H, which then “opens up” the chromatin, allowing the key DNA repair proteins to bind to the site of injury. The key DNA repair proteins include 53BP1, RPA, BRCA1, and CtIP (not shown). Collectively, these proteins repair double-strand DNA breaks by the more accurate repair program called “homologous recombination”, or HR.  The other DNA DSB break repair pathway, NHEJ, is not as accurate a repair pathway as the HR pathway, which is the pathway shown in the diagram.

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Image and highlights source SIRT6 Recruits SNF2H to DNA Break Sites, Preventing Genomic Instability through Chromatin Remodeling  “Highlights: •SIRT6 arrives to DNA break sites within 5 s, •Lack of SIRT6 and SNF2H increases sensitivity to genotoxic damage, •SIRT6 directly recruits SNF2H, which in turn opens chromatin at break sites. •SIRT6 and SNF2H are necessary to recruit 53BP1, RPA, and BRCA1 to the damage sites”

With DSBs, SIRT6 plays crucial roles in both the nonhomologous end joining (NHEJ) pathway and the homologous recombination (HR) pathway.  With HR, SIRT6 deacetylates a key protein called CtIP which works with the BRCA1 protein to generate single stranded DNA (aka “DNA end resection”) at the site of the DSB. Unless CtIP and BRCA1 can form a single strand of DNA at the break site by “DNA end resection”, then the non-homologous end joining (NHEJ) method of DNA repair takes over, which does not repair DSBs nearly as accurate as HR.  In addition to the effects of SIRT6 on HR and NHER repair of DSBs,  SIRT6 activates PARP1 directly as well.  This increases DNA repair for single stranded DNA breaks (SSBs) by increasing base-excision repair (BER) pathway for SSBs and also increases double stranded DNA repair by both HR and NHEJ pathways.  All of these effects have a net effect of increasing genome stability within the cell.

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Image and legend source: 2011 Repairing split ends: SIRT6, mono-ADP ribosylation and DNA repair  “SIRT6 regulates genomic stability. SIRT6 promotes genome stability by regulating DNA single-strand and double strand break repair pathways and by facilitating telomere maintenance. The deacetylase and the mono-ADP ribosyltransferase activities of SIRT6 both contribute to this function.”

Based on the three mechanisms illustrated above, SIRT6 plays a key role in both SSB and DSB repair.  As a result, when SIRT6 knock-out mice are created, they have an accelerated aging-like phenotype.

The Other Effects of SIRT6 besides Genome Stability

Other than genomic stability, the other primary effect of SIRT6 are mediated by histone protein modifications of specific lysines found on Histone 3 (H3K9 and H3K56).  Although SIRT1 has some histone protein deacetylase function, it has a much greater mono-ADP-ribosyltransferase activity and an even greater “deacylation” function. “Deacylation” refers to the removal of long chain fatty acids (palmitic acid, myristic acid, oleic acid, and linoleic acid) from lysine amino groups on proteins.  One of the SIRT6 targets for ADP-ribosylation is PARP1 (see above).  PARP1 is mono-ADP-ribosylated by SIRT6 which activates PARP1.  The “deacylation” function of SIRT6 was just recently discovered. Here is a diagram that shows the effects of SIRT6 deacetylase function and the effects of SIRT6 on CtIP which also occurs via its effects on gene expression in the nucleus of every cell.  (The mono-ADP-ribosyltransferase and deacylation function of SIRT6 are not shown in the diagram).

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Illustration  and legend reference “SIRT6 is a critical regulator in genome stability, metabolism, and inflammatory response. By deacetylation of H3, SIRT6 regulates metabolic homeostasis and inflammatory response in peripheral tissues, while functioning as a central regulator of somatic growth.”

As you can see, most of the effects of SIRT6 are mediated by histone protein deacetylation of two specific lysine residues found on the histone 3 (H3) subunit of the histone “spools” that wind up DNA and compact it into the nucleus.  The H3 subunit has two specific lysine residues found on the tail of “H3” protein called H3K9 and H3K56.  When these two sites are deacetylated by SIRT6, this inhibits the type of metabolism seen in cancer cells called “Warburg metabolism” where the cells are totally dependent on sugar for generating energy and cannot burn fats.  Also, SIRT6 inhibits the inflammation caused by the major inflammatory “on switch” called NF-kB.  The other mechanisms of action of SIRT6 have not been fully elucidated, such as the effects of SIRT6 on growth hormone, IGF-1 and telomere stability.  What appears clear, however, is that all of the effects of SIRT1 and SIRT6 occur in the nucleus of the cell and all of the activity of both SIRT1 and SIRT6 are dependent on the availability of NAD+ in the nucleus of the cell.

The effects of SIRT7 within the cell, SIRT7 activators and SIRT7 inhibitors

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Image source: A Big Step for SIRT7, One Giant Leap for Sirtuins… in Cancer

Outlier Research

Unfortunately, there are research results that challenge some of the basic assumptions of those who are looking for health creation through enhancing body NAD+ levels. And, they chellenge some of our predictions listed above.   For example there seems widespread agreement that:

1. If you want to activate SIRT1, you must have adequate levels of nuclear NAD+.

2,  Having adequate levels of nuclear NAD+ is the best way to keep SIRT1 active.

2. The impact of SIRT1 is universally health-producing.

Unfortunately, there appears to be research that suggest that these broad assertions ain’t always necessarily so.  We have above discussed how SIRT1 phosphorylation can increase or decrease SIRT1 activity and a number of proteins that bind to SIRT1 and increase or decrease its activity.  Cold is another example.

Cold can activate SIRT1 in a way that is completely independent of NAD+

You can use cold to activate SIRT1 and PGC1alpha regardless of your nuclear NAD+ status. The December 2011 article The cAMP/PKA Pathway Rapidly Activates SIRT1 to Promote Fatty Acid Oxidation Independently of Changes in NAD+ reports: “Highlights

• Stimulation of the cAMP/PKA pathway results in phosphorylation of SIRT1 serine 434
• SIRT1 S434 phosphorylation increases intrinsic deacetylase activity
• SIRT1 activation by S434 phosphorylation is rapid and independent of changes in NAD+
• S434 phosphorylation induces PGC-1α deacetylation and increased fatty acid oxidation”

“The NAD+-dependent deacetylase SIRT1 is an evolutionarily conserved metabolic sensor of the Sirtuin family that mediates homeostatic responses to certain physiological stresses such as nutrient restriction. Previous reports have implicated fluctuations in intracellular NAD+concentrations as the principal regulator of SIRT1 activity. However, here we have identified a cAMP-induced phosphorylation of a highly conserved serine (S434) located in the SIRT1 catalytic domain that rapidly enhanced intrinsic deacetylase activity independently of changes in NAD+ levels. Attenuation of SIRT1 expression or the use of a nonphosphorylatable SIRT1 mutant prevented cAMP-mediated stimulation of fatty acid oxidation and gene expression linked to this pathway. Overexpression of SIRT1 in mice significantly potentiated the increases in fatty acid oxidation and energy expenditure caused by either pharmacological β-adrenergic agonism or cold exposure. These studies support a mechanism of Sirtuin enzymatic control through the cAMP/PKA pathway with important implications for stress responses and maintenance of energy homeostasis.”

We have discussed cold as a simple and practical hormetic healh intervention in previous blog entries(ref)(ref)(ref)(ref).

SIRT1 turns off Nrf2 and its health-producing affects

We mentioned this above.  We have published several blog entries on the numerous impacts of activating Nrf2 which turns on hundreds of positive “antioxidant response genes.” See this list.  One of the last things we would expect to find is that SIRT1 which also produces numerous health benefits actually turns Nrf2 off. but that appears to be the case. It tends to keep Nrf2 in the cytoplasm rather than allowing it to migrate to the nucleus where it can do good.  Even worse, resveratrol, our favorite SIRT1 activator actually inhibits expression of Nrf2.

SIRT1 “turns off” Nrf2-mediated antioxidant gene expression?

The 2010 publication: Acetylation-Deacetylation of the Transcription Factor Nrf2 (Nuclear Factor Erythroid 2-related Factor 2) Regulates Its Transcriptional Activity and Nucleocytoplasmic Localization reports: “Activation of Nrf2 by covalent modifications that release it from its inhibitor protein Keap1 has been extensively documented. In contrast, covalent modifications that may regulate its action after its release from Keap1 have received little attention. Here we show that CREB-binding protein induced acetylation of Nrf2, increased binding of Nrf2 to its cognate response element in a target gene promoter, and increased Nrf2-dependent transcription from target gene promoters. Heterologous sirtuin 1 (SIRT1) decreased acetylation of Nrf2 as well as Nrf2-dependent gene transcription, and its effects were overridden by dominant negative SIRT1 (SIRT1-H355A). The SIRT1-selective inhibitors EX-527 and nicotinamide stimulated Nrf2-dependent gene transcription, whereas resveratrol, a putative activator of SIRT1, was inhibitory, mimicking the effect of SIRT1. Mutating lysine to alanine or to arginine at Lys⁵⁸⁸ and Lys⁵⁹¹ of Nrf2 resulted in decreased Nrf2-dependent gene transcription and abrogated the transcription-activating effect of CREB-binding protein. Furthermore, SIRT1 had no effect on transcription induced by these mutants, indicating that these sites are acetylation sites. Microscope imaging of GFP-Nrf2 in HepG2 cells as well as immunoblotting for Nrf2 showed that acetylation conditions resulted in increased nuclear localization of Nrf2, whereas deacetylation conditions enhanced its cytoplasmic rather than its nuclear localization. We posit that Nrf2 in the nucleus undergoes acetylation, resulting in binding, with basic-region leucine zipper protein(s), to the antioxidant response element and consequently in gene transcription, whereas deacetylation disengages it from the antioxidant response element, thereby resulting in transcriptional termination and subsequently in its nuclear export.”

A brand new 2015-dated publication seems to say the direct opposite to the just-cited publication Polydatin promotes Nrf2-ARE anti-oxidative pathway through activating Sirt1 to resist AGEs-induced upregulation of fibronetin and transforming growth factor-β1 in rat glomerular messangial cells.  “Highlights:

• PD increased Sirt1 levels, promoted Nrf2-ARE pathway activation, and reduced ROS levels in AGEs-treated GMCs. (PD is a resveratrol glycoside)
• PD resisted AGEs-induced upregulation of FN and TGF-β1 by activating Sirt1-Nrf2-ARE pathway.
• PD ameliorated DN in a STZ-induced diabetic rat model.”

“Sirt1 and nuclear factor-E2 related factor 2 (Nrf2)-anti-oxidant response element (ARE) anti-oxidative pathway play important regulatory roles in the pathological progression of diabetic nephropathy (DN) induced by advanced glycation-end products (AGEs). Polydatin (PD), a glucoside of resveratrol, has been shown to possess strong anti-oxidative bioactivity. Our previous study demonstrated that PD markedly resists the progression of diabetic renal fibrosis and thus, inhibits the development of DN. Whereas, whether PD could resist DN through regulating Sirt1 and consequently promoting Nrf2-ARE pathway needs further investigation. Here, we found that concomitant with decreasing RAGE (the specific receptor for AGEs) expression, PD significantly reversed the downregulation of Sirt1 in terms of protein expression and deacetylase activity and attenuated FN and TGF-β1 expression in GMCs exposed to AGEs. Under AGEs-treatment condition, PD could decrease Keap1 expression and promote the nuclear content, ARE-binding ability, and transcriptional activity of Nrf2. In addition, PD increased the protein levels of heme oxygenase 1 (HO-1) and superoxide dismutase 1 (SOD1), two target genes of Nrf2. The activation of Nrf2-ARE pathway by PD eventually led to the quenching of ROS overproduction sharply boosted by AGEs. Depletion of Sirt1 blocked Nrf2-ARE pathway activation and reversed FN and TGF-β1 downregulation induced by PD in GMCs challenged with AGEs. Along with reducing HO-1 and SOD1 expression, silencing of Nrf2 increased FN and TGF-β1 levels. PD treatment elevated Sirt1 and Nrf2 levels in the kidney tissues of diabetic rats, then improved the anti-oxidative capacity and renal dysfunction of diabetic models, and finally reversed the upregulation of FN and TGF-β1. Taken together, the resistance of PD on upregulated FN and TGF-β1 induced by AGEs via oxidative stress in GMCs is closely associated with its activation of Sirt1-Nrf2-ARE pathway.”

How do these and possibly many other contrarian or contradictory findings play out in-vivo? We simply don’t know.  We don’t know how all the regulatory feedback inhibition loops will affect each other when we make a major intervention like enhancing body levels of NAD+.  And we don’t know what the effect of many other variables will be, such as initial oxidative state, circadian state, relationship to stressors, disease states, hormonal states, age, sex, etc. Our theories which look at one pathway at a time under standardized conditions cannot tell us that. We will only know the effect of NAD+ enhancement therapy through observing net health effects and key health surrogate biomarkers. And we have to do that on and highly individualized basis That is why we regard the issue of individual biomarkers to have such great importance.

By Vince Giuliano with inputs and assistance from Melody Winnig and James P Watson

INTRODUCTION

The consumer electronics industry is giving a tremendous boost to public and individual health – perhaps the most important boost in the first half of this century. The digital health movement could result in a number of important but unforeseen consequences, such as the emergence of powerful personal electronic health biomarkers which were hitherto unavailable.   The movement is already contributing significantly to changing our health paradigm by allowing individuals to document their day-to-day health-related behavior patterns.

2014 has been a huge year for health tech according to digital health incubator startup health, “Digital health funding in the first three quarters of 2014 has already surpassed $5 billion, close to double what was invested in all of 2013 ($2.8 billion). “Digital health funding for the year is on track to double last year’s total,” said Unity Stoakes, co-founder and president of startup health. “Some trends we’re watching include a growing corporate interest in digital health, more global cross-pollination of ideas, as well as increasing health consumerism as people move into the driver’s seat when it comes to their care.  With this kind of capital pouring into the market, the health tech space should be exciting to watch in the coming years.” (from mashable.com 5 digital health trends you’ll see in 2015.

This Part 1 blog entry provides a panoramic snapshot view of one area of digital health: the rapidly changing landscape of consumer health and fitness smart wearables like smart watches, online and mobile health and wellness applications, and the associated emergence of online and mobile software platforms that can integrate such applications together.  I also discuss a couple of devices I am personally familiar with.

Smart wearables include electronics-infused clothing and body devices that enhance our perceptions and allow us to do things we cannot normally do – such as know our actual heart rate at any time, or experience virtual reality.  Consumer digital health and fitness is a central theme of smart wearables,  The expectation has been  that 90 million SMART wearables would be sold in 2014(ref),  Accordingly, tremendous capital has been flowing into this area as can be seen from the amazing new array of health-measuring wearables now being sold by leading retailers.

Morgan Stanley has projected that the smart wearables market will soon be worth $1.6 trillion(ref). “Wearable devices will far surpass market expectations, and become the fastest ramping consumer technology device to date, in our view,” a group of Morgan Stanley analysts wrote in a note on Thursday. The analysts add that wearable devices will have “far-reaching” impacts by creating a new category and disrupting or even accelerating change within industries outside of technology.” (I think one of these industries will be medicine – VG) “The analysts project sales of wearable devices will grow at a 154-percent annual compound rate through 2017, where 248 million devices will be sold. The figure will grow even further where sales of wearable technologies will reach one billion in 2020.”

THE QUEST FOR SIMPLE, SENSITIVE AND EASILY-MEASURABLE GENERAL STRESS BIOMARKERS

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Quest for the Holy Grail   Image source

A key fantasy I and other longevity researchers have long had may soon start becoming reality – the ability for ordinary people to monitor readily accessible biomarkers that will allow us to evaluate the impacts of day-to-day health and longevity interventions and stress events.  Such an intervention could be a simple change in diet, exercise or sleep patterns, or consuming a new supplement or drug.  Stress events could be a disease, of an emotional or traumatic nature, or as simple as disruption of the normal sleep pattern. These biomarkers could conceivably provide easy and very accessible answers in a few days to very basic questions such as “is my new approach (to a new pattern of exercise/taking a new supplement or drug/sleeping longer or differently/etc.) really working?”  “Comparatively speaking, how well am doing now compared to before?”  “What price am I paying for staying up drinking and carousing until 4 AM last night?”  “How long will it take for me after returning from China to re-establish my normal circadian rhythms?” “What has been the health impact of my partying for several nights in a row over Christmas, drinking more than usual and eating large late meals?”  “I had to take a strong prescription antibiotic.  How long will it take for my gut biome to recover?” Or “I have added a high intensity 8-minute segment to my daily exercise regimen, designed to send my heart rate above 125.   What is this doing to me or for me?”   I now believe the fantasy of ordinary people being able to answer such questions for themselves without professional help is rapidly becoming real.  It will be realized through a powerful wave of developments in consumer electronics powered by billions of dollars in investment.

In fact, during the period of generating this blog entry I have been doing some initial personal research that leads me to think I may already have discovered reliable metrics of constitutive  stress that can be easily computed from measurements made by an existing wearable device.     I expect to report on that personal research very soon in a Part 2 blog entry. Wearables - measuring practical stress biomarkers.

Because new improved products are coming on the market at an incredible rate, it is likely that some devices described here will be becoming obsolete in six months or even less.  That is why I expect to continue reporting on health and fitness wearables on an ongoing basis in this blog.

What is exciting from the viewpoint of this longevity science blog is not that the existing or even the latest generation of consumer devices and software can measure traditionally-identified  biomarkers, such as those identified in the previous blog entry NAD+ an emerging framework for life health and life extension — part 2: deeper into the nad world, hopeful interventions. Rather what exciting to me is the extremely massive public scale and rapidity of adoption of consumer health wearables and apps, the deep pocket companies investing in them, the variety of physiologic biomarkers that are turning out to be electronically measurable by wearables, the possibilities inherent in 24-7 gathering of personal health data, and the extremely low cost to consumers of starting to participate in this movement.

THE SITUATION IS BIG, RAPIDLY DEVELOPING, ALREADY INVOLVING MORE THAT A HUNDRED MILLION PEOPLE, HIGHLY FACETED, AND CHAOTIC

The situation is one of chaos typical in the early stages of a rapidly developing new family of electronic gadgets aimed at almost everybody. There is an avalanche of new products together with much confusion about what they really do or whether they even really work.  A wide variety of products with differing capabilities was already on the marketplace for Christmas 2014.  I understand there were more than 300 booths at the 2015 Consumer Electronics Show in Las Vegas featuring new health and fitness product.  It is impossible to know which product offerings are best and who the winners and losers will eventually be.  The situation is like that we have seen repeatedly over the years for personal computers, digital cameras, personal PDA assistants, mobile music devices, digital pads and smart phones. Seemingly-sophisticated digital watches that can measure exercise and heart rates are now being sold for $5.99 and up. We seek here to provide a snapshot of the changing picture as we see it now.

SOME RECENT HISTORY

A variety of stand-alone health monitoring devices and kits have long been found on the shelves of drugstores. They measure a number of biomarkers that once required going into a doctor’s office or laboratory for testing.  E.g. they measure blood pressure, blood glucose, pulse oximetry, heart rate, heart rate variability, cholesterol, and many are designed to detect presence of certain diseases.  They are designed for personal use and are available to all without prescription.   Likewise, some treadmills and other machines in sports facilities and homes have had built-in indicators of exercise heart rates and elaborate displays showing stress levels, heart rate and likely calories burned in the course of an exercise session.

What we are seeing today is the miniaturization and personalization of such devices – the continuing upward integration of historical lines of consumer electronic health and fitness devices and applications, including: a) functionalities of such stand-alone personal testing devices, b) fitness measuring devices that first appeared in sports facilities connected to machines, and c) consumer health and wellness computer applications, particularly mobile applications (over 100,000 of them),. Most current attention is on an almost-here generation of smart watches and wrist straps that monitor movement, exercise patterns, calories burned, heart rate, sleep parameters and other bio-indicators.  However, I believe we are still in a very early stage of the wearables revolution, and new technologies and devices will continue to emerge that are not imagined now.  This health-wearables revolution is being propelled by new technologies, by increasing consumer interest in health and well-being, and by a maturing baby boomer generation of mostly healthy people in the middle and late 60s who want to stay healthy, and by a consumer electronics industry that sees personal health as an ultimate Killer App.

Here, we look at some of what is going on with

• Fitness trackers
• Smart watches and bracelets
• Advanced body electronic sensor devices and smart clothing
• Health and fitness apps
• Integrated health and fitness display software platforms
• A couple of wearables that I have personal experience with

The consumer wearables area of digital health we discussed here is very large but the entire field of digital health is even larger. According to a recent report by Startup Health.  “The top 10 most active subsectors include big data/analytics, navigating the care system, practice management, sensors/diagnostics and patient engagement.” We don’t touch the first three of these areas here and focus on consumer electronic monitoring. Also we touch only lightly on the tremendous area of associated software apps and online services. Perhaps in a subsequent blog entry we will try to make sense of the some 100,000  health and fitness apps currently available on iOS and Android.

ACTIVITY TRACKERS

A few years back, a first generation of personal health and fitness wearables appeared, like the first FitBit and Jawbone movement-tracking devices.  They provided crude but effective constant real-time measurements of number of steps taken, intensity of exercise, flights of stairs climbed, calories burned, and different stages of sleep. They communicate by bluetooth with computers and smart phones and it is possible to see how these indicators change throughout the day and night, compare them from day-to-day, and even share them with other people in a personal network.  I have been using a FitBit One for about a year now and have found it quite useful for telling me at the end of the day whether I have had sufficient exercise, and, when I wake in the morning a rough indicator of how well I have slept.

From Wikipedia: “The term “activity trackers” now primarily refers to wearable devices that monitor and record a person’s fitness activity. The concept grew out of written logs that led to spreadsheet-style computer logs in which entries were made manually, such as that provided in the us by the president’s council on physical fitness and sports as part of the president’s challenge.^[1] improvements in technology in the late 20th and early 21st century have made it possible to automate the monitoring and recording of fitness activities and to integrate them into more easily worn equipment. Early examples of this technology include wristwatch-sized bicycle computers that monitored speed, duration, distance, etc., available at least by the early 1990s. Wearable heart rate monitors for athletes were available in 1981.^[2] wearable fitness tracking devices, including wireless heart rate monitoring that integrated with commercial-grade fitness equipment found in gyms, were available in consumer-grade electronics by at least the early 2000s. Wearable fitness tracking computers with tightly integrated fitness training and planning software were available as consumer products by at least 2006.^{[2][3]?\ —} Electronic activity trackers are fundamentally upgraded versions of pedometers; in addition to counting steps, they use accelerometers and altimeters to calculate mileage, graph overall physical activity, calculate calorie expenditure, and in some cases also monitor and graph heart rate and quality of sleep.^[4][5][6] some also include a silent alarm.^[5][7] some newer models approach the us definition of a class ii medical monitor, and some manufacturers hope to eventually make them capable of alerting to a medical problem, although fda approval would be required.^[8] early versions such as the original FitBit (2009), were worn clipped to the waist;^[4] formats have since diversified to include wristbands, armbands, and smaller devices that can be clipped wherever preferred.^[7][9] Apple and nike together developed the nike+ipod, a sensor-equipped shoe that worked with an ipod nano. In addition, logging apps exist for smartphones and facebook;^[6] the nike+ system now works without the shoe sensor, through the gps unit in the phone. The forthcoming Apple watch and some other smart watches offer activity tracker functions.^[8]in the us, bodymedia has developed a disposable activity tracker to be worn for a week, which is aimed at medical and insurance providers and employers seeking to measure employees’ fitness.^[10] other activity trackers are intended to monitor vital signs in the elderly, epileptics, and people with sleep disorders and alert a caregiver to a problem.^[8] –

Earbuds and headphones are a better location for measuring some data, including core body temperature; valencell has developed sensor technology for new activity trackers that take their readings at the ear rather than the wrist, arm, or waist.^[11] — There are collar-mounted activity trackers for dogs.^[12][13][14] — Much of the appeal of activity trackers that makes them effective tools in increasing personal fitness comes from their making it into a game, and from the social dimension of sharing via social media and resulting rivalry.^{[4][6][15][16]} the device can serve as a means of identification with a community,^[17] which extends to broader participation.”

The December 2015 issue of PC magazine report The Best Activity Trackers for Fitness provides a lot of comparative data on some current trackers.  This is what they look like.  As you can see, the most popular ones are worn on the wrist

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DEVICES FOR THE HIGH-END SPORTS AND FITNESS MARKET

One of the families of the new wave of wearables is devices designed to be worn in gyms and exercise clubs or by serious workout training addicts.  While this category is quite important, it is not the central focus of this blog entry.  The AMIGO is an example. It is a combination of a wristband and shoe lace device for measuring both upper and lower body movement.  You train the Amigo to recognize patterns of movement that occur in specific exercises. This product, like many others reported here, is scheduled for delivery in 2015.  Of many other devices in this category I mention the Polar H7 Bluetooth Smart Heart Rate Sensor, available as a bluetooth-enabled strap that measures heart rate quite accurately.  This can be used as input for measuring Heart Rate Variability (HRV) on your smartphone, a potentially useful stress indicator.  Much more on this in the Part2 blog entry.

MEDICAL ESTABLISHMENT WEARABLES

Another important category of wearables that we do not focus on here are high-end devices and systems designed to be worn in hospitals or healthcare institutions as well as by individuals at home under medical care. An example is the ZypherLIFE system. This system is designed to measure a significant portfolio of biomarkers and integrate them into medical systems as illustrated in the following diagram.

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WHERE CONSUMER FITNESS ACTIVITY TRACKERS STAND NOW

We are currently seeing a transition from the first generation of consumer fitness trackers to a variety of second-generation devices with differing form factors and expanded functionalities.

The first generation are devices like the Fitbit One , the Nike+ Fuelband SE, and the Jawbone Up and many similar devices combine a sensitive electronic  accelerometer-pedometer into a little bracelet or pendant which monitors movement. This movement can be interpreted by software to provide logging of: steps taken per day, total distance traveled, intensity of walking or running exercise during various times of the day, periods of sleep and rough indicators of depth of sleep, and calories burned per day. Other options, such as inclusion of an altimeter, allow logging of flights of stairs climbed.   Following is one of several screenshot of a typical day of my FitBit results. I have been logging activity with this device for about a year.  I have found it particularly useful for assessing periods of sleep and telling me at the end of the day whether I have exercised sufficiently.

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Most of the shown boxes in the online or cell phone display are expandable to show more detail, and it is also possible to view weeky and monthly comparative data.  The weight is from a FitBit wireless scale which also records computed BMI and lean-to-fat ratio

My FitBit 1 communicates with my Samsung S5 smart phone or my computer via bluetooth.  Current and historical records are uploaded to the cloud and available on any Internet or mobile-connected platform. Further, my records can be shared with other FitBit users at my option. The software display platform is proprietary and cannot accept inputs from non-FitBit products. Other first-generation trackers generally provide similar capabilities and sharing of performance records with other users of the same device, a very useful feature for encouraging social support of exercise patterns.

Many smartphones came with built-in accelerometers and can do similar tracking.  The Samsung S5 is an example and the phone comes with a similar activity tracking app, Samsung’s S Health.  The phone itself contains a pedometer like the FitBit and can make other health measurements like instantaneous heart rate.  Limits of the first-generation trackers and smart phones include:

• Measurements of movement depends on where the device is located on the body. If it is clipped to a belt or in a pants pocket, for example, the device may not provide a good indicator of my upper body exercise. If the device is a smartwatch on my wrist, it cannot detect the exercise movement when I am on a treadmill holding the rail.  Or, while I am pushing a shopping cart.  At best, it provides an approximation of whole body movement.  You cannot rely on your smartphone for activity tracking if you don’t carry it all the time.
• The measurements do not include any that indicate how my body is responding to the exercise. They do not include anything we would normally regard to be a biomarker.
• Most basically, the devices are limited by the sensors they contain, in the case of a FitBit, an accelerometer/pedometer and an altimeter.
• The software platforms and display apps are highly specific to the manufacturer and are incompatible among each other. Users of one tracker cannot automatically share performance records with users of a different tracker.  And if you carry two or more wearable trackers from different manufacturers, you can’t display the results together so you can compare them.  Each of the three tracking apps now on my S5 smart phone only accepts device specific input and none can display an integrated picture.
• Although the market is crowded with new devices, hard technical and reliable performance specifications are generally unavailable. Information available from manufacturers is generally high-level hype, and it is quite impossible to tell which devices deliver on their promises and which deliver junk results.  Reliable reviews are few and far between, and industry-level standards are missing.   It is a general case of “buyer beware.”  This situation appears to persist today for smartwatches and other new devices coming onto the market.  Having said this, I can say that my limited personal experience with my FiytBit and Basis Peak devices up to this point has been generally positive.

CURRENT MARKET TRENDS

What we have seen in the market since before Christmas 2014 is a) strong price competition for activity trackers of the first generation described above – many new low-cost competitors, b) rapid emergence of second-generation and third-generation devices with much enhanced functionality and bio-measurement capabilities including smartwatches and smart wrist straps, c) a flood of new products being sold, and d) announcements of several new kinds of bio-measuring wearables that go far beyond traditional activity tracking. ..

There are some traditional products such as from FitBit, Misfit and Shine, selling in the $50 – $150price range.  These are from FitBit:

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Image Source: Fitbit.Com

Likewise, other reputable first-generation products like those from Jawbone are dropping their prices, but not by a lot.

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Image Source: Amazon.Com

A recent review article is compare before you wear: the most popular fitness trackers.

THE NEW GENERATIONS OF WEARABLES – CHOICES GALORE

Smart wearables and wrist straps in the second generation include high-end devices costing $500 and more and low-end devices costing as little as $5.99.   This link goes to the first of 13 pages listing smartwatches and tracker-related such devices for sale at Walmart,  This link is to a Cruncheara site offering smart wearables ranging from eyeglasses, to polo shirts to insoles.

Who is in the game?  Wearable health and fitness monitoring devices are available or announced from Polar, Garmin, Mio, Basis, Withings, FitBit, Timex, New Balance, Wahoo, Suunto, Jawbone, Pebble, Androset, Scosche. LL Bean, Radius, Jarv, Pyle, TomTom, New Arrival, Tune Belt, Luxsure, SNEER, New Arrival and several others,  And, oh yes, also in this game are lurking giants like Microsoft, Intel, SONY, Motorola, Samsung and Apple.  Browse for yourself!  Here is a REI shopping site for watches and straps with heart rate monitors,  Here is an Amazon.com shopping site for health and fitness monitors,  Here is a listing of LL Bean offerings.  Here is a different amazon.com catalog listing.  Here is a listing of just SONY smart watches. This page from saleguru.net shows price comparisons for 49 different Apple-compatible models of smartwatches.

At the lower-end price points, such devices are highly likely to become must-have items for middle school and high school kids and young adults.  Many cheaper ones were stocking stuffers for Christmas.  For this reason alone, it is likely that tens of millions of people were introduced to health and fitness capable wearables just during the 2014 Christmas season,  The images shows some of the cheaper devices.

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Smartphone sensors

Many smartphones have built-in sensors that allow them to function as first-generation trackers.  Such as:

– accelerometer pedometer-type motion sensors which allow them to monitor movement and even sleep – if you are willing to sleep with your cell phones

– cameras which can double as optical heart rate monitors.  They can also serve as bar code scanners for screening packaged foods at supermarkets or some restaurant menus that print barcodes.  Software apps can then give you nutritional values and calorie counts.

HIGHER-END CONSUMER FITNESS TRACKERS AND SMART WATCHES

The higher-end devices are embodying the functionality of the first generation, in some cases doing a better job at that, plus additional functionality due to the incorporation of additional sensors.  They are evolving from being basically movement trackers to more general health biotracking devices.  Again, the first generation was based on pedometer/accelerometer and altitude sensors. The higher- end second-generation trackers variously embody additional other sensors such as for heart rate measurement, skin temperature, 02, skin galvanic resistance, and GPS capability. Some of these are sold in the form of wrist straps, and some in a new generation of smart watches,   In general, these devices offer a higher level of integration with smart phones, and come with broader and more comprehensive but still proprietary health and wellness apps. Estimates of levels of exercise and associates stress can be based on combinations of the sensor outputs.  The following as representative of the higher-end second generation trackers/smartwatches with heart rate measurement capabilities:

• The Jawbone UP3 $180
• The Microsoft Band $199
• The Fitbit Charge HR $150 or the FitBit Surge $250
• The Garmin Forerunner 310XT $299
• The Apple Watch (annouced but possibly not available until Spring 2015) Very exciting for Apple and iOS followers, with many health and wellness and advanced features.  Probably starting at $350)  See this review.

How can one make sense of this avalanche of offerings?  I don’t know. My personal choice a few weeks ago was based on my health/longevity biomarker primary focus of interest – rather than a strongest interest in  fitness/exercise, elegance, or general smartwatch capability.  After limited research I ordered a Basis Peak.  That and my trusty old FitBit One are the only devices I am personally familiar with and will discuss here in any detail.

THE BASIS PEAK

The Basis Peak arrived at my doorstep about three weeks ago, so I can tell you what motivated me to buy it but am not yet quite ready to report on my experience with it.  The package claims “The ultimate fitness and sleep tracker.” Image may be NSFW.
Clik here to view.Based on its published specifications and positive reviews, it indeed may be that right at the moment. However, there is no guarantee on how long it will remain that way.  A November 2014 review in PC world  This is the band to buy if you obsess over heart rate and sleep tracking reports: “this is a second generation device, an improvement in multiple dimensions over the first generation device which appeared about a year ago. Among the sensors it encompasses are a FitBit-type accelerometer, an optical sensor that measures heart rate through the skin 24-7, a galvanic skin resistance sensor and a skin temperature sensor. Basis was recently acquired by Intel and has a history of earlier devices basically marketed to the fitness community.  As I write this, smart watches with heart rate measurement capabilities are announced and will be available in the coming months from Apple,  FitBit, and Jawbone among others, but these will be first generation devices which will no-doubt need improvement.  Now being an Intel company, I expect a continuing process of improvement in the Basis products.”

So far, I am very happy with the choice.  Here is the Peak’s record of my sleep for a typical night..

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I do not know how the Peak can distinguish REM sleep from light sleep and cannot vouch for the accuracy of its findings. The FitBit was on my wrist next to the Peak.  It is interesting that the estimates of both devices for any period of sleeping are off by only a few minutes, although the details of the sleep is reported differently.  The FitBit does not try to distinguish among depth or type of sleep.  Here is A FitBit sleep record for me for a week:

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Here is a day’s(24 hours) activity record from the Peak:

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I have been logging these Basis Peak patterns every day on a spreadsheet since the watch arrived and studying them to see what their implications might be for my health.  After just 24 days of doing this I can say the results so far are quite exciting.  I am coming to believe these activity indicators can be combined with the sleep indicators to produce interesting new constitutional stress-state measures,  I expect to report on my observations after a full month of data gathering and analysis in the Part 2 Blog Entry for the wearables topic.

A preliminary observation relating to the device is that the existing software could be more comprehensive in providing consumer advice,   Also, I strongly suspect that the sensor data might be combined in different ways to provide measures of other physical conditions such as hydration and heat exhaustion stress.  My sense is that the Basis Peak is probably a superior hardware platform but that its software and user-friendliness integration might be significantly enhanced.  My hope is that the Basis company will move in this direction.  Earlier, the company promised an API for Peak outputs that would allow new customized forms of use data analysis.  And the company promised a software upgrade for December 2014 that would include smartwatch notifications on the Peak.  But so far these have not been forthcoming.

A more basic downside of the Basis Peak or any other single smart wearable available today is that its measurements are limited by the sensors it contains.   Ideally, my smartwatch would provide good measurements of heart rate variability, breathing regularity, blood pressure, body posture, glucose levels, blood oxygen, ECM, and embody a GPS capability.  At present the Basis Peak does not measure any of these although other new-generation consumer wearables do. (None measures all or even most of these).  Further, it is doubtful that third-party applications from either iOS or Android will be able to compute adequate heart rate variability (HRV) measurements from the physical sensor inputs the Peak watch provides.

NO SINGLE SMARTWATCHCAN PROVIDE THE BIOSENSING WE WILL WANT

Different smartwatches and other biosensing wearables typically embody different combinations of possible sensors and none have even most of them. This is understandable because  the number of sensors that can be put in any single device is limited for reasons of size, power consumption and location of that device on the body.  If you want to measure blood flow, near the ear may be a much better location than on the wrist.  If you want to measure body movement during exercise, it is best to have multiple sensors on the arms, legs and torso. If you want to measure brain waves, it is best to have sensors on the head, etc.

For these reasons, I expect the long-term  trend not to be cramming more and more sensors into a smartwatch, but rather the emergence of body networks of sensors all communicating with a central processing and data storage and external communications capability.  Today,  the smartphone is a far better candidate for that capability than any smartwatch.

This observation is also disappointing news for those expecting that the Apple Watch will be the ultimate sensing device with regard to health and fitness.  It can’t possibly be that.  However, it and watches like the Peak might provide a good start.  What can be put in any smart watch is a matter of compromise.  For example, the Apple Watch’s brilliant reconfigurable color display uses so much power that I suspect the watch must be taken off and recharged daily, while the black-and-white display trackers like the Peak can go for a few days between charges.

In the longer term I expect to see a wider variety of specialized and communicating wearables and direct-to skin-contact sensor devices, all kinds of smart clothing, and ultimately subcutaneous and possibly deeper body implants of many kinds.  These sensors will measure a growing list of bio indicators, communicating locally to a central processor, probably in the smart cell phone, which in turn will communicate with database and processing resources on the web.

BEYOND SMARTWATCHES – THIS YEAR

The article  5 digital health trends you’ll see in 2015 list some interesting predictions of what is likely to come in 2015. I quote from this article here.

“Wearables for the ear”

Despite that current craze for wrist straps and smartwatches, the ear may be the next favorite place..

“Due to the proximity to the temporal artery, devices worn on the ear can conduct completely unobtrusive, passive monitoring and offer far more precise measurements,” says Dr. Vahram Mouradian, founder and CTO of Sensogram Technologies. “Moreover, they can deliver a wealth of wellness information, including real time blood pressure, respiration rate and oxygen saturation, in addition to the typical readings of heart rate or steps taken.” — We’ve already seen a few of companies introduce ear buds with basic health monitoring, such as Iriveron and Freewavz. Watch for increasing sophistication in ear-based devices over the coming year.  For example, Sensogram’s Sensotrack — slated for general availability in March 2015 — is an elegantly designed device that fits snugly on your ear, where it measures heart rate, blood pressure, oxygen saturation and respiration rate. It also counts steps and calories burned, while sensing your speed, activity level, geolocation, altitude, body posture and pace.”

Notice that this device adds oxygen saturation, respiration rate and body posture to the measurements made by the latest smartwatches.

Continuing, “also keep an eye out for Bitbite, the first ear-based device that automatically tracks your eating habits and helps you improve them with real-time dietary advice. The Bitbite device fits comfortably in your ear and learns when, where and how you eat. It then analyzes this data and gently nudges you to make adjustments, such as slowing down your eating pace or drinking more water. Bitbite doles out its advice either by “whispering” in your ear or by alerting you through the bitbite smartphone app.  Bitbite just launched its Indiegogo crowdfunding campaign on November 11, with general availability expected in Q2 of 2015.”

So, some of the devices are becoming more bossy and nanny-like.  Some may end of virtually screaming at you if you start doing something healthwise foolish.  For now, they’re mostly simply flashing reminders on your cell phone or smartwatch screens or giving you a gentle buzz.  The Pavlok, a wrist wearable, is more aggressive and gives you electrical shocks.

“2. Sweat sensor strips”

“Want even more insight into what’s going on in your body? You’ll soon be able to track your internal biochemistry with a simple biosensor strip.  Electrozyme is developing a printed, flexible strip sensor that inserts into the back of your wearable device and measures the metabolic substances secreted in your sweat, allowing you to track your electrolyte balance, hydration level, muscle exertion and physical performance. According to electrozyme, the chemical analysis enabled by its disposable biosensors can give people actual insights into their metabolism that go way beyond steps and heart rate.”  Ok.  Another bunch of indicators.  “see also: Is that App FDA approved? Mobile health tech falls into gray area.  “The advantage of tracking sweat using chemical sensors is that it gives insight into your body chemistry and how it is responding to your workout that is not available using traditional sensors,” says Jared Tangney, Ph.D., co-founder and COO of Electrozyme, llc. “Chemical sensors open up a whole new world of information that was previously never available in wearable devices.”  “Tangney sees a range of different applications for this technology, including letting you know when it’s time to drink some water — something most of us probably need: according to some reports, up to 75% of americans may be chronically dehydrated.”

“3. Smartphone case devices”

“You’re already carrying around a smartphone with a protective case. Why shouldn’t it do double-duty as a medical device?  “We’re starting to see some initial forays into using smartphones and their cases to measure medical conditions that previously required specialized equipment,” says Joanne Rohde, CEO and founder of Axial Exchange. “imagine an electrocardiogram anywhere — not just at your doctor’s office — or a dyi blood test to check your glucose right in your pocket. Some of these innovations are already available, but there are many more to come.”  One of the first to hit the market was the Alivecor heart monitor, an FDA-approved iPhone case that allows you to record ECGS and heart rate on the go. You can rest it on your fingers or chest to record an ECG in 30 seconds, and know right away if atrial fibrillation is detected, which could be an early indicator of stroke.”  This could be valuable because the pulse rate monitors currently being marketed fall far short of providing ECG levels of information.”

” in 2015, watch for more such devices to become available as they pass through the FDA approval process. — For example, Azoi’s Wello is a mobile health tracking device which doubles as an iphone case and is currently in the process of getting FDA approval. It can measure vitals such as ECG, heart rate, blood oxygen saturation levels, respiration and temperature.”

“4. Prescription-only apps”

“There are already thousands of health apps you can find on Google Play or iTunes. Soon, some of these apps may require a prescription.  One early example is Welldoc’s Bluestar, the first “mobile prescription therapy” for people living with type 2 diabetes. The prescription-only app allows people to input data about their glucose levels, diet, exercise, well-being and other factors, which Bluestar automatically analyzes to give the patient immediate guidance and feedback. Bluestar also analyzes the data for the patient’s physician and allows the patient to provide a detailed summary of their progress to the physician for review prior to or during office visits. — While you might not see a flood of prescription-only apps hit the app store in January — launching one requires FDA approval, clinical trials, insurance reimbursement and more — Welldoc’s co-founder and chief medical officer Dr. Suzanne Sysko Clough says we should “expect to see more mobile prescription therapies for many major chronic diseases over the coming years.”

“5. Healthier lighting

“Finally, ever wonder why “you have trouble drifting off after staring at your iPAD in bed? The culprit may be the blue light emitted from your device — the part of the light spectrum that causes the biggest changes to your internal circadian rhythm, which can disrupt your sleep and impact your health.”

(Check out our blog entry Blue light, sleep, mental alertness and health)

“People who don’t get enough sleep have trouble being productive, controlling their emotions and coping with change. They’re also at greater risk of major health issues such as heart disease, diabetes and stroke,” says Cameron Postelwait of Sewell Development Corporation, developer of the low-blue-light Drift light bulb, which launched in May of this year. Postelwait believes that in 2015, we’ll see a much bigger focus on the effects that artificial light has on people’s health, as well as new product innovations to address the problem, particularly in clinical environments and hospitals.  “A patient in unstable condition requires nurse visits all through the night. Every time the nurse enters the room, he activates some kind of lighting to help him check on the patient’s condition and to give medication. This throws off the patient’s natural circadian rhythm, which not only disturbs sleep, but may also impact immune response,” explains Postelwait. “There’s a huge need for lighting that either has an absence of blue light or a way to change the amount of blue light that the patient receives during the day or night.”  But while too much bright light might be harmful at night, too little during the day can also bring you down.  Luckily, there are folks hard at work to add some sunshine to your day.  For example, Goodlux Technology recently launched Sunsprite, the first wearable device to track daily bright light intake. According to Goodlux, scientific studies have linked bright light exposure to health benefits such as better energy, mood and sleep.”

“There’s so much focus on nutrition and fitness that mental wellness is often overlooked. Bright light sets the body’s internal clock, which controls essential components of mental wellness: hormones, energy levels, mood, digestion and sleep,” says Goodlux CEO Ed Likovich. “Three out of four of our early adopters report improvements in these essential components to mental wellness.”  “The solar-powered Sunsprite boasts dual sensors that measure visible and UV light and lets users know when they’ve absorbed just the right amount of bright light to maintain health, while also providing tracking for monitoring UV exposure.” (Quotes above are from ref)

HEALTH AND FITNESS MEASURING DEVICES VS. MEDICAL MEASURING DEVICES

As the consumer wearables move increasingly towards measuring traditional medical indicators like heart rate and blood pressure, the distinction between sports-and-fitness aids and medical devices becomes increasingly blurred.  The former are not subjected to regulation but the later are regulated by the FDA, including being subject to approval.  For now, manufacturers of most fitness-oriented devices and consumer smart wearables have not sought such approval.  At the same time, a number of other manufacturers have sought and sometimes have received approval.  See the December 2014 article Round-up: 31 FDA clearances for digital health in 2014  “In August, the FDA proposed to largely deregulate a sizable list of Class II and Class I medical devices and no longer require their makers to go through the 510(k) process. These devices included thermometers, smart body scales, stethoscopes, and some ophthalmic cameras. — Still, there were many devices that did get clearances this year — by MobiHealthNews’ count there were at least 31 new FDA 510(k) clearances for digital health. == Devices cleared this year include an app that uses the iPad’s camera to estimate the amount of blood lost during a surgery, a smartphone-connected thermometer, an iOS application that treats a medical condition called tinnitus, and a vitals sensing chair. There was even one mysterious clearance from South Korean technology company LG Electronics for something called “LG Smarthealth”.

The clearances are listed in the article.  Most relate to applications in medical practice. Yet the distinction of what constitutes a medical device remains fuzzy. “Swiss remote cardiac monitoring company LifeWatch received FDA 510(k) clearance for LifeWatch VSP (Vital Signs Patch), the company’s adhesive patch for remote patient monitoring. The device is a disposable adhesive strip which contains sensors to monitor ECG, heart rate, respiration rate, temperature, saturation, and movement. It also contains a battery which allows the device to collect data continuously for five to seven days.”  This description of functionality sounds identical to that for some sports-and-fitness wearables.

SMART CLOTHING

Building sensors into clothing and shoes seems to be a sensible approach and is already starting to happen.  From the Gizmag article Top personal health and fitness gear of 2014: .  “The Omsignal biometric smartwear is created from a stretchable, machine-washable fabric designed to compress the user’s torso in order to encourage blood flow both during exercise, and after, to expedite recovery. The shirts feature an inbuilt accelerometer and electrocardiogram sensors that monitor variations in the wearer’s heartbeat throughout the workout, while other sensors work together to calculate calories burnt. Data collected by the smart shirt is recorded by a detachable data module and relayed in real time via a bluetooth to the Omsignal companion app (currently only available for iOS) on the user’s smartphone. — The collection currently features only men’s smart shirts, which range from us$100 to $130 in price, with women’s shirts slated to become available in early 2015.  —  Not content with its own offerings, Omsignal also partnered with Ralph Lauren to design the Ralph Lauren Polo Tech Shirt. Complete with polo player logo, the shirt features an electrocardiogram, a breathing sensor, a gyroscope and an accelerometer that monitors and analyzes stress levels, energy output, heart rate, heart rate variability, breathing rate, breathing depth, activity intensity, steps and calories burned and provides real-time training feedback. For those looking to keep fit in colder climes, the Hexoskin Arctic Biometric Smartshirt (us$199) features sensors knitted into a new textile developed from research on polar bears. It tracks metrics like heart rate, steps and calories burned, as well as cadence, activity level, acceleration, breathing rate, sleep duration, heart rate recovery and variability, and more (in all, it tracks some 42,000 data points per minute). — from shirts to shoes – or at least, insoles. The moticon opengo sensor insoles (price on application) turn your footwear into a wireless performance-tracking system with 13 pressure sensors that monitor foot pressure, movement, acceleration patterns and gait, with the data stored on a usb stick for post-workout computer analysis, or transmitted wirelessly via ant+ for real-time feedback while working out.” I think it will only take a few years for costs of sensor-ladened clothing and shoes to come down to ordinary consumer price points.  And the polo tech shirt already appears to have sensors beyond those available in any smartwatches

HEALTH AND FITNESS APPS

Starting perhaps four or five years ago, health and fitness applications began to appear for both the Apple iOS and the Android smartphone  platforms.  They are available in the Health and Fitness departments if the iTunes Store and of Google Play. These are variously devoted to activity and sleep tracking, management of diet, calorie counting, weight tracking, connecting you to fitness communities, personal training, customized workouts,  specific athletic activities such as running, and countless specialized functions.  Some make use of your phone camera and offer voice commands or virtual personal trainers,  many allow you to compare week-by-week progress.  Some are based on or can connect to motion trackers, or use your phone’s GPS, to determine where you have been, the distance covered and the level of exercise. Some are friendly companions, others are more like judgmental or bossy coaches.  Some award you points for healthy behavior, some give you constant reminders, some network you with possibly supportive people.  Some track your activity and tell you when you have fulfilled your daily quota.  Some apps can be customized to personal activities like rock climbing or playing ping-pong.   Some interface with pedometers, either in devices like a FitBit or built into your smartphone.  Some perform highly specialized functions, like variable heart rate analysis when connected to a chest strap with sensors.  Some have you play health and fitness games.   The variety is almost endless.  Zombie movement tracking, anybody?  The more complex of these apps cover several approaches and increasingly offer integrated personal views of health from multiple viewpoints.

To get a more concrete sense of these apps, here are short reviews of the “best 64″ such apps for 2014.  Here is another list of the “best 100 android health and fitness apps” for 2014.    One noticeable characteristic of these reviews is the general absence of hard technical or performance specifications that allow any real comparison.   The reviews tell you what the apps look like and are generally supposed to do, but not how well they actually work and leave you little sense of what is best to buy.

Here is a news story from June 2014, and so already hopelessly obsolete.  “health and fitness apps finally take off, fueled by fitness fanatics — “over the past six years, we have seen mobile and its apps disrupt and transform many industries. The healthcare industry, while named early on as an industry that would be totally transformed by smart devices, seemed to have lagged behind. In 2013 while the overall mobile app industry grew 115% in terms of average daily usage, the health and fitness category only grew 49%. This appears to be changing rapidly in 2014. We are not even halfway through the year (and usage normally accelerates in the summer and during the holidays) and the growth in health and fitness app usage has been stunning. We have studied the usage of over 6,800 iphone and iPAD apps listed in the health and fitness category on Flurry’s platform and we have seen a 62% increase in usage of health and fitness apps over the past six months. This compares to 33% increase in usage, measured in sessions, for the mobile app industry in general. Growth in health and fitness is 87% faster than the industry, which is itself growing at an astounding rate.” according to Nielsen: 46 million people used fitness apps in January  2014(ref).  The current number is of health and fitness apps users is without doubt more than double that.

The good news is that these apps tend to be cheap – typically free or less than $5, and increasingly comprehensive and multi-platform accessible, working interchangeably on a smartphone, a PC or a pad.  And they offer a multiplicity of displays of health data.  The bad news is that the vast numbers of them make it very difficult for a novice to figure out which ones to download and use.  Another piece of bad news is that those that connect to wearables tend to be very device specific.  The information offered about them online tends to be very limited.  And almost every app stands alone.

INTEGRATED CONSUMER HEALTH AND WELLNESS PLATFORMS

Given that there are many dozen kinds of of biometric sensors coming into use, be they in smart phones, wristbands, earbuds, clothing, shoes, or patches, question that arise include:

1. What specific patterns of data among all those collectible by today’s and emerging wearables are useful either to individuals or health practitioners for determining states of health, possibly predicting diseases, and suggesting actions?
2. As a prerequisite, how can this data be collected, viewed and analyzed together in ways that make sense for analyzing or predicting health and suggesting individual actions that will lead to longer healthier lives?

I do not know, nor do I think that anyone knows, the best answers to the first question. What I can say is that I have started to pay very careful attention to the personal data I am gathering now from my Basis Peak and am looking for patterns and correlations that I think may be meaningful. I believe I am finding interesting correlations between Peak data patterns and experienced stress events.  I will also be conducting tiny personal experiments, such as determining how my nightly dose of melatonin or the time I have dinner or what I eat for dinner may affect my sleep patterns.

Integrated data analysis platforms

With regard to the second question, we already seeing the emergence of software platforms that will receive data from multiple sources and display them integrated together. As can be expected, the main competition now seems to be between Apple and Google and their respective operating systems iOS and Android.

In the Apple world, “Apple’s new mobile operating system, iOS8, released in September included a new feature in the Health app that displays various bits of health data collected by various apps in one place. In the same vein, the app’s Medical ID feature lets you input various pieces of health information that could be important for emergency services workers if you are involved in an accident. This includes such things as allergies, blood type, medical conditions and emergency contacts. Importantly, this data can be accessed even if your phone is locked.(ref)”  The healthkit wellness app appears to be the main current Apple effort. “Healthkit will allow a user to view a personalized dashboard of health and fitness metrics, which conglomerates information from a myriad of different health and wellness apps, helping them “communicate” with one another.  With this technology, it’s easy to envision hospitals, clinics, pharmacies, laboratories, and even insurers integrating bilaterally with any patient information housed on healthkit, at the discretion of the user. ” Mayo Clinic, Cleveland Clinic, Kaiser Permanente, Stanford, UCLA, And Mount Sinai Hospital are all rumored to be working with Apple to figure out how to exchange relevant patient information to enhance the continuity of a patient’s care. In addition to these potential collaborators, electronic health record providers epic systems and allscripts are rumored to be working with Apple in some sort of partnership.^3,4”  at least 56 apps to start with will connect to healthkit(ref). The consumer interface of healthkit, called Health is preloaded on all iphones running iOS

In the Android universe, it appears that Google Fit is the main competitor to Apple’s Healthkit. “Mobile fitness apps are a dime a dozen these days, but Google is trying to add value by letting fit act as a hub for third-party apps like those from Strava, Withings and Runkeeper. Fit users can access data gathered by those apps within the Fit app, instead of having to switch between them. That functionality makes Google Fit the prime competitor to Apple’s Healthkit, a software platform for iOS 8 that lets third-party apps share their data with Apple’s health app. Google Fit is available for devices running Android 4.0 (Ice Cream Sandwich) and above. Whether Google Fit catches on likely depends on the number of integrations it will support with other apps, and how well it presents the combined data. It’s unclear if other popular apps from device makers like FitBit or Jawbone will be integrated with Fit, and Google didn’t immediately comment.  In addition to it being an app, Google Fit is a software development kit. Its apps aim to let developers access data from other sources to make their own apps more powerful. Fit, therefore, could be a win for Google by strengthening the broader ecosystem of health apps, and then weaving them into Fit.  Fit API partners include Basis, Adidas And Motorola.” (As of the writing of this blog the Basis Peak data cannot be imported into Google Fit)

Both the Healthkit and Googlef Fit apps have had their own problems.  See the October 2014 articles Apple’s health app is an embarrassment and Google Fit has its own ailments.  I had no problem downloading the  Google Fit app yesterday but found it quite useless in its present state for what I wanted it to accomplish,  I wanted it  to offer me an integrated view of data from my three current health and fitness devices.  Those are my FitBit 1, my Basis Peak and my Samsung s5 smartphone which also has a pedometer and instantaneous heart rate measurement capabilities. As far as I can tell, there is no way right now to import data from the FitBit 1, or the Basis Peak into Google Fit.  Apparently, neither the FitBit nor the Basis organization have released the necessary APIs (application program interfaces).  So, a serious impediment to the emergence of software platforms that offer integrated data views from multiple devices could be the device manufacturers themselves who want to hold on to their own users via their own software.  It could be that both Healthkit and Google Fit are actually on the right track but just not there yet.  A Google fit webpage soliciting participation of sensor-makers lists 20  “partner” organizations including Intel, Basis and Withings. A list of apps that are presumably now compatible with Google Fit is here, as of this writing 13 in number.

MEDICAL AND RESEARCHER SKEPTICISM

The health and wellness consumer movement is seen by some members of the Health Care and Medical Establishment as possibly unsanitary trespassing on their pre-owned territory.

They rightly point out that the devices mentioned here are not regulated and who knows what these gizmos really can and cannot measure.  And human behavior being as it is, information feedback may or may not be enough to influence personal health related behavior.  Already we are seeing a few warning rockets being fired.  For example, check out this research report: Wearable tracking devices alone won’t drive health behavior change, according to researchers. “New Year’s weight loss resolutions are in full swing, but despite all the hype about the latest wearable tracking devices, there’s little evidence that this technology alone can change behavior and improve health for those that need it most, according to a new online-first viewpoint piece in JAMA. The paper, written by researchers at the Perelman School of Medicine, the Penn Medicine Center for Health Care Innovation, and the LDI Center for Health Incentives and Behavioral Economics at the University of Pennsylvania, points out that even though several large technology companies are entering this expanding market, there may be a disconnect between the assumed benefits and actual outcomes.”  Personally, I think this kind of engagement of serious researchers and questioning in the Wild-West health-wearables market could be a very good thing and should be welcomed.

SOME QUESTIONS AND SPECULATIONS

Will the area of consumer health wearables continue to take off as seems to be happening now or will it turn out to be a current fad as people do not find the health-measuring facilities of their smart watches to be that useful?  

I expect the wearables trend will continue to take off and become mainstream, although there are likely to be fits and starts and many products and concepts that go nowhere.  The trend will be part of and empower a broader trend of consumer health consciousness and individuals increasingly taking responsibility for their own health and longevity

Where is body sensor tracking going?  What, beyond smartwatches and straps will be the next big way of embodying consumer health- measuring sensors?

I do not see the present major trend continuing – that is integrating more and more sensors and measuring capability into a single device, be it wristband or smartwatch. Every added sensor, especially if it is constantly active in measures 24/7, takes space, has weight and consumes power. There are only so many sensor devices that can be compacted into the case of a smartwatch.  Nobody wants a 2 pound smartwatch.  That is why the different fitness band and smartwatch offerings available today are all compromises and none have all the functionality that is possible. Some leave out the altimeter, some leave out the pulse oximeter, some leave out the continuous pulse rate monitor, some cannot measure O2, etc.  I see the emergence of numerous specialized measuring devices which may be associated with different parts of the body, which are integrated together by third-party multi-sensor tracking systems.  The earbud sensors and leg motion sensors mentioned above are examples.  Others might be in the form of patches, pills that are swallowed, shoelaces, pieces of clothing, shoe inserts, and subcutaneous implants.

Specifically, how popular can we expect clothing that embodies biosensors to become? 

My guess is, in the longer run, very popular, along with smart shoes, hats, scarves, gloves, earrings, rings and pins. wallets, jewelry, skin patches and what-have you.

Where will the point of integration for all these sensors be on the body? 

For now the best answer seems to be in the smart phone where there is plenty of processing power, memory, and external communications capability.

What about industry standards?

Standards will have to be developed, getting us beyond the current Wild West of proprietary software platforms associated with individual pieces of proprietary hardware and the over 100,000 health and wellness apps out there.

To what extent will there be a confluence of consumer health-and-fitness and medical sensing devices?  Will the consumer health systems connect with or become integrated with or evolve into professional ones utilized by the medical profession? Or will they continue to evolve in their own trajectories and be regarded by medical professionals as “toy” systems. How and when will mainline medical practitioners tune into and adopt their practices to what health-conscious consumers are doing? 

This is very hard to predict.  I expect the consumer and medical devices and systems to remain in separate categories for some time.  I foresee a slow and bumpy process of reconciliation of consumer-driven health initiatives and medical-establishment protocols and initiatives, one that may require 30, 50 or more years.  In the shorter term, the consumer-related technologies and personal use patterns are likely to evolve very quickly in comparison to the hardened institutionalized patterns of the medical establishment, big pharma and the FDA.  There will be controversy.  There will be continuing efforts to regulate the consumer products as medical devices, but these will be mostly resisted. If history is any predictor, I expect soon to hear solemn warnings from medical device doctors in white coats not to trust health measurements made by consumer devices.  A few forward-looking HMOs or medical insurance companies may move to accelerate a process of reconciliation, however.  It will be fun to watch how this all unfolds.

ON TO PART 2

I will publish a Part 2 blog entry soon related to the likely emergence of practical health stress biomarkers, based on both theory and very recent personal experience.  I acquired a Basis Peak smartwatch a little more than three weeks ago and have been building a log of daily and nightly experience with the parameters it can measure – continuous heart rate, movement, skin temperature, ambient temperature and perspiration.  Based on these measurements I have been looking for overall health measurements – basically measures of overall constitutional condition of stress.  I believe I have been successful at identifying such, but don’t want to publish my conjectures until I have at least a month of data and personal experience to report on.  I am very excited by this effort.  In addition, in a Part 3 blog entry Jim Watson will report on  what is generally  thought to be a good measure of constitutional health – HRV, Heart Rate Variability,

DISLAIMER

None of the authors has or has had a personal or commercial relationship with manufacturers or sellers of any of the devices mentioned here.   In mentioning personal choices and experience, we acknowledge that our choices were necessarily relatively uninformed, given a general lack of reliable technical and performance specifications.  More capable products might appear on the market at the same or lower price point within six months, and any existing device may be obsolete in a year.  These are characteristics of a fast-paced consumer electronics market with multiple competitors at its current stage of development – like the situation was with consumer digital cameras about seven years ago.

By Vince Giuliano

This entry is different from any other published so far in this blog  – it describes an original research experiment rather than characterizing a whole area of research or being an editorial.

This is the second blog entry related to electronic wearable devices capable of making personal behavioral and physical parameter measurements that bear on health and wellness and likely longevity.  The first blog entry was Digital health – health and fitness wearables, apps and platforms – implications for assessing health and longevity interventions – Part 1 Flux in the market.  It provides a snapshot view of one area of digital health – the rapidly changing landscape of consumer health and fitness wearables like smart watches, online and mobile health and wellness applications, and the associated emergence of software platforms that can integrate such applications together.  I say snapshot because of the rapidity with which the market for these device is evolving.

This current blog entry reports on initial personal experience related to identifying what appear to be effective daily stress biomarkers that are derivable from measurements made by my Basis Peak smartwatch.  I report here on the results of logging data measurable by the watch for 30 days, analyzing it, identifying what I believe are critical constitutional stress biomarkers, and confirming that values of those biomarkers indeed correlate well with known stress events I have gone through over a period of 30 days.

A third blog entry on this series written by James P Watson, is currently in the draft stage and will be published soon.  This will be on Heart Rate Variability, a well-established and well-studied constitutive stress biomarker different from the new ones that I propose and discuss here.

THE DESIRABILITY OF AN EASILY MEASURED CONSTITUTIONAL STRESS BIOMARKER

First of all, I reiterate why I think this research could be quite important.

The Part 1 blog entry describes a fantasy enjoyed by some health and longevity researchers including myself – the availability to ordinary people of constitutional stress biomarkers that will help us understand the effectiveness of the multiple interventions we are entertaining for maintaining health and active longevity.

As I said there, “Such an intervention could be a simple change in diet, exercise or sleep patterns, or consuming a new supplement or drug.  Stress events could be of an emotional or traumatic nature or as simple as disruption of the normal sleep pattern.  The desired biomarkers could conceivably provide easy and very accessible answers in a few days to very basic questions such as “Is my new approach (to a new pattern of exercise/taking a new supplement or drug, sleeping longer or differently/etc.) really working?”  “Comparatively speaking, how well am doing now compared to before?”  “What price am I paying for staying up drinking until 4 AM last night?”  “How long will it take for me after returning from China to re-establish my normal circadian health patterns?” “What has been the health impact of my partying for several nights in a row over Christmas, drinking more than usual and eating large late meals?”   Or “I have added a high intensity 8-minute segment to my daily exercise regimen, designed to send my heart rate above 125.   What is this doing to me or for me?”  This fantasy of being able to answer such questions with measured physiological data may well be realized through a powerful wave of developments in consumer electronics powered by billions of dollars in investment.”

Based on the limited observations reported in this current blog entry, I think that I have identified a couple of readily measurable and indicative day-to-day stress biomarkers of exactly the kind we have been looking for.  These can be derived from measurements from a smartwatch, a Basis Peak in my specific case. No need for chest straps, electrodes and electrode gel required for heart rate variability measurements.  On the one hand, this blog entry is about a highly personal exploration.  On the other hand, it might also characterize stress biomarkers that can be measured and utilized by everybody.  Only a much longer period of systematic study and tests by additional people will tell for sure.

Some background personal experience over the last year

 Skeptics ask the question of “what good are the measurements made by health and fitness wearables anyway?  What is their practical value?  The skeptics point out that there has been a very high rate of abandonment of use of the first-generation trackers(ref).  People have been discovering that they are not conveying useful new information after a while, and stop using them

I don’t have definitive answers to this question yet. I don’t think anyone has. Yet I remain excited about the question and am happy to report here on my personal experience with exciting new stress bioindicators.

The personal usefulness to me of my first the-generation health wearable, a FitBit One, has been providing me ongoing support and impetus for my daily exercise regimen.  For about a year now, it has provided me a good approximation of the degree to which I have moved my body and calories expended, so that in late afternoons and evenings I have a good assessment of how much more exercise I need to fill in to meet my daily exercise quota.  I have been looking to move 3.5 miles or more a day and burn at least 1750 calories.  Some days I will go out on several shopping expeditions or engage in physical work such as mowing the lawn or raking leaves or shoveling snow, perhaps climbing 18 flights of stairs in the course of the day.  On those days I will easily make the daily exercise quota just by what I have been doing. Other days, particularly ones when I have been sitting at the computer studying and writing, I have sought to make the quota by filling in exercise later in the day on the treadmill or possibly by swimming or other physical work around the house.  The FitBit One measurements have been very useful to help me make those decisions.  They let me know how much I had to fill in.  And, the weekly comparisons have been useful to tell me whether I have been following my exercise  intentions.  And the FitBit scale measurements of weight and BMI have been helpful for letting me know if my eating is getting out of hand.

NEW MEASUREMENTS WITH THE BASIS PEAK

I have had the Basis Peak smartwatch for a little more than a month now and have been in the process of discovering what it is good for. The device provides very interesting records of activities and sleep.  The device includes sensors for movement (an accelerometer), heart rate, skin and ambient temperature, and galvanic skin response (for measuring perspiration). It logs these measurements from my wrist 24-7 when it is not in its charging station.  The following two diagrams illustrate a typical day’s activity record and sleep record

Image may be NSFW.
Clik here to view.

Taypical Basis Peak daily activity record

Image may be NSFW.
Clik here to view.

Typical daily Basis Peak sleep record

The Peak software is constantly recording, except when the watch is in its charging station – for about an hour every other day. New records like the above are produced every day and the historical ones are available online or on my smartphone.  The Peak also produces a display that illustrates levels of daily activity by the hour for a week.  That looks like this:

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Clik here to view.

CHART A Activity during week

Depth of color represents level of activity in terms of calories burned per hour..  Note that January 7 was different than the other days; I comment later here on what happened that day..

DERIVING DAILY STRESS BIOMETRICS FROM DATA PROVIDED BY THE PEAK

Since receiving the watch, I have been very concerned about what useful biometrics might be based on this kind of data.  Based on theory, actual measurements and correlating them with personal stress events as reported below, I am coming to believe that there are easily derived  measurables that together can constitute indicators of daily and even hourly stress, and constitutional stress recovery capability. My confidence in this statement and excitement about this has continued to grow as I have tracked these measurables for 30 days now.

About resting heart rate RHR

Key to my observations are measurements of resting heart rate RHR (ref). RHR is normally taken to be an indicator of overall health, level of fitness and capability to handle stress(ref).  The lower RHR the better, assuming there is no pathology or drug use effect. And lower RHR correlates with higher heart rate variability HRV, which is seen as another important measure of fitness and ability to handle stress.  An objective of exercise training is to lower our RHR.  Beats per minute (BPM) up in the 70s are too high, and possibly indicating a pathology if RHR is up in the 80s.  RHR down in the 50s is good and in the 40s for trained athletes is even better.  This chart rates RHR to fitness, from a sports and fitness science site Top End Sports

Note in this chart that RHR is not very sensitive to age but is quite sensitive to fitness condition.

However, RHR is not one thing: it varies day to day, according to the time of day and according to individual state of constitutional stress.  Mine normally falls in the “excellent” range for people my age 56-61BPM.  But some times when I am stressed it can be in the upper 60s.  So, the normal advice is to measure RHR right after you wake up in the morning while you are still resting in bed.  Still, it is not at all the same day-to-day.  Worse, actual measurements with the Basis Peak indicates that there is an immense catch in that you can’t reliably measure RHR in that usual way described commonly in the literature. Again normally, RHR is supposed to be determined by measuring pulse immediately after you wake up before you get out of bed.  According to my Basis Peak, that is like getting out your camera and photographing a lightning strike when you see one.  There is no stability in heart rate during that interval according to the Peak.  After waking up, heart rate instantly starts to spike upwards as does perspiration even before I am fully awake.  By the time I could start taking my pulse, it is too late.  So, if the Peak is measuring correctly, the conventional way of measuring RHR yields unreliable results.  That is the bad news about RHR, but there is also seems to be some very good news.

The next observation is that over all the 30 days of measurement so far, that there are more or less stable plateaus of heart rates every night and every morning in which heart rate remains quite constant for at least an hour and a quarter.  One plateau occurs shortly after sleep starts and the other plateau occurs just before sleep ends.  These plateau intervals, according to the Peak are during sleep and only observable when awake by examining records.  I call these;

ERHR for evening resting heart rate. In measuring this for a given evening, I access the Peak online records, take four measurements of heart rate during the just-after-going-to-sleep plateau, and average these.  These are averages over about an hour and 15 minutes, and each Peak measurement included is also an average over a number of minutes.  So, the values truly represent stable plateau values.

MRHR for morning resting heart rate. In measuring this for a given morning, I access the Peak online records, take four measurements of heart rate during the before-waking-up sleep plateau and average these.  These are also averages over about an hour and 15 minutes, and each Peak measurement included is also an average over a number of minutes.  So, the values again truly represent stable plateau values.

So, I believe MRHR gives me resting heart rate measurements that are more reliable than can be obtained the conventional way. But this is just the beginning of the good news. There are other interesting and consistent day-to-day observations.

Here are some of my current speculations.

1. MRHR is a measure of how prepared the body is to deal with stress in the coming day, stress responsiveness. The lower the MRHR the more relaxed and refreshed the body is and prepared to deal with stress. This is based on the literature of what is normally called “resting heart rate.” As mentioned, MRHR is more accurately and consistently measured during the final stage of sleep instead of after waking when heart rate is no longer stable and invariably is spiking up.
2. ERHR is a measure of the accumulated stress at the end of the day, accumulated constitutional stress, some of which may have been accumulated in the course of several days. The greater the ERHR, the greater is the need for sleep recuperation regeneration or other measures that restore stress responsiveness.
3. The overnight ratio MRHR/ERHR is a measure of the degree of recuperation and regeneration in the body that has happened in the course of the period of sleep. The lower this ratio, the better. This ratio appears to be always less than 1 and averages about 90% for me over the 30 days of measurement,
4. Another measurement of the degree of the overnight degree of recuperation and regeneration is the difference of the two beat rates ERFR-MRGR. The bigger, the better the restoration of stress response during sleep. This number appears to be always positive and ranges from near-zero for me to over 12BPM.  Over the 30 days it averages 6.15BPM.

MRHR LEVELS AND ERHR-MRHR CORRELATE WELL WITH PERSONAL STRESS EVENTS I HAVE EXPERIENCED DURING THE PERIOD OF MEASUREMENT

Here in Chart B is a graph showing my MRHR and ERHR  for the 30 days of measurements I have taken so far.

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Clik here to view. CHART B  Blue is night-before ERHR and red is MRHR for the same night of sleep, in BPM.

Chart B graph is a good dynamic portrayal of the three measures MRHR, ERHR and ERHR-MRHR.  The blue line shows ERHR, stress on going to sleep and the red line shows MRHR, stress the morning after just before waking up.  The gap between the red line and the blue line is a measure of overnight stress recovery ERHR-MRHR.  The bigger the gap for a given day between the two lines, the more the sleep-related stress reduction and the better.  When the gap is tiny or vanishes, accumulated constitutional stress is not worked off very well by sleep.

Now, let’s look day-to-day at what is shown in Chart B as correlated with stress events in my life.  To help you understand what is going on, I allow myself to relate some personal matters.

Up through day 10 I am living my life more or less regularly with regular sleeping and no special stress events.  Then what happens on days 10-13? MRHR peaks and overnight sleep stress recuperation almost vanishes (as measured by ERHR-MRHR being very close to zero on day 11. There is a pretty good explanation. Days 11 and 12 are Christmas Eve and Christmas Day, times of irregular schedules, heavy late meals, multiple parties round-the-clock family events, birthday parties. a certain amount of drinking, and the emergency hospitalization of my first wife. I speculate that these stress events worked not only to raise MRHR but also to compromise night-time recovery of heart rate homeostasis over sleep

It appears that I have had very good stress recuperation starting on overnights 13-17.  And the morning of the 15, MRHR was 54bpm, the lowest measured. Again, my life was getting back to regular and I was feeling good about things.

Note that on day 18 the gap ERHR – MRHR closed and stayed closed through day 21.  What happened then, what stresses led to that? Here are some explanations: A possible factor affecting day 18 may be cold stress. Before retiring the night of the 18th, my wife, granddaughter and I were out walking in the bitter cold in downtown Boston for more than an hour and a half to see the First Night celebration events and fireworks, I was thoroughly chilled, Hoever, probably the key factor applicable during this period is nutritional stredss. I had a colonoscopy scheduled for day 21, so following the orders of my gastroenterologist, I literally had to start wrecking my gut biome on day 16. That is I had to go on a no-fiber diet featuring white bread, no fresh fruits or vegetables, no oatmeal, no nuts, and empty-carbs food and drinks. I also had to quit a couple of favorite supplements – Fish Oil Omega 3s and glucosamine. You can see how this and possibly the cold stress caught up with me by looking at what happened on Chart B on day 18. This is New Years Eve where the gap ERHR-MRHR nearly vanishes again as it did a week earlier. Yes dear reader, diet does have an immediate impact on stress responsiveness!, Without the butyrate and other good responses produced by the fiber-responsive gut bacteria, there goes my overnight stress reduction capabilities

That crummy situation persisted through to prepping for the colonoscopy procedure on the eve of  day 21. The sleep on the night of the 20-21 was particularly bad due to being mostly awake running back and forth to the bathroom   You can see that night was characterized by unusually high activity, by looking at the above weekly activity of Chart A for Wed Jan 7.  Note from Chart A that I got up that day two hours earlier than normal that day to go to the hospital, and there was an hour of quiet starting at 9AM.  That is when I was under anesthetic for the procedure in the hospital.  You can see on Chart B that the accumulated impact on day 21 is that the stress indicator MRHR of 67bpm is the highest on record.  And there was virtually no stress recovery ERHR-MRHR during the stressful night before.

You can see from Chart C below how my pattern of types of sleep started to go screwy the night of day 16 with no REM sleep then, and stayed screwy through the time of the colonoscopy on day 21.

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CHART C  Green is light sleep, red is REM sleep and blue is deep sleep according to the Peak, all in percentages.

What happened on night 16 with no REM sleep? Perhaps it was the dawnin on me then that my first wife Lois would very soon die. We have always been good friends, we share two sons, and her family and mine share holidays and events together. She has been there in my life and part of my extended family for the last 65 years.

Going backto Chart B, days 22 and 23 show recovery of ERHR- MRHR and some but not complete recovery of MRHR.  What happened then? After the colonoscopy I resumed a normal diet and had had a good 2 hour nap. That night’s sleep was long and sound with ample REM and deep sleep and you can see significant changes in the charts toward a normal pattern on day 22. MRHR/ERHR is back down to 86%. You also see normalization of the sleep chart type curves in the Chart C

The gap ERHR – MRHR Chart B for day 24 seems to be closed again, indicating no overnight stress recovery, and MRHR shows no recovery to previous lows.  What happened then? Day 23 was a time of grief stress for me, with the funeral for Lois, ceremony, family wake events, and sharing of the loss with loved ones. That night I had horrible dreams. And body stress as measured by MRHR has still not recovered to the lower rates in the 50s before the colonoscopy

Days 25-30 show a steady trend to stress response recovery with slowly lowering MRHR, with Chart C showing declining oscillations between the different types of sleep.  Day 25 shows (Chart B) improvement: the gap ERHR-MRHR starting to open up again and a little lessening of the stress as seen in MRHR.  Day 26 of Chart B shows stress recovery ERHR-MRHR is there but not up to what it could be and stress level MRHR is stubbornly refusing to drop to its lower levels of before. Why is MRHR so slow in recovery to its possible low of 55?. I can think of two possible reasons: continuing sadness associated with Lois passing away, and a slow rate of recovery of my gut biome after the colonoscopy.

You can see days 26-30 reflect continuing normalization of all patterns to resemble those of days 1-10.  The large oscillations in the different categories of sleep shown in Chart C are markedly less and it appears that the impact of the previous stress events is slowly vanishing.

In summary, looking at the timing of stress events in my life over the last 30 days and correlating these day-by-day with measurements, it indeed appears highly plausible that MRHR is a measure of how prepared my body has been to deal with stress in the coming day.  And, that ERHR-MRHR is a measure of the overnight sleep-related degree of recuperation and regeneration in resting heart rate.

This is exciting for me because we have a long been searching for measurements of constitutional stress and measurements of stress-recovery capability. We are interested in these because we would like to know what our health interventions do, if anything.  The measurements MRHR and ERHR-MRHR might provide part of the answer.  In a next phase of this research as outlined below, now having established base-line measurements for me, I will be looking to see if some simple health interventions produce noticeable effects in these indices.  I will also be researching heart rate variability HRV during this period and seeing how those measurements fit in. My gut feel is that we might be getting a handle on measuring constitutional stress and overnight stress recovery.

ABOUT HEART RATE VARIABILITY

I am proposing that MRHR/ERHR and ERFR-MRGR are measures of overnight sleep-related stress homeostasis recovery.  This is new and fairly out-of-the-box.  The usual approach for using cardio signaling to estimate constitutional stress homeostasis recovery is via measuring heart rate variability (HRV).

So I comment here on HRV.  Heart rate variability (HRV) is generally acknowledged to be a good and well-studied measure of constituitive stress homeostasis recovery capability(ref).  So if my proposal is correct, people with large HRV should show larger and/or more frequent values of ERFR-MRGR and recover from stress events more quickly.  This idea is yet to be tested.   I have just started personal HRV measurements, but do not yet fully understand how to interpret them. They are much more complex measurements than those described above.

HRV as a health index goes back to the ancient Greeks(ref) and there are 17,895 Pubmed research publications related to it.  By contrast the measures proposed here are quite new. Use of these measurements were suggested to me by a reader comment in the Basis Peak blog about the sleeping heart rate plateaus, although as far as I know I am the first to formalize and systematically use them. Nonetheless at this moment I think they should be entertained.  For one matter, given the current state of technology, the measurements proposed here can be more easily measured 24-7 than HRV.  It is not now clear that HRV measurement can be accomplished by a smartwatch or wrist band.  To measure it reliably appears to require wearing a special belt or electrode array and use of electrode gel.  Also HRV is notoriously sensitive to the state of the individual, time of day, particular breathing patterns and other variables such as whether the individual is standing, sitting, or supine.  Despite its very long history, HRV remains a controversial measurement.  And comprehending it fully and understanding how to interpret it requires a level of expertise that lay people don’t possess.  James P Watson has drafted a comprehensive blog entry on HRV and I am currently helping prepare that for publication.  It will be appearing as Part 3 of this Wearables blog series very soon.

While the Basis Peak does not now measure HRV, it does contain sophisticated second- generation heart rate sensors and could possibly do so with proper software support.    If so, this could possibly be an excellent direction for the Basis company to move toward.

BEYOND STRESS TO ASSESSING IMPACTS OF HEALTH INTERVENTIONS

The personal events reported here are stress events. I expect that the proposed stress measures MRHR and ERHR-MRHR will be equally valuable for showing constitutional state impacts for important health and longevity interventions.  I intend to extend my program of deriving biomarkers from wearables and recording of correlated experiences.   During the coming month or two, I expect continue my existing 30 day logging of Peak data and in addition::

• To periodically measure my blood pressure using a cuff and add this to tracked biomarkers
• To log pulse rate measurements using a sensitive sensor on a chest strap, a Polar H7,  and alternative software to validate the Peak’s HR accuracy
• To measure and also log heart rate variability HRV using the same heart strap and Android-based software. I am currently using HRV Expert by Cardio Mood, sophisticated software created by a team in Moscow
• To see what correlations might exist between the stress-related indices reported here and HRV indices
• To look into alternative possible stress and other indices based on the collection of measurements made and known stress events and health interventions in my life, deriving inputs from the Basis Peak, the Polar Belt, my FitBit and possibly additional wearables
• To experiment with variations in my health and longevity interventions to see what impacts they will have on the indices.  For example, I have started to modify my daily treadmill exercise regimen to include 7-minute segments with higher heart rates on different days, starting with instantaneous rates of115 BPM ramping up to over 130 BPM
• I may also experiment with stopping one or more of my longevity interventions for a period of a week to see what the stress consequences are
• To experiment with doing my treadmill exercises at different times of day to see what impacts timing of this may have
• To network actively with other people involved with stress monitoring via sharing experience and data.  As of the moment I have started doing that with two colleagues
• To follow this blog entry up with others reporting more hypotheses and experience.

What exciting times we live in!

By James P Watson with contributions and editorial assistance by Vince Giuliano

This is the third blog entry related to electronic wearable devices capable of making personal behavioral and physical parameter measurements that bear on health and wellness and likely longevity. The first blog entry was Digital health – health and fitness wearables, apps and platforms – implications for assessing health and longevity interventions – Part 1 Flux in the market.  It provides a snapshot view of an important area of digital health – the rapidly changing landscape of consumer health and fitness wearables like smart watches, online and mobile health and wellness applications, and the associated emergence of software platforms that can integrate such applications together.  I say snapshot because the technology and market for these devices are evolving very rapidly..

The second blog entry is Digital health – health and fitness wearables, Part 2: looking for practical stress biomarkers.   It reports on initial personal experience related to identifying daily stress biomarkers that are derivable from measurements made by Vince’s Basis Peak smartwatch.  Vince reports there on the results of logging data measurable by the watch for 30 days (since expanded to 47 days), analyzing it, identifying a few simple but critical stress biomarkers, and correlating the biomarker indications with known stress events he has gone through. That blog entry describes original research by Vince, defining and testing constitutional stress biomarkers. Those biomarkers are simpler and more easily, consistently, and reliably measurable using consumer wearables technology than HRV. While grounded in a history of research having to do with resting heart rates, these biomarkers are subject to validation via additional research and testing by additional people. I share Vince’s excitement about them.

This Part 3 blog entry relates to Heart Rate Variability (HRV), a well-researched biomarker for constitutional stress recovery capability.  The entry is in two Sections:  Section I Which is a primer about the science and usages of HRV on medicine and sports, “HeartRate Variability 101,” and Section II which is about practical HRV-measuring wearables and associated sofware.  Vince and I have independently been taking HRV measurements on ourselves.   Up to this time, this personal experience has been initial and inconclusive

We will probably communicate further about the theoretical bases for Vince’s biomarkers and about our practical personal experience measuring both HRV and Vince’s biomarkers in a soon-to-be-published Part 4 wearables blog entry.

Section I:  Heart Rate Variability “101”

HRV refers to the fact that there are minor variations from one heart beat to another.  The pulses may look the same on an emergency room monitor, but if you look carefully you will see they are slightly different, e.g. the periods between the spikes are sometimes longer or shorter and other minor differences are also likely to be present.

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Image source

Not only are the pulses different, but heart rate is constantly going up or down a little.  Actually this variability is a good sign because the differences between beats in a healthy person represents a complex coordination between heart actions, breathing, nervous system and other body systems.  Under conditions of high stress and some pathologies these differences are suppressed and your heart beats more or less like a mechanical metronome.  A rock steady beat (low HRV) is predictive of diseases and cardiovascular problems.

“Various models propose that HRV is an important indicator of both physiological resiliency and behavioral flexibility, reflecting the individual’s capacity to adapt effectively to stress and environmental demands. It has become apparent that while a large degree of instability is detrimental to efficient physiological functioning, too little variation can also be pathological. An optimal level of variability within an organism’s key regulatory systems is critical to health. This principle is aptly illustrated by a simple analogy: just as the shifting stance of a tennis player about to receive a serve may facilitate swift adaptation, in healthy individuals, the heart remains similarly responsive and resilient, primed and ready to react when needed(ref).”

Here are some summary facts about HRV:

• HRV, a measure of cardiac autonomic function, is a proxy for autonomic nervous system function, one of the fundamental physiologic systems governing organismal homeostasis.
• HRV can be measured non-invasively and safely using a diverse array of tools at little variable cost.  All you need is a bluetooth communicating heart monitor strap costing as little as $50, a smartphone and computer, and to license a HRV app which could cost as little as $1.99 or be free.  You may be able to get a measurement of it using your smartphone’s camera and an aoo.  Section II of this blog entry lists practical alternatives.
• HRV measurement tools (including those available on personal mobile devices) already have a global footprint around the world.
• Autonomic dysfunction, as assessed by too little HRV, is associated with the panoply of aging diseases.
• Therapies that ameliorate the diseases of aging, reduce mortality rates, and improve longevity such as exercise, sleep, smoking cessation and caloric reduction have been shown also to improve HRV.
• Therapies that are known to promote health outcomes such as meditation, yoga, acupuncture and other alternative medicine treatments have been shown to improve HRV.
• Chronic stress, which has been shown to exacerbate diseases of aging, increase mortality rates, and shorten longevity, lowers HRV.
• HRV measurements via continuous fetal scalp monitoring have been used to prognosticate fetal distress and risk of mortality since 1965.
• HRV, blood pressure and heart rate statistics can be powerful predictors of all-cause mortaity

The March2015 publication Blood pressure and heart period variability ratios derived from 24-h ambulatory measurements are predictors of all-cause mortality reports “CONCLUSION: The 24-h BP to heart period variability ratios are powerful independent predictors of all-cause mortality, especially for elderly hypertensive patients with slow heart rate. The results support their interpretation as integrative indices of cardiovascular function and markers for cardiovascular dysregulation during low DBP states, with potential use in clinical practice.”

I think that HRV is a great concept, considering that it can now be measured so easily with a “ECG-quality R-to-R interval measuring, Blue tooth chest strap” and a smart phone.  I describe options in Section II below

The history of HRV is very venerable.  Heart Rate Variability – A Historical Perspective points out that HRV was noticed by the ancient Greeks ”  ” Archigenes (1st century AD)  apparently described eight characteristics of the pulse, including observations on its regularity and irregularity.”

A great deal has been learned about HRV since its discovery, and it has become an important tool of sports medicine and for sports endurance training.  there has been much research on the topic  Pubmed.org currently lists 18,188 research publications related to the topic. 

HRV analyses are based on measuring frequencies of heartbeat events, a variant of classical mathematical Fourier analysis.  By looking at the relative power present in different frequency bands of a heartbeat signal, a great deal can be learned about the functioning of the autonomic nervous system and general stress.  This was known back in 1981.  The publication Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control then reported “Power spectrum analysis of heart rate fluctuations provides a quantitative noninvasive means of assessing the functioning of the short-term cardiovascular control systems. We show that sympathetic and parasympathetic nervous activity make frequency-specific contributions to the heart rate power spectrum, and that renin-angiotensin system activity strongly modulates the amplitude of the spectral peak located at 0.04 hertz. Our data therefore provide evidence that the renin-angiotensin system plays a significant role in short-term cardiovascular control in the time scale of seconds to minutes.”

This kind of analysis, conducted by sophisticate software, can yield much more physiologic information than a simple number which quantifies total variation.

Here are some of the things that have been learned about HRV:

1. HRV spectrum: Respiratory Sinus Variation (High Frequency), Parasympathetic activity (High Frequency and Low Frequency), vs Sympathetic activity (Very low frequency)

As background, “The parasympathetic nervous system (PNS) controls homeostasis and the body at rest and is responsible for the body’s “rest and digest” function. The sympathetic nervous system (SNS) controls the body’s responses to a perceived threat and is responsible for the “fight or flight” response. The PNS and SNS are part of the ANS, or autonomic nervous system which is responsible for the involuntary functions of the human body.”:  The quote is from the webpage Parasympathetic vs. Sympathetic Nervous System which compares specific aspects of the two systems.

Here is a diagram of brain-heart communications that bear on HRV showing sympathetic and parasympathetic channels:

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Image and legend source: A healthy heart is not a metronome: an integrative review of the heart’s anatomy and heart rate variability, “Figure 3. The neural communication pathways interacting between the heart and the brain are responsible for the generation of HRV. The intrinsic cardiac nervous system integrates information from the extrinsic nervous system and from the sensory neurites within the heart. The extrinsic cardiac ganglia located in the thoracic cavity have connections to the lungs and esophagus and are indirectly connected via the spinal cord to many other organs such as the skin and arteries. The vagus nerve (parasympathetic) primarily consists of afferent (flowing to the brain) fibers which connect to the medulla, after passing through the nodose ganglion. Credit: Institute of HeartMath.”

HRV power spectrum analysis can differentiate between HRV changes that are due to breathing (called Respiratory Sinus Variation) and HRV changes that are due to the autonomic system (ANS). 

“The normal variability in heart rate is due to the synergistic action of two branches of the ANS (the sympathetic and parasympathetic branches), which act in balance through neural, mechanical, humoral and other physiological mechanisms to maintain cardiovascular parameters in their optimal ranges and to permit appropriate reactions to changing external or internal conditions. In a healthy individual, thus, the heart rate estimated at any given time represents the net effect of the parasympathetic (vagus) nerves, which slow heart rate, and the sympathetic nerves, which accelerate it. These changes are influenced by emotions, thoughts and physical exercise. Our changing heart rhythms affect not only the heart but indirectly also the brain’s ability to process information, including decision-making, problem-solving and creativity. They also directly affect how we feel. Thus, the study of heart rate variability is a powerful, objective and noninvasive tool to explore the dynamic interactions between physiological, mental, emotional and behavioral processes(ref).”

• Very low frequency variations (power) in HRV are primary sympathetic nervous system activity and include frequencies in the range of 0.04 – 0.15 Hz
• Autonomic system variations in HRV include both sympathetic and parasympathetic activity, but there is a lot of overlap in the spectrum between these two divisions of the autonomic system
• High frequency (HF) variations (power) are due to respiratory variations and are mediated by the parasympathetic system.  HF are frequencies in the range of 0.25 Hz.  HF power can be improved with exercise and deep breathing.  Because HF power (respiratory changes) is driven by parasympathetic tone, HF power is dramatically increased in endurance athletes who have a resting bradycardia (low resting heart rate).
• Low frequency variations (power) in HRV are due primarily to the parasympathetic system and include frequencies in the range of 0.15 – 0.4 Hz.
•  However, less sophisticated HRV analysis systems lump VLF and LF into one power spectrum.  When this is done, they typically do a ratio of LF to HF (LF/HF), which is normally about 3.6 + 0.7

Here is a graph of these spectrums:

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“Figure 2. Power spectrum of HRV (PSD = power spectral density).”  Graph and legend from Heart Rate Variability Summary prepared by Ichiro Kawachi in collaboration with the Allostatic Load Working Group. Last revised 1997.   “The approach uses Fourier transforms. The HRV spectrum contains two major components: the high frequency (0.18-0.4 Hz) component, which is synchronous with respiration and is identical to RSA. The second is a low frequency (0.04 to 0.15 Hz) component that appears to be mediated by both the vagus and cardiac sympathetic nerves. The power of spectral components is the area below the relevant frequencies presented in absolute units (square milliseconds). The total power of a signal, integrated over all frequencies, is equal to the variance of the entire signal. Some investigators have used the ratio of the low-to-high frequency spectra as an index of parasympathetic-sympathetic balance; however, this remains controversial because of our lack of complete understanding of the low frequency component (which seems to be affected by centrally generated brainstem rhythms, baroreceptor feedback influences, as well as both sympathetic and vagal input).”

Another diagram is

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Image source

Other publications describing the overall HRV approach include:

• Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control 1981
• Heart rate variability in athletes 2003
• Sympathetic control of short-term heart rate variability and its pharmacological modulation 2007
• Low-frequency oscillations in arterial pressure and heart rate: a simple computer model 1989
• Autonomic differences between athletes and nonathletes: spectral analysis approach 1997
• Supine vs. Standing HRV Measurement: Is one better than the other? 2012
•  Power spectral analysis of heart rate and arterial pressure variabilities as a marker of sympatho-vagal interaction in man and conscious dog 1986

As you can see from the references, HRV has been of great interest in the sports medicine and training fields.

Practical conclusions:

• We need a sensor and software that can tell us the “power spectrum” of HRV, including the HF power (0.25 Hz),  LF power (0.15-0.4 Hz) and hopefully the very LF power (0.04-0.15 Hz)
• The ratio of LF to HF is normally about 3.7 and ideally, we need a software program that computes this as well.
• We want to decrease sympathetic tone (VLF or LF),  increase parasympathetic tone (HF and LF),  slow down heart rate,  and increase deep breathing (HF) to improve HRV.  These are not going to be easy to do in a rat or a mouse.  But the good news is that if we are healthy, HRV measurement can guide us for doing them for ourselves

Ideally, we will simultaneously use a sensor that senses respiration, since this is such an important aspect of HRV

2. HRV Physiology: What “drives” Heart Rate Variability?

There are several anatomic structures in the brain, the brainstem, the aorta (pressure and chemoreceptors),  carotid artery (pressure and chemoreceptors in the carotid body),  and the heart (SA node) that “drive” heart rate variability.  Stretch receptors in the muscles also “drive” HRV.

References:

• Power spectral analysis of heart rate and arterial pressure variabilities as a marker of sympatho-vagal interaction in man and conscious dog 1986

• Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control 1981

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Reference for diagram:  Supine vs. Standing HRV Measurement: Is one better than the other?

HRV changes with Posture: Supine vs Sitting vs Standing position

HRV dramatically changes with body position – this is something we will NOT have to deal with in mice or rats (they don’t walk upright)

In the supine position, heart rate and blood pressure are low.  In the supine position, sympathetic activity is low and parasympathetic activity is high.

Just changing from the supine to sitting position changes these parameters somewhat, which can be measured in the power spectrum of HRV (i.e. LF vs HF).  In the supine position, the HF power spectrum is high. In the sitting position, the HF power spectrum of HRV decreases from 25% to 6.2%  (p < 0.005).  In the supine position, the LF power spectrum is low.  In the sitting position, the LF power spectrum of HRV increases.  One practical implication is that if you are older with a relatively lower HF power spectrum, you might get better HRV measurements while supine.

With standing, there is a dramatic withdrawal of parasympathetic activity and an increase in sympathetic activity.  This is not surprising, considering that these autonomic refexes are necessary to maintain blood pressure to the brain, in light of the standing position (i.e. gravity)

What is surprising is that endurance exercise training increased HRV in the HF power spectrum, but did not change HRV in the HF power spectrum with subjects in the supine position (Kiviniemi, 2007).

Conclusions:

• Because body position makes such a big difference on the HRV power spectrum and overall HRV values, measuring HRV in the same position is key to avoiding false positives and false negatives and getting consistent day-to-day HRV measurements.
• Standing position measurements may be a better reflection of the value of physical exercise if you are an athlete with a low heart rate.  If you are older or have a higher heart rate, on the other hand, the supine position might be better.
• Postural changes will not be a factor in mice or rats.  This is why the field is “wide open”, when it comes to doing HRV work in four legged creatures that do not walk on two legs

References:

• Supine vs. Standing HRV Measurement: Is one better than the other?
• Standing vs. Supine ithlete HRV Measurement 2012
• Effects of high altitude acclimatization on heart rate variability in resting humans  1996.
• Autonomic nervous control of heart rate at altitude (5050 m)  1994

4. HTN, CHF, and MI => increased sympathetic afferent output: Reducing autonomic (sympathetic) afferents from the brain, via the cardiac sympathetic nerves

Patients with hypertension (HTN) and chronic heart failure (CHF) have reduced HRV due to many factors.  After a myocardial infarction (MI), there is also a decrease in HRV.  HRV can predict mortality after an MI and in patients with CHF.

• In these patients, there is strong evidence that reduced parasympathetic activity contributes to the decrease in HRV with CHF
• In these patients, there is strong evidence that reducing cardiac sympathetic nerve activity increases HRV
• Major factors are salt intake, Angiotensin II, decreased endothelial NO, and increased sympathetic afferent output

I do not think HRV would improve that much by consuming plant polyphenols, but reducing the output from the CNS to the cardiac sympathetic nerves (CSNs) should make a big difference.  CSN output is a separate effect from respiratory sinus arrhythmia (RSA).  ” Respiratory sinus arrhythmia (RSA) is heart rate variability in synchrony with respiration, by which the R-R interval on an ECG is shortened during inspiration and prolonged during expiration. Although RSA has been used as an index of cardiac vagal function, it is also a physiologic phenomenon reflecting respiratory-circulatory interactions universally observed among vertebrates(ref).”

RSA is involved in the high frequency spectrum of HRV, whereas CNS activity is involved with the slow frequency HRV with a period of about 10 seconds.

Obviously exercise and sleep will help, but I do not think that yoga, meditation, or Tai Chi is very feasible in mice or rats.  We may be able to reduce CNS output with an RF interference electrode that could be “trained” to inhibit CNS output.  In rodents with aging that have low eNOS activity and low nitric oxide levels in their blood vessels, chemical sympathectomy with 6-hydroxy dopamine restores HRV. We may want to do this as well.

References:

• Sympathetic control of short-term heart rate variability and its pharmacological modulation 2007
• Nitric oxide and cardiac parasympathetic control in human heart failure
• Cardiac sympathetic nerve stimulation does not attenuate dynamic vagal control of heart rate via α-adrenergic mechanism 2004
• Dynamic characteristics of heart rate control by the autonomic nervous system in rats 2010
• Chemical sympathectomy restores baroreceptor-heart rate reflex and heart rate variability in rats with chronic nitric oxide deficiency 2014
• Sympathetic predominance an essential hypertension: a study employing spectral analysis of heart rate variability 1988
• Heart rate variability as an index of sympathovagal interaction after acute myocardial infarction 1987

Conclusions:

• Controlling HTN is clearly important for keeping HRV high.
• There may be a possibility of doing a  “chemical sympathectomy” with 6-hydroxy dopamine in our “reference mammals.”

5. Reducing Renin-angiotensin signaling

There is some evidence that reducing Renin Angiotensin II signaling (RAS) has a beneficial effect on HRV,  This primarily improves low frequency power of the HRV power spectrum,  Many studies of sleep have shown that Angiotensin II levels decrease at night in a circadian fashion (no surprise!).

At night, there is an increase in baroreceptor reflex sensitivity, which is the highest during dreaming.  “Baroreceptors (or archaically, pressoreceptors or baroceptors) are sensors located in the blood vessels of all vertebrate animals. They sense the blood pressure and relay the information to the brain, so that a proper blood pressure can be maintained(ref).”  With CHF, the circadian changes in HR and the baroreceptor reflex (BRS) is blunted.  This explains part of the mortality of CHF that is independent from LVEF

Angiotensin Type I receptors are the main way that this harmful effect of Ang II is mediated.  This may be why the ATR1 receptor blockers seem to have a longevity effect. See our blog entry ACE and Angiotensin II: The “Double Agents” that Play Multiple Roles in the Molecular Story of Life.

References:

• Angiotensin II disproportionally attenuates dynamic vagal and sympathetic heart rate controls 2008
• Reflex Regulation of Arterial Pressure during Sleep in Man – A Quantitative Method of Assessing Baroreflex Sensitivity 1969
• Disruption of cardiovascular circadian rhythms in mice post myocardial infarction: relationship with central angiotensin II receptor expression 2014
• Abnormal baroreflex function is dissociated from central angiotensin II receptor expression in chronic heart failure  2012
• Role of AT1 receptors in the central control of sympathetic vasomotor function 1996
• Effect of losartan, an angiotensin II type 1 receptor antagonist on cardiac autonomic functions of rats during acute and chronic inhibition of nitric oxide synthesis 2012

Here is a diagram of what happens with Renin-Angiotensin system blockade and HRV.

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Reference for diagram: Power spectrum analysis of heart rate fluctuation: a quantitative probe of beat-to-beat cardiovascular control

Conclusion:  Blockade of the Renin-Angiotensin system increases the area under the curve of the low frequency HRV power spectrum.  Using ATR1 blockers appears to be the best way to do this.

6. Exercise and HRV – HF power increases, LF power decreases in endurance athletes. Supine position with exercise.

Endurance/Aerobic exercise

Endurance, aerobic exercises increases HRV in the high frequency (HF) spectrum (respiratory sinus arrthymia) but actually lowers the low frequency (LF) power spectrum.  Endurance athletes therefore have higher HF power spectrum and lower LF power spectrum

This drop in LF power spectrum may represent an imbalance between parasympathetic activity and sympathetic activity, due to the resting bradycardia seen in endurance athletes.  This resting bradycardia is due to increase in parasympathetic outflow.

Weight lifting exercise (supine position)

Because the sympathetic system signaling is dramatically decreased when the body is in the supine position, weight lifters working out in on a bench in the supine position display a dramatic increase in HRV in the low frequency (LF) power spectrum.  This means that there is a decrease in sympathetic activity when in the supine position.

References:

• Assessment of training-induced autonomic adaptations in athletes with spectral analysis of cardiovascular variability signals 1995
• Heart rate variability in athletes 2003
• Autonomic differences between athletes and nonathletes: spectral analysis approach 1997
• Heart rate variability and autonomic activity at rest and during exercise in various physiological conditions 2003

7. Sleep and HRV -

Sleep has a beneficial effect on HRV.  At night, Angiotensin II levels go down, increasing HRV.  Baroreceptor reflex sensitivity goes up at night.  It is the highest during dreaming – REM sleep.

Here is a diagram showing typical behaviors of heart rate, the parasympathetic and the sympathetic nervous systems during wakefulness, non-REM and REM sleep.

Image may be NSFW.
Clik here to view.

Image and legend source  “(A) Modulation of cardiac activity during wakefulness: reflex loops [baroreflex (BaroR), respiration (Resp), chemoreflex (ChemoR)] including brainstem centers (BS) and central autonomic network including midcingulate cortex (MCC), insula (INS), amygdala (AMY) contribute to cardiac activity, leading to increased heart rate (HR), increased sympathetic activity (SNS), and decreased parasympathetic activity (PNS). (B) Modulation of cardiac activity during non-REMS: The drop in brain activity, with predominant contribution of reflex loops on ANS activity, leads to decreased HR, with parasympathetic predominance, and decrease in sympathetic modulation. (C) Modulation of cardiac activity during REMS: autonomic cardiac regulation is shared between central control in relation with the insula and amygdala and homeostatic control of the cardiovascular system by reflex loops, leading to decreased HR with sympathetic predominance and decreased parasympathetic activity. Red circles indicate increase and blue circles decrease in autonomic cardiac activity.“

Reference: Reflex Regulation of Arterial Pressure during Sleep in Man 1969

HRV has been suggested to be an excellent tool for the analysis of sleep and for distinguishing between healthy and pathological sleep.  See these additional references.

Conclusions: 

• Baroreceptor sensitivity can be rapidly “reset” at night.  This is very important for controlling blood pressure
• HRV results are likely to be different during during REM and non-REM sleep
• HRV can be  a useful tool for distinguishing between sleep disorders.

8. Oxidative stress and HRV

Rodents have accelerated telomere shortening that is NOT due to replicative senescence,  This means they probably have a lot of oxidative stress.  Although I could not find any articles specifically linking oxidative stress to HRV, I found some articles that showed that ROS-producing chemotherapy (alkylating agents) decreased HRV

Polyphenols and MitoQ may help some, but I think the following would be better:

1. Bucky Balls – See our blog entries on C60 fullerenes(ref)(ref),
2. Methylene Blue(ref)(ref)(ref), and
3. a few of the other potent mitochondrial-specific antioxidants like MitoQ that change the membrane potential across the inner mitochondrial membrane.

I speculate that these could potentially make a huge difference in HRV

References:

• The influence of oxazaphosphorines alkylating agents on autonomic nervous system activity in rat experimental cystitis model 2013
• Cardiac autonomic function in acutely nitric oxide deficient hypertensive rats: role of the sympathetic nervous system and oxidative stress 2011

9. Gut bacteria/Celiac disease

Some fascinating information is coming out about the effect of wheat intake in patients with celiac disease.  Patients with celiac disease have a much lower HRV than controls.  Also, patients with celiac disease have less HRV with deep breathing.

• 20% of the patients with celiac disease had parasympathetic dominance, whereas
• 36% of the patients with celiac disease had sympathetic dominance
• 44% of the patients did not show parasympathetic or sympathetic dominance

Measurements can show decreases in HRV with all three of these groups of celiac disease patients.

Reference: Disturbances of autonomic nervous system activity and diminished response to stress in patients with celiac disease 2014.

10. Stress and Glucocorticoids - glucocorticoids, epinephrine (from adrenal gland), and norepinephrine (from sympathetic nerves) all decrease HRV

Stress reduces HRV by several hormones and neurotransmitters, including glucocorticoids, epinephrine, and norepinephrine.  Glucocorticoids (cortisol, synthetic steroids, etc.) decrease heart rate variability.   Glucocorticoids also increase systolic blood pressure.  The mediator of this is impaired hydrogen sulfide signaling.

References:

• Vascular Disease: Biology and Clinical Science — Session Title: Microcirculation and Cerebral/Coronary/Peripheral Circulation II 2014
• Glucocorticoids modulate baroreflex control of heart rate in conscious normotensive rats

High sodium diet - This decreases HRV

A high sodium diet decreases HRV,  The exact mechanism is not clear, but it probably involves blood pressure changes and changes in nitric oxide (NO) synthesis.

Reference:  Contribution of nitric oxide to arterial pressure and heart rate variability in rats submitted to high-sodium intake 2001

12. Gasotransmitters – Nitric oxide (NO), Hydrogen Sulfide (H2S), and Carbon Monoxide (CO)

Much has been learned about HRV from patients with congestive heart failure (CHF) and patients with coronary artery disease (CAD).  In CHF, there is a dramatic decrease in heart rate variability (HRV) and an increase in low frequency systolic blood pressure variability (SBPV).  CHF also results in increases in breath interval variability (i.e. snoring, sighing, etc.), and an increase in apneic episodes (like in obstructive sleep apnea).

These effects in CHF are thought to be mediated by the carotid body (CB), which senses oxygen levels.  Hypoxia causes an increase in CB signaling.  The molecular mediators of these changes in CHF include sympathetic dysregulation, renin-angiotensin dysregulation, increased atrial naturetic peptide (ANP), increased brain naturetic peptide (BNP), and increase in Hypothalamic-pituitary-adrenal axis hormones (cortisol, epinephrin, aldosterone).

However, the most recent molecular mediator discovery in CHF is the gasotransmitters – they play a major role in the pathogenesis of CHF.  These gasotransmiters and their dysregulation plays a major role in the decrease of HRV that occurs with these diseases.

Nitric Oxide is “good”, but NO dysregulation is “bad“- acute exogenous NO administration is ‘good”, but chronic exogenous NO administration results in “nitrate tolerance”.

Inhibition of endogenous NO production decreases HRV in some circumstances.  In others, inhibition of endogenous NO production increases HRV

Nitric oxide has “good” and “bad” effects that are very hard to understand.  Nitric oxide is thought to play an important role in the tonic control of carotid body (CB) chemosensitivity.  Both eNOS and nNOS are present in the CB.  NO inhibits CB activity by suppressing the CB chemoreceptors during normoxic periods.

Basal NO production, eNOS and nNOS expressio in the CB are depressed with CHF.  Thus there is no inhibition of the CB in CHF, due to a decrease in NO.

Part of the Paradox of NO is due to the differences between endogenous production of NO and exogenous production of NO.  In addition, part of the paradox is due to the differences between “acute” exogenous NO administration vs “chronic” exogenous NO administration.

• Acute NO administration reduces sympathetic activity and increases vagal outflow
• Chronic NO administration results in a loss of these effects and the development of “nitrite tolerance”

The easiest way to understand this is that “nitric oxide acts at distinct levels in the autonomic system to control cardiac rate, with opposing effects at different sites

In general, endothelial nitric oxide has mostly “good effects.”  For instance, neuronal nitric oxide inhibition decreases HRV 3-fold by inhibiting vagal stimulation,  whereas increased neuronal NO increases HRV.  For instance, endothelial Nitric oxide synthase 3 increases NO after an MI and this increases survival from an MI – this also increases HRV

• Nitric oxide increases in response to a high sodium diet – this reduces HRV.
• With CAD, nitric oxide levels from eNOS fall.

References

• Contribution of nitric oxide to arterial pressure and heart rate variability in rats submitted to high-sodium intake 2001
• Endothelial Nitric Oxide Synthase and Heart Rate 2002
• Effects of nitroglycerin treatment on baroreflex sensitivity and short-term heart rate variability in humans 2002
• Nitric oxide and autonomic control of heart rate: a question of specificity 2002
• Nitric oxide and cardiac parasympathetic control in human heart failure 2002
• Nitric oxide synthases in vagal neurons are crucial for the regulation of heart rate in awake dogs 2001
• Role of the Carotid Body in the Pathophysiology of Heart Failure 2013

Conclusions:  Administration of exogenous NO (acutely for a short period of time) may increase HRV, whereas chronic administration would not.  Administration of an exogenous NO donor (such as L-arginine) would also increase HRV, but probably not continuously on a permanent basis.

Hydrogen Sulfide is “good”, but dysregulation of H2S is “bad” – H2S is involved with carotid body chemoreceptor sensitivity and with breathing control

Inhibition of hydrogen sulfide restores normal breathing, but increased hydrogen sulfide increases survival after an MI.  Hydrogen sulfide also increases antioxidant gene expression via an Nrf2-mediated pathway.  H2S product by the enzyme CSE is not decreased in CHF – CSE inhibition with CHF reduces carotid body afferent responsiveness

There are a lot of puzzling paradoxes about H2S that I do not understand.

• Hydrogen sulfide signaling upregulates antioxidant gene expression by the transcription factor, Nrf2 – this is a “good thing”
• Hydrogen sulfide also increases survival after an MI via a nitric oxide synthase 3 dependent mechanism – this is a “good thing”
• However, H2S production by the carotid body chemoreceptors can also have “bad consequences”
• H2S is known to be an important signaling molecule in the carotid body (CB). H2S is synthesized by cystathionine gamma-lyase (CSE) in the CB
• Carbon monoxide (CO) inhibits CSE, thereby increasing HRV.
• Exogenous CSE inhibitors such as PAG also increase HRV
• Evidently in response to hypoxia, hydrogen sulfide signaling reduces HRV and causes abnormal breathing (apnea and increased breath intervals)
• Increased hydrogen sulfide signaling from the carotid body hypoxia sensor is part of the pathology of CHF.
• These effects in CHF are thought to be mediated by the carotid body (CD), which senses oxygen levels.
• In CHF, there is an increase in carotid body (CB) chemoreceptor outflow due to hypoxia.  The “signaler” is the gasotransmitter, hydrogen sulfide
• Thus CHF results in increased CB hydrogen sulfide synthesis, which reduces HRV. increases breath interval variability (BIR), and increases apneic episodes
• Inhibitors of hydrogen sulfide synthesis (by cystathionine gamma-lyase) like the drug di-propargylglycine (PAG) inhibit H2S synthesis by the CB.  In rodents, this reduced the apnea index by 90%, reduced breath interval variability by 40-60%,

Hydrogen sulfide signaling is a major reason why HRV decreases with aging.

References”

• Vascular Disease: Biology and Clinical Science — Session Title: Redox Signaling and Oxidative Stress  Abstract 17514: Hydrogen Sulfide Levels and NRF2 Activity are Attenuated in the Setting of Critical Limb Ischemia (CLI)
• Hydrogen sulfide improves survival after cardiac arrest and cardiopulmonary resuscitation via a nitric oxide synthase 3 dependent mechanism in mice 2010
• Chemical sympathectomy restores baroreceptor-heart rate reflex and heart rate variability in rats with chronic nitric oxide deficiency 2014
• Baroreceptor reflex function in rats submitted to chronic inhibition of nitric oxide synthesis 1994
• Nitric oxide synthesis blockade reduced the baroreflex sensitivity in trained rats 2009
• Inhibition of hydrogen sulfide restores normal breathing stability and improves autonomic control during experimental heart failure 2013
• Role of the Carotid Body in the Pathophysiology of Heart Failure 2014

Conclusion:  Since CSE is not down regulated in CHF, hydrogen sulfide probably plays a supportive role, rather than a causal role in CHF.  I am not sure if we want to inhibit it or activate hydrogen sulfide signaling.  In CHF, we probably want to inhibit it.  Under normal conditions, I am not sure. If we want to improve breathing, however, we will need to inhibit H2S production.

If we want to increase Nrf2 gene expression, however, we will want to activate H2S production or give exogenous H2S.  To improve HRV, we may need to use a H2S synthesis inhibitor like PAG to down regulating H2S signaling from the carotid body

Carbon Monoxide is “good”, but dysregulation or too much CO is “bad” - CO production in the CB is due to HO-2 gene expression and is reduced in CHF

Carbon monoxide (CO) is the 3rd and last gasotransmitter to be discovered.  CO is very important to health.  The first case of a human to have a gene mutation in HO-1 (heme oxygenase-1, the enzyme that synthesizes CO) died at age 6.  HO-1 gene knock-out models in mice and rats also die at a young age.  It came as a surprise to many that the body synthesizes CO – to be exact, it makes 16.4 micro moles per hour of CO – all from heme breakdown.  The total amount of CO produced per day is 12cc (see ref below)

In the pulmonary artery, CO relaxes the pulmonary vasculature under normoxic conditions.  In the carotid artery, CO regulates (decreases) the sensitivity of the carotid body to hypoxia.  However, CO plays a much greater systemic role in the body – CO is synthesized during heme breakdown by heme oxygenase 1 (HO-1), heme oxygenase 2 (HO-2), and heme oxygenase 3 (HO-3).

HO-1 is the inducible form of the enzyme and is primarily found in the spleen, but is also expressed in other tissues in lower amounts.  The spleen is the only organ where HO-1 overpowers HO-2.  HO-1 is NOT expressed in the carotid body (CB).  HO-2 is the constitutively activated form that is mostly expressed in the brain and testes – it is expressed in the carotid body (CB).  HO-3 is another constitutive form of the enzyme that has only been found in rat tissues so far.

With CHF, the expression of the constitutively activated HO-2 in the CB is decreased.  This means there is less CO produced by the CB with CHF.

References:

• Role of the Carotid Body in the Pathophysiology of Heart Failure 2013
• Carbon Monoxide: Endogenous Production, Physiological Functions, and Pharmacological Applications 2005
• Disturbed heart rate variability: a dose-dependent response to elevated nitric oxide and carbon monoxide in exhaled breath 2013
• Chapter 2 The Role of Carbon Monoxide as a Gasotransmitter in Cardiovascular and Metabolic Regulation

Conclusions:

• Exogenous administration of low dose CO (acutely only) may have a beneficial effect on HRV, since this inhibits CSE and thereby H2S production
• Exogenous administration of low dose CO (acutely) also induces the enzyme that makes endogenous CO, HO-1.
• For this reason, exogenous CO administration should have both a direct and indirectly beneficial effect on HRV, but no one has measured this yet

13. Chronic Hypoxia, Rapid ascent to altitude, High Altitude Acclimatization, and High Altitude Training in Athletes

Chronic hypoxia does not affect heart rate that much, but it has a dramatic negative effect on HRV.  This is why obstructive sleep apnea and COPD are so bad for health.

Rapid ascent to altitude decreased HRV in both the LF and HF power spectrums. In these individuals who ascended rapidly to altitude, 48% of them developed acute mountain sickness (AMS).  This decrease in HRV happened in individuals who developed AMS and in those who did not develop AMS (no difference).

Acclimatization has been reported to have conflicting results on HRV.  One study found that acclimatization at low altitudes (2,000-3,000 meters) and high altitude (5,000 meters) did not modify HRV in any statistically significant fashion.  Another study done in tourists at 2,700 meters and at 3,700 meters showed a decrease in HRV at both altitudes.  This decrease in HRV occurred in both the HF and LF power spectrums. At 3,700 meters, the sympathetic system was dominant over the parasympathetic system.  Another study done in high altitude mountaineers  found that at sea level, postural changes are mainly controlled by increases in sympathetic tone with sitting and standing,  whereas at high altitude, postural changes are mainly controlled by a decrease in parasympathetic activity and not an increase in sympathetic activity (5,000 meters).  However, high Altitude does not necessarily impair HRV, even though there is continuing exposure to low oxygen tension.

In trained Andean participants, completing a marathon at 4,220 meters elevation transiently increases sympathetic predominance of HRV after the marathon for less than 1 day.  Then their HRV returns to a healthy, baseline where parasympathetic tone dominates over sympathetic outflow.

References:

• Heart rate variability in rats with chronic hypoxic pulmonary hypertension 2006
• Autonomic adaptations in andean trained participants to a 4220-m altitude marathon 2005
• Effect of rapid ascent to high altitude on autonomic cardiovascular modulation 2008
• Effects of high altitude acclimatization on heart rate variability in resting humans 1995
• Alterations in autonomic nervous control of heart rate among tourists at 2700 and 3700 m above sea level 2001
• Autonomic nervous control of heart rate at altitude (5050 m) 1994

Conclusion:  I do not see any way how manipulating atmospheric pressure by reducing it would help HRV

14. Air pollution

Air pollution has been shown to decrease HRV (that’s bad!)  The best study on this was done at Harvard’s School of Public Health.  They found that pollution in general reduced HRV and Blood Pressure Variability (BPV), but that ozone (O3) and moving averages of particulate pollution in the air (PM2.5) were most associated with a decrease in HRV and BPV.  The decrease in standard deviation of normal-to-normal HRV (SDNN) and low frequency power spectrum (LF) were greatest in diabetics.

FReference:  Effects of air pollution on heart rate variability: the VA Normative Aging Study.  A new study dated January 15 2015

Section II:  Consumer HRV Hardware and Software 101

The marketplace is offering a wide variety of consumer hardware and software options for measuring HRV, particularly newer options where bluetooth measuring devices link to smartphones with apps that link to personal HRV measurements databases on the web.  We list some of these options here.

A.   HRV Hardware

Chest Strap systems:

Chest straps are the most well-developed devices that give “ECG-quality R-to-R interval measurements” and wirelessly communicate to your smart phone.  The trend is to go to Bluetooth straps, rather than the old ANT+ standard that the fitness HR straps used in the past.  A new Bluetooth standard used in some belts, Bluetooth® 4.1, the latest standard, is a low energy one that increases battery life dramatically.  This low energy Bluetooth allows you to “sync” your HR strap to a smart phone or tablet PC.    Here is a list of several Bluetooth straps and a few ANT+ straps that can be used for HRV.

Polar H7 Bluetooth strap – Bluetooth 4 low energy

hardware compatibility – compatible with either iPhones (IOS) or Android phones (Android 4.3 or greater)

• transmits the data in real time to a phone or to a Polar watch (like the M400)
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: 350 hours – need to unsnap device from belt after use to get this.
• cost – $50-70
• nice feature – can wirelessly connect to a lot of gym equipment.
• Both Vince and I have been using this sensor for our personal HRV measurements.

Wahoo TICKR heart rate monitor strap  – this is the entry level Wahoo strap – it has both ANT+ and Bluetooth wireless links

• phone compatibility – compatible with either IOS (iPhone 4S or later) or Android phones (Android 4.3 or later version)
• software compatibility – compatible with Apple Healthkit and over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: 350 hrs
• cost – $58-70
• nice feature – has a free 8 week Wahoo Fitness Burn and Burst Training app

Wahoo TICKR Run strap – this is Wahoo’s more advance version that has an accelerometer built into it – its wireless links also have both ANT+ and Bluetooth 4.0

• phone compatibility – compatible with either IOS (iPhone 4S or later) or Android phones (Android 4.3 or later version)
• software compatibility – compatible with Apple Healthkit and over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – yes
• battery life: 350 hrs
• cost – $68-80
• nice feature – has a Wahoo Running Smoothness Metrics program/feature

Head-to-head review of Polar H7 and Wahoo TICKR straps – BATTLE! – Wahoo Fitness TICKR RUN Heart Rate Monitor vs Polar H7 Heart Rate Sensor + Polar Stride Sensor

60beat BLUE strap – Bluetooth 4 low energy

• hardware compatibility – compatible with either iPhones (IOS) or Android phones (Android 4.3 or greater)
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• cost – $35-60
• nice feature – comes with several free apps called MapMyRun, RunMeter, Endomondo, LogYourRun, and RunKeeper

Cardiosport Bluetooth SMART strap – Bluetooth 4.0 low energy

• hardware compatibility – compatible with either iPhones (IOS)
• not compatible with Android phones
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• cost – $35-60
• nice feature – comes with several free apps called MapMyRun, RunMeter, Endomondo, LogYourRun, and RunKeeper
• Review of this strap: ithlete Cardiosport Bluetooth SMART Heart Rate Monitor Strap – In Depth Review

smartLABhBeat HRM strap – Bluetooth 4.0 low energy with Bluetooth Smart technology

• phone compatibility – compatible with iPhones or iPads
• Works with several APPs that support HRM on iOS, Android and Windows mobile devices
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: rechargeable battery with 26 hrs battery life
• cost – $39-40 (big price drop recently)
• nice feature – cheap!

Zephr HxM Smart HRM strap – Bluetooth 4.0 low energy (no rechargeable battery on this model)

• phone compatibility – compatible with Android phones (Android 4.3 or later version), and with Windows 8 phone devices
• not compatible with iPhones or iPads
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: rechargeable battery with 26 hrs battery life
• cost – $50-60
• nice feature – waterproof up to 1 meter

Zephr HxM BT strap – Bluetooth 4.0 low energy and a rechargeable battery

• phone compatibility – compatible with Android phones (Android 4.3 or later version) and Windows phones
• no compatible with iPhones or iPads
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: rechargeable battery with 26 hrs battery life
• cost – $60-80
• nice feature – charging cradle to recharge the battery (most straps do not have rechargeable batteries)
• review of this strap:  Keep the beat with the Zephyr hxm bluetooth heart rate monitor

4iii Innovations V100 viiiva strap – ANT+  and  Bluetooth Smart wireless linkages

• phone compatibility – compatible with iPhones
• not compatible with Android phones
• can send real time info to ANT+ watches or bicycle computers
• software compatibility – compatible with any iPhone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: 200 hrs
• cost – $80
• pros – has both ANT+ and Bluetooth technology
• cons – not compatible with Polar devices and doesn’t work with legacy Bluetooth standard
• online review of this strap: 4iiii’s Viiiiva ANT+ to Bluetooth Smart Bridge & Heart Rate Strap In-Depth Review

Pear Sports Mobile Training Intelligence strap – This one of those that can be used with either iPhone or Android phones

• Bluetooth 4.0 low energy with Bluetooth Smart technology
• phone compatibility – compatible with iPhones, iPods,  and iPads (4 or later model)
• compatible with Android phones
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: 400 hrs
• cost – $80-100
• optional foot pod
• Marketed as a comprehensive training system.  Real-time coaching while exercising is via heart rate feedback.
• Nice features – 1. Realtime Audio training coaching, 2. comes with PEAR “Stride” earphones with Earlock ear pieces

Online reviews:

• Product Review: PEAR Sports Smart Training System for Android and iOS
• Pear Training-Intelligence (for iPhone)
• Pear Sports Mobile Intelligence Training System for iPhone Review

Under Armour Armour39 strap – ANT+

• phone compatibility – compatible with iPhones, iPods,  and iPads (4 or later model)
• not compatible with Android phones
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: 400 hrs
• cost – $75-80
• nice feature – transmits your data in “real time” into MapMyFitness Apps, or into the Armour39 App, or to the Under Armour Watch

Jarv Premium Bluetooth 4.0 strap – Bluetooth 4.0 low energy with Bluetooth Smart technology

• phone compatibility – compatible with iPhones, iPods,  and iPads (4 or later model)
• Works with most Android 4.3 or later devices
• Software compatibility – not compatible with Apple Healthkit, but compatible with over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: 400 hrs
• cost – $35 (big price drop recently)
• nice feature – comes with several free software apps – MapMyRun, RunMeter, EndoMondo, LogYourRun, and RunKeeper
• Plus, it is cheap!

Xyzer HRM strap – Bluetooth 4.0 low energy with Bluetooth Smart technology – one of those that are both iPhone and Android compatible

• phone compatibility – compatible with iPhones, iPods,  and iPads (4 or later model)
• not compatible with Android phones
• software compatibility – not compatible with Apple Healthkit, but compatible with over 50 Smartphone apps
• onboard memory – no
• accelerometer/pedometer – no
• battery life: 400 hrs
• cost – $29-40 (big price drop recently, perhaps the cheapest belt sensor system)
• nice feature – compatible with either phone platform

Ear and Finger sensor systems

These systems are not as well-developed (none are wireless) or are not as “user friendly.”  However, these have been around longer and the software that comes with them seems to be better than that for the chest straps.

HeartMath earlobe sensors – 6400 Inner Balance, EmWave Pro Model 6030 – HRV sensor that mounts on earlobe.  A $299 stand-alone package with an earbud and hand-held monitor

The Journey to Wild Divine finger sensors (DeePak Chopra endorsed) – Biofeedback device that senses HRV from 3 finger sensors

Tink Wellness Sensor – this is a finger sensor that picks up HRV, respiratory rate, and pulse oximetry (blood oxygen saturation) – a combination not now available in chest or wrist sensor systems

Wrist-based HRV sensors

Including  the “Breathing Coach” (W/Me) and the two 24/7 Automatic activity/sleep trackers (Reign and Basis Peak)

There is a growing interest in developing a HRV device that automatically tracks both activity and sleep with no straps, no wires, and no need to have a phone constantly linked.    This currently means that the device must be wrist mounted and have an internal data storage feature that can be downloaded to a smart phone.    If such a device is going to be created in the future, the future is already here!

There are at least three devices that fit into this category.

The first one mentioned here is not yet for sale – it is a proposed product created by a Kickstart, crowd-funded company.  This device is not a 24/7 device, from what we know,  The second and third ones mentioned here are both “24/7 activity/sleep trackers” that are waterproof and stylish.  The Jaybird Reign is a wrist band, whereas the Basis Peak looks more like a watch.

The Jaybird Reign measures HRV, whereas the Basis Peak does not, however, there is hope that the new owner of Basis Peak (Intel) will add this feature.

W/Me – The Wrist band HRV monitor designed as a breathing coach

This is a Kickstarter company that has been funded with $140,000 of crowd sourcing money.        They don’t yet have the wrist sensor in the market, but their prototypes and marketing information look great!

• Technology:  a wristband style of wearable
• Claims to state, your agility score, and your autonomic system age

What it does for you:  this is a breathing coach program

• See the Kickstarter writeup Finally, a wearable device that can improve your life.  A  HRV sensor sends your measurements via Bluetooth to your phone.  The info is then downloaded into a spectrum analyzer, which then figures out your mental state and helps you as a breathing coach.
• compatibility:  iOS with Bluetooth 4.0 (iPhone 4S or higher)
• battery:  rechargeable via USB port.  7 day battery life
• cost: $55 for early access.  $139 once it comes out
• features: no chest strap, ear lob device, or finger device needed
• what it tells you:  It purports to calculate your mental state
• pros: very modern and stylish
• cons:  Based on limited existing capitaliation, company must be in a very early stage.  Unclear if the device  delivers on its marketing promises.  Unclear if it gives you any info on HRV power spectrum (i.e. numbers on LF, HF, etc.).  Unclear if and when the product will actually hit the marketplace.  “Tis many a slip twix the cup and the lip.”

Jaybird Reign – The Wrist band HRV monitor, Activity tracker, Sleep monitor, with unique feedback features for sleep decision making

• This device is from Austrailia and is already on the market that targets HRV for training recovery decisions (i.e. a rest coach) and for deciding how much sleep you need (i.e. a sleep coach)
• This is a stand-alone wrist band HRV monitor that has been designed primarily to help you determine when your body is fatigued and should rest – They call it the “Go-Score”, which is when you have fully recovered and are ready to do strenuous workouts again.  It also tells you how much sleep you need and whether your sleep was fitful.
• compatibility: iOS with Bluetooth 4.0 (iPhone 4S or higher) now
• Android software will be available in 2015 for all Android 4.3 or higher devices
• battery life:  Lithium batter that lasts 5 days
• water?  lifetime waterproof warranty
• cost: $180-200
• features: no chest strap, waterproof, stylish magnetic pin-locking technology for strap
• more info here.
• Pros:  The Jaybird Reign won the Outdoor Retailer 2014 “Gear of the Show” award
• It also won the “Best Digital Health and Fitness Product” award at CES 2014
• It also won SlashGear’s “Best Wearable Tech” award at CES2014
• Cons:, does not display HRV power spectrum, missing many practical features commonly available such as a display, alarms, “Can’t see trends over time. Doesn’t track heart rate. No calorie-logging system. Rudimentary mobile app. No desktop or Web app. No integration with third-party apps. No real display; LED indicators difficult to read. Difficult to fasten. Sensor pops out easily. So-so battery life.(ref)”
• online review: From PC magazine   Rated only fair because of negatives just listed.

Basis Peak Ultimate Fitness & Sleep Tracker – The Wristwatch HR monitor (does not measure HRV at this time), Activity tracker, and Sleep monitor that also has a skin temperature sensor and a sweat sensor- Bluetooth 4.0

• This is a second-generation wristband devices made by a company called Basis that was recently cquired by Intel Corp,
• It is a specialized “smartwatch” designed as a “24-7 automatic fitness and sleep tracker”
• The sensors it embodies include a 24-7 continuous heart rate monitor, an accelerometer, a galvanic skin resistance sensor, and skin and ambient temperature sensors
• Since the Basis company is now owned by Intel, they may possibly update the device with software or hardwired versions that offer HRV
• It can measure stress and various stages of sleep using its accelerometer – measures REM sleep, light sleep, and deep slee
• The constitutional stress biomarkers proposed by Vince in the Wearables Part 2 blog entry were based directly on data from his Basis Peak
• compatibility: iOS with Bluetooth 4.0 (iPhone 4S or higher) now
• Android software for all Android 4.3 or higher devices
• battery:  rechargeable battery with magnetic charging cradle, 4 day battery life (Vince gets only 3 days)
• screen: touch screen
• cost: $200
• water?:  waterproof to 5 atmospheres
• features: no chest strap, waterproof, stylish magnetic pin-locking technology for strap
• more info: https://www.mybasis.com
• Pros:  The Basis Peak watches have won several editor’s choice awards, including PC magazine
• A recent Basis Peak software upgrade allows the Peak to show smartwatch-type notifications originated from the companion smartphone
• It zeroes in on habits, rather than raw numbers.
• Excellent web interface
• Cons:  It does not measure distance traveled, stairs climbed, breathing, oxygen saturation or many other biomarkers measurable using wearables..  It also does not now measure heart rate variability
• Online review of the Basis Peak

A comparison on the Jay Bird Reign and the Basis Peak can be found here, including “The top 20 reasons for the Basis Peak.”

Vince’s comments;  See the Part 1 and Part 2 Wearables blog postings for my experience with the Basis Peak and what I believe are good constituitive stress biomarkers that can be derived from its measurements.  I find these very exciting and believe that they could be practically superior to HRV in practice by virtue of their stability, reliability and ease of measurement. It looks like the Jaybird Reign may be a good competitor to the Peak, and currently possibly superior in reporting in some respects.   As I point out in these blog entries my impression is that the Basis Peak offers an excellent hardware platform but that its software may not yet come close to providing the biomarker measurements that might be derived from its sensor outputs.  In particular, I strongly suspect its second-generation heart rate measurement sensors could be used to create HRV measurements if the Basis company saw this as an objective.  I mention also that a few competing products have just come on the market that are promoted as  generating measurements similar to those obtainable from the Basis Peak.  I am thinking particularly of the FitBit Charge HR, the Fitbit Surge and the Jawbone UP3.  The above comments regarding the Peak may also apply to these products.

Using the iPhone or Android phone camera to measure HRV

An iPhone camera can be used as a photoplethysmography (PPG) device.  This means it detects changes in blood volume during the cardiac cycle. Using the iPhone camera and the PPG technique, you can calculate HRV

Here is an article on this:  Heart rate variability using the phone’s camera.

The correlation between an iPhone camera based HRV monitor and a Polar H7 is very close (0.97).  Here is the diagram to show this:

Image may be NSFW.
Clik here to view.

• This approachbasically eliminates the need for any heart rate sensor hardware
• You just put your finger up to the iPhone camera and it senses your HR and calculates your HRV
• The measurements may not be that accurate, but clearly the simplest, easiest, and cheapest way to measure HRV
• Here are some places you can download an app to do this
  • Camera Heart Rate Variability – this one is $3.99
  • Stress Check Pro by Azumio – this is a $1.99 app
  • Instant Heart Rate by Azumio – this one is for free

Azumio Instant Heart Rate Pro is also available for use with selected Android phones on Google Play

More Sophisticated Sensors

These devices are much more sophisticated than the above devices.  They have more sophisticated capabilities, such as measuring respiration (breathing), skin temperature, and, reportedly, HRV.

Zephyr BioModule BH3 removable sensor system - This is a strapBluetooth 4.0 low energy strap that is much more sophisticated than the straps above, for Android devices

• The sensor is removable and can be mounted on a strap, a compression shirt with a pocket for the sensor, or a loose fit shirt
• The sensor has a respiration sensor, a 3-axis accelerometer, and HR/HRV sensor
• Sold as a performance monitoring device for serious athletes

Vital Connect’s Health Patch – This is a strapless Bluetooth 4.0 low energy patch

• It can measure HR, HRV, respiratory rate, skin temperature, body posture, steps, sleep staging
• However, they do not have software out for this yet, so you have to use Sweetbeat’s software, which is what I am using now with the Polar H7 chest strap.
• See the Sweetwater HRV site and the Vital Connect store site

Actiheart system by Camntech – This is a strapless (or strap) system that can be stuck on the skin with standard ECG electrode patches

• One electrode goes over the sternum and the other electrode is 10 cm away over the “V5″ or “V6″ position of an ECG (this gives better data)
• It can also be used with a strap and can record heart rate and activity for up to 21 days.
• Unfortunately,  it is not a Bluetooth 4.0 low energy system
• It can also record “Inter-beat-Interval”, which is used to calculate HRV.  Here is some more info on this:

HRV Software

Software for Bluetooth HRV devices -

BioForce – This is a highly rated HRV app for Blue tooth HR sensor devices – Android

Senseview – This is a free software that is designed for the Android 4.0 dual core processor

Sweetbeats – This one is $4.99.  It is the one I am using.  Currently Apple iOS only but an Android version has gone into alpha testing.  My rating: not that good

ithlete - This one is expensive.  Android and Apple iOS

Heart Rate Variability Logger – this one is free – Apple iOS

SelfLoops HRV – this one is free  – Apple iOS

HRV tracker – this one is also free – Apple iOS

Heart Rate + Cardiorespiratory Coherence – this one is $4.99. My rating: from what I can see, this may be  the best one out there

MyCalmBeat – this one is free

Buddha Mind – this one is $2.99 – Apple iOS. My Rating: I like this one, from what I have read.  There are 120,000 downloads, but 58% say there are problems with it

Expert by CardioMood – $1.69 – Android only

May be the best non-professional Android HRV app. Offers much information including spectral power breakdowns.  This is one of the HRV  apps that Vince has been using with his Polar H7 belt.   Vince likes the wealth of readouts, but has been unable to generate measurements with consistency and reliability.  Vince relates that on five  occasions he has made three measurements in a row while in the same sitting position without touching the belt or changing position or modifying anything else.  The way Vince puts it is that one of the back-to-back measurements shows a  50% or more high frequency HRV power measurement, stress of 60 and the profile of a healthy athlete; another of the measurements shows 75% of the HRV power is in the low and ultralow frequency spectrum and the stress index is over 300.  And the third measurement is so screwy that if Vince were to take it seriously, he would have his wife call an ambulance to rush him to an emergency room.  These variations seem to appear in most multi-reading session Vince has done with the software.  So something is seriously wrong in the software, the belt, or in what Vince has been doing.

Elite HRV – free – Android

Has a neat and simple dashboard and rated with 4 stars.  Produces only two numbers: heart rate and a HRV reading.  Company is in development,  Does not appear to offer a web data interface yet.  This is another HRV  app that Vince has been using.  Produces back-to-back HRV measurement that are not always consistent.   Three readings taken  in a row for Vince may show stress indices of 36, 76 and 58.

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Vince and I expect to report on our personal HRV measuring experiences and measurements in a subsequent blog entry.  There we expect to compare the ease and efficacy of measuring HRV as against Vince’s stress biomarkers MRHR (morning resting heart rate just before waking up) and ERHR-MRHR (drop in sleeping resting heart rate overnight).

By James P Watson, with contributions and editorial assistance by Vince Giuiliano

 INTRODUCTION AND OVERALL PRINCIPLES

This is the first of a pair of blog entries concerned with dementias – neurological diseases including Alzheimer’s Disease (AD) and its cousins.  This Part 1 write-up was inspired by a recent small, non-randomized clinical trial done by Dr. Dale Bredesen that showed true “Reversal of Cognitive Decline” in 9 out of 10 patients with documented cognitive decline (Bredesen, 2014).  Not all of these patients had AD, but all had cognitive decline.  Five had AD, two had SCI (subjective cognitive impairment), and two had MCI (mild cognitive impairment).  Although this study was too small to allow any statistical conclusions, it is the most positive report in a series of disappointing reports on the recent failures of Big Pharma’s monoclonal antibodies against amyloid-beta.  Dale Bredesen’s approach was a multifactorial one – utilizing 24 different approaches to halt or reverse cognitive decline.  We explore those 25 interventions here, focusing on the first 19.  They do not depend on drugs.  . The focus of this blog entry is “What can be done about dementias now?”

The forthcoming Part 2 blog entry will provides a detailed discussion of some of the key science related to AD and dementias.  This is the “What is science telling us about dementias?” part which gets quite complex.  We review major theories related to AD there including the Hardy Hypothesis related to amloid beta, the GSK3 theory and more detail on the neuroinflammation theory which we introduce in this Part 1 blog entry.  We expect to emphasize the emerging importance APP (Amloid Precursor Protein).  And we will describe some very recent research that appears to establish that a basic cause of AD is the proliferation in aging of vestigal DNA segments in our genomes (known as LINEs which are long interspersed nuclear elements and SINEs which are short interspersed nuclear elements) with encode over and over again for the production of APP and for the failure of its clearance.  This could well finally explain the role of beta amyloid in AD.

We have published a number of earlier blog entries relating to AD and dementias.  For example, you might want to review my August 2014 blog entry The Amyloid Beta face of Alzheimer’s Disease.

About dementias

Dementia only happens to a minority of the population with aging, but is becoming an ever increasing problem with the explosion in longevity occurring world-wide

Cognitive decline is the major “fear” people have of getting old.  Even individuals with the feared “ApoE4 polymorphism” are not “predestined” to develop Alzheimer’s Disease (AD).  The ApoE4 allele is only a “risk factor” for AD, not the cause of AD.

A common error is that most people view “dementia” and “Alzheimer’s disease” as synonyms, but this is incorrect.  Alzheimer’s disease is only responsible for 60% of cases of dementia in the US and even less of the cases in Japan.  In the US,  Vascular Dementia (VaD) is the second-most common cause of dementia (20%), whereas in Japan, the incidence of AD and VaD is almost the same.  In the US, the remaining 20% of dementia cases are due to several other neurodegenerative diseases such as Lewy Body Dementia (LBD), Parkinson’s disease with dementia (PDwithD), Frontotemporal dementia/ALS spectrum disorder (FTD/ALS), and mixed dementia (which is usually a mixture of AD and VaD).

A portrayal of the breakdown follows.

Image may be NSFW.
Clik here to view.

Image source

In the Middle East and China, VaD is more common than AD.  This was true in Japan two decades ago, but now the ratio of AD to VaD is 1:1.  Since AD and VaD are clearly the leading causes of dementia world-wide, we will focus mostly on these two types of dementia.  Also, the risk factors for AD and VaD overlap and there are cases of “mixed dementia” which include features of both diseases.  AD affects 5.4 million Americans and 30 million globally.  By 2050, these numbers will be 13 million (US) and 160 million (world-wide) (Ferri, 2005). Many experts now regard dementia from neurodegenerative diseases as the 3^rd leading cause of death after cardiovascular disease and cancer.  Despite millions of dollars being spent annually on research, the exact causes of these dementias are still unknown, but the number of clues to the causes is growing and we will explore some of the main ones in our Part 2 blog entry.

Neuroinflammation is the most universally accepted explanation for AD

What is clear is that the “universal sign” of all neurodegenerative disease is “neuroinflammation”, which under the microscope is manifested as “gliosis” and is seen with AD, VaD, PD, FTD/ALS, and the type of dementia seen after multiple concussions, which is now called “Chronic Traumatic Encephalopathy” (CTE).  Although they all have different “triggers” for each disease, they all have “neuroinflammation” and histologic signs of gliosis.  We return to neuroinflammation several times as a central theme here and in the Part 2 blog entries.

Another “universal feature” is that all of these disease have familial cases with as few as 5% being genetic (AD) and as many as 50% being genetic (FTD).  In these familial cases, there is most often a genetic mutation that is causal in nature (early onset disease) or a single nucleotide polymorphism (SNP) that is not causal in nature, but predisposes the patient to the disease.   With the exception of CTE (where the primary cause is multiple concussions) and PD (where pesticide exposure, family history of PD, and depression combine to produce an odds ratio OR = 12.0), most of the cases of neurodegenerative dementias remain largely sporadic with unknown specific causation.

Environmental risk factors for neurodegenerative diseases are discussed in the 2005 publication Neurodegenerative Diseases: An Overview of Environmental Risk Factors  and in publications in this list.

Despite millions of dollars being spent annually on research, the exact cause of these dementias are still unknown, but the number of clues to the cause is growing.  What is clear is that the “universal sign” of these neurodegenerative diseases is “neuroinflammation”, which under the microscope is manifested as  “gliosis” and is seen with  AD, VaD, PD, FTD/ALS, and the type of dementia seen after multiple concussions, which is now called “Chronic Traumatic Encephalopathy” (CTE).  Although they all have different “triggers” for each disease, they all have “neuroinflammation” and histologic signs of gliosis.  Another “universal feature” is that all of these disease have familial cases with as few as 5% being genetic (AD) and as many as 50% being genetic (FTD).  In these familial cases, there is most often a genetic mutation that is causal in nature (early onset disease) or a single nucleotide polymorphism (SNP) that is not causal in nature, but predisposes the patient to the disease.

With the exception of CTE (where the primary cause is multiple concussions) and PD (where pesticide exposure, family history of PD, and depression combine to produce an odds ratio OR = 12.0), most of the cases of neurodegenerative dementias remain largely sporadic with unknown specific causation.

Failure of Monotherapeutic Approaches to Neurodegeneration – It is time to consider multiple component therapies

The development of drugs to treat neurodegeneration has probably been the biggest failure of the pharmaceutical industry.  Although there are three FDA-approved drugs for AD, none of them produce anything other than a marginal, unsustained effect on symptoms.  Hundreds of clinical trials for AD have failed over the past two decades, most recently being the large Phase III trials of monoclonal antibodies that target amyloid-beta.  As of today, no drugs have been approved for Frontotemporal dementia, Vascular dementia, and Lewy body dementia.  Only one drug has been approved for Amyotrophic lateral sclerosis (ALS).  All of the clinical trials done for these diseases have largely been with monotherapeutic drug approaches.

We know from the field of cardiovascular disease, cancer, and HIV that single drug therapy for these diseases largely fail.  .  It is now clear that cancer is “incurable” with chemotherapy unless multiple drugs are used.  Combination therapies have become the standard for treating these conditions.  The requirement to combine drug therapies appears to pertain as well to diseases that we cannot “cure” but that are are yet treatable:  we can control the disease and prevent premature death from the disease.  This includes cardiovascular disease, HIV, and a few other glaring chronic diseases.  These diseases like dementias involve simultaneous upregulation or downregulation of hundreds or thousands of genes including protein-producing ones, and simultaneous activation or inhibition of a large multiplicity of pathway.  It is a very tall order to find a single molecule that can have the right effects on so very many different upregulated and downregulated molecules and pathways at the same time.  Yet, Big Pharma by tradition and because of patent law likes to look for single molecules that can be patented and that can make a big differences in a key step in a highly specific disease processes.  But most serious aging-related diseases and dementias don’t offer such an opportunity.

The Multi-factorial approach rather than “single target” approaches to Treating Alzheimer’s Disease

For the same reasons, it makes sense that a single drug made by “Big Pharma” could NOT solve the problems with these neurodegenerative diseases.  Here is a list of 25 different interventions that were combined into one effective program that “reversed” AD in 9 of 10 patients treated in a pilot study at UCLA and the Buck Institute.  None of these involve drugs.  I will include in black, the ones that were recommended by Dr. Dale Bredesen in what he calls the “MEND” program, which is an acronym that stands for “Metabolic Enhancement for NeuroDegeneration”.  You can check out his 2014 paper Reversal of cognitive decline: A novel therapeutic program.

SECTION I PRACTICAL INTERVENTIONS

1.  Eat a low glycemic, low inflammatory, low grain diet – Since sugar triggers insulin release and the insulin receptor triggers brain aging, this is easy to understand. For several complex reasons, certain proteins found only in grains (such as wheat germ, wheat gliadins) also triggers inflammation. The foods that have a high glycemic index or have lots of wheat in them include the following:

2.   Enhance autophagy – This can be done without fasting all day.  Research has shown that fasting for at least 12 hours per day (evening and night) is sufficient to activate autophagy.  Not eating for at least 3 hours before bedtime also activates autophagy.  Eating the evening meal earlier in the day also helps.  For those who do not want to fast for at least 12 hours, there may be little hope of “cleaning the cobwebs out of the brain”.  Studies have shown that eating too much or eating late at night completely shuts off autophagy.  This is probably the #1 reason why most people have so much “proteotoxicity” in the brain, the pancreas, and other organs.  You can review our blog entry Autophagy – the housekeeper in every cell that fights aging.

There are some natural compounds and some drugs that stimulate autophagy, however. They include the following:

• mTOR inhibitors – The mTOR pathway is “downstream” from the Insulin/IGF-1 pathway. The mTOR pathway completely “shuts off” autophagy and stimulates protein synthesis. This is the primary “danger” of eating too much meat or protein (i.e. stimulating the mTOR pathway).  Continually inhibiting the mTOR pathway is probably not a good idea either, since it is very important to synthesize proteins.  However, intermittent mTOR pathway inhibition has been shown to be a very effective way of stimulating “cellular housekeeping” in the brain. The best-known drug that inhibits the mTOR pathway ia rapamycin.  Low glucose levels and low amino acid levels in the blood also inhibit mTOR.  It is interesting that at least one big pharma company, Novartis,  is interested in marketing rapamycin as an anti-aging drug(ref).
• AMPK activators – The AMPK pathway is one of the major pathways that activates autophagy. AMPK is activated by both exercise and fasting. The AMPK pathway is a “cross-talk” pathway between mTOR and the Insulin/IGF-1 pathway.  Activating AMPK inhibits both of these “bad” pathways. (They are only bad in certain contexts of aging and still serve important functions in aging people.  We could not be alive without them.  In the Part 2 blog entry we will talk about how some times IGF is the good guy we don’t want to be without.)  Besides exercise and fasting, AMPK can be stimulated by three hormones, some drugs and many natural compounds. The most potent AMPK activator is muscle contraction (i.e. exercise). The three hormones that stimulate AMPK are thyroid hormone and two hormones secreted from fat: leptin and adiponectin. Next to this, the most potent chemical activators of AMPK are probably AICAR and ZMP. These are synthetic compounds that are the only true “exercise mimetics”.  ZMP is a derivative of AICAR.  AICAR has been shown to increase endurance in rodents by 44% without exercise.  This is amazing.  Combining AICAR with exercise makes the drug even more effective. Unfortunately, AICAR is very expensive ($350-450/gram).  Common drugs that activate AMPK include metformin and aspirin.  Natural compounds that activated AMPK include resveratrol, pterostilbene, curcumin, EGCG,  betulinic acid, Gynostemma Pentaphyllum, Trans-Tiliroside (from rose hips), and 3-phosphoglycerate.  See this list for articles in this blog that deal with autophagy or describe autophagy activators.
• Sirtuin activators – The 3^rd major family of pathways that activates autophagy is for the Sirtuin enzymes (SIRT1-7). Sirtuins are enzymes that remove acetyl groups from proteins. The most important ones it deacetylates for autophagy are 3 proteins that are crucial to the autophagy system of “cellular housekeeping”.  These 3 proteins are Atg5, Atg7, and Atg8. There are many practical reasons why activating Sirtuin-induced autophagy is critical to health.  For instance, SIRT1 activation protects cells in human degenerative discs from death by promoting autophagy.  This is why fasting has been shown to eliminate back pain. The most well-known SIRT1 activator is resveratrol, the active ingredient in red wine.  However, both red wine and white wine have been shown to activate Sirtuin enzymes.  NAD+, NMN, and NR all activate Sirtuin enzymes (all 7 of them), whereas resveratrol only activates SIRT1.   You can see our blog entry NAD+ an emerging framework for health and life extension — Part 1: The NAD World

3.   Reduce stress – psychological stress, depression, worrying, and being obsessive compulsive all increase the risk of Alzheimer’s disease. The most effective ways to reduce “cellular stress” are as follows:

• Yoga – yoga has been scientifically proven to reduce stress. The mechanism may be multifactorial, but studies suggest that activating stretch receptors in the muscles induces the SIRT3 gene.  The Sirtuin pathway is a major pathway activated by fasting, caloric restriction, red wine, NAD+, NMN, NR, and certain other natural compounds.
• Meditation – meditation has been scientifically prove to reduce stress. However, 3 minutes of prayer is NOT meditation. Meditation requires 30-60 minutes of time. The MEND program recommends 20 minutes of meditation twice a day (No one prays that long).
• Tai chi – this ancient Chinese form of exercise has been shown to reduce stress
• Exercise followed by rest – exercise alone does not reduce stress, but exercise followed by a good night’s rest is very effective at reducing stress
• Stretching exercises – These have a special beneficial effect on stress, especially back stretching exercises for back pain.

Self-monitoring of daily stress and exercise can be helpful for knowing what your stress levels are and how good a job you are doing at keeping stress at non-harmful levels.  A great many of the upstream conditions that can lead to dementias mentioned here (sedentary life style, improper diet, inadequate sleep, etc) are likely to induce constitutional stress which can be picked up by such monitoring.  A host of new wearable devices can keep track of exercise and its consequences.  See the blog entry Digital health – health and fitness wearables, apps and platforms – implications for assessing health and longevity interventions – Part 1.  Vince has identified two constitutional stress measurements in his blog entry that can be tracked starting with smartwatch heart rate and sleep measurements, MRHR (morning resting heart rate before awakening), and ERHR-MRHR (difference between evening resting heart rate and morning resting heart rate during sleep, a measure of overnight sleep-related constitutional stress recovery),.  These are described in the blog entry Digital health – health and fitness wearables, Part 2: looking for practical stress biomarkers.  Also, heart rate variability is another personally trackable constitutional measurement of stress,  See my recent blog entry on heart rate variability, Digital Health Part 3.

4.    Optimize sleep – At least 8 hours of sleep at night is very effective in preventing Alzheimer’s disease.

Daytime sleeping probably is not as effective, but is probably not harmful provided that a person is not too sedentary with daytime sleeping (i.e. short naps).  Adding 0.5 – 3 mg of melatonin and 500 mg of tryptophan is also very helpful in getting a good night’s sleep.  One of the biggest problems with getting a good night’s sleep is sleep apnea, which is actually very common as we get older.

Image may be NSFW.
Clik here to view.

“A Simplified schematic of the proposed interventions that may have potential to delay AD pathogenesis — The green arrows indicate pathways for improved circadian regulation and sleep quality, ultimately delaying AD pathogenesis. According to this model, chronobiotics (i.e., bright light therapy (BLT); melatonin; exercise; and food restriction) and good sleep hygiene could be used individually—but preferably in combination—to improve circadian regulation and sleep quality, decrease inflammation and Aβ deposition, and thereby delay AD pathogenesis.”  Image and legend source

5.   Exercise – The World Health Organization recommends 150 minutes of exercise per week, but the best scientific evidence suggests that this is NOT enough. The best scientific evidence suggests at least 450 minutes of exercise per week.  That is 60 minutes per day and an extra 20 minutes on one of those days.  If you want to skip Saturday, that means 75 minutes per day (1hr 15 minutes).  The exercise should include the following for preventing Alzheimer’s disease:

• Swimming, outdoor hiking, calisthenics, aerobic fitness classes, spinning classes, etc.
• 30-45 minutes of aerobic exercise where the heart rate is 60% of training heart rate.  This can be on a stationary bicycle, an elipical machine, a “hand bicycle”, a stair climber,
• 1 mile per day of walking outside (the speed is not important)
• Resistance exercise – this includes weight lifting, machines, stretch bands, push-ups, etc.
• Stretching – stretching activates stretch receptors which activates the SIRT3 gene, which is key for mitochondrial function and decreasing free radicals in the muscles (which cause pain
• Listening to relaxing music – classical music listening is a good way to relax.

Watching TV or looking at a computer screen and “surfing on the computer” probably does NOT work to reduce cellular stress.  Here are some of the blog entries we have published relating to exercise.

6.   Brain stimulation – The Mayo Clinic did a study in 487 patients where they participated in a computerized cognitive training program called “Brain Fitness Program” by Posit Science. This computer training required 1 hour of time per day, 5 days per week for 8 weeks (totaling 40 hours). This was called the IMPACT study.  This program increased their auditory processing speed by 131% and improved their memory an equivalent of approximately 10 years!  Here is some information on this inexpensive computer program:

• Posit Science Brain Fitness Program for One Person
• PositScience Brain Fitness Program Classic (PC Version)

Some of us think that we may keep our brains fit by constantly trying to figure out the mechanisms of aging.

7.  Keep your homocysteine low – High homocysteine levels seem to correlate with inflammation and also with deficiencies in folate cycle intermediates. The MEND program recommendation is to check your homocysteine levels and if it is > 7, then to take methyl-B12, methyltetrahydrofolate, pyridoxal-5-phosphate, and trimethylglycine (if necessary). The dosages are: Methyltetrahydrofolate – 0.8 mg/day and Pyridoxine-5-phosphate –  50 mg/day

8.   Keep your vitamin B12 high – Vitamin B12 is very important in memory and prevention of dementia. Vit B12 deficiency alone can cause dementia. It is easier to prevent than to reverse.  The MEND program recommends taking methyl-B12, not regular B12. They recommend basing the dose of methyl-B12 on serum levels of B12, which they recommend keeping above 500 with 1mg of methylB12/day.

9.  Keep your C-reactive protein low – CRP is a measure of inflammation. This correlates very well with inflammation in the brain (called neuroinflammation).  They recommend keeping the CRP levels below 1.0 and the Albumin/globulin ratio > 1.5.  There are no FDA-approved drugs that lower this which are safe to be used on a chronic basis.  However, there are several natural products that are effective in reducing C-reactive protein (CRP).  They include curcumin (400 mg/day), Fish oil (DHA & EPA), and an anti-inflammatory diet that is low in sugar and wheat products.  The MEND program recommends 700 mg of DHA twice a day (total 1400 mg) and 500 mg of EPA twice a day (total 1,000 mg).  Since most Fish oil capsules are only about 1/3^rd omega-3 fatty acids, that means you need to take about 7,000-8,000 mg (i.e. 7-8 one gram capsules) per day of Fish oil.

10.   Keep your fasting insulin low – Most people develop insulin resistance with aging. Unfortunately, this is rarely diagnosed until they have already suffered the consequences of insulin resistance, which include metabolic syndrome, hypertriglyceridemia, hypercholesterolemia, Alzheimer’s disease osteoarthritis, accelerated hearing loss, accelerated visual impairment (including presbyopia, cataracts, and age-related macular degeneration, aka AMD).  Once these things occur, then reducing your fasting insulin no longer is useful – the cells are already dead!  The MEND program recommends keeping your fasting insulin to < 7.0.  The best way to do this is to eat a low glycemic index diet, encourage ketogenesis by 12 hours of fasting per day, exercise, sleep, and in some cases the drug metformin.  We have found that the NAD precursor, NMN is effective in reducing fasting insulin levels.  Other supplements designed to enhance NAD+ may help as well.

11.   Hormone balancing – The MEND program recommends normalizing thyroid hormone levels (free T3, free T4, estrogen, testosterone, progesterone, pregnenolone, and cortisol). For most people, cortisol levels are way too high.  The best way to reduce cortisol is to reduce stress, improve sleep, and also possibly to supplement with NMN or NR.  The rest of the hormones decline with aging and often need replacement. Here are some ways to make this safe:

• Testosterone replacement therapy – this is risky in older men, due to the risks of accelerated coronary artery narrowing due to neointimal hyperplasia, as well as benign prostatic hypertrophy worsening or by making prostate cancer grow. For this reason, a thorough work-up for prostate cancer must be done before starting testosterone. In addition, testosterone dosing should be based on testosterone levels.
• Progesterone – This is primarily for women, but also helps men in low doses. Any progesterone replacement therapy should also be based on blood levels of progesterone.
• Pregnenolone – This helps both men and women for the brain.
• Estradiol (E2) – This should also be done based on blood levels of E2

12.   Healthy gut bacteria – Most people have very unhealthy gut bacteria due to the use of antibiotics, due to general anesthesia, and due to dietary factors such as a high sugar diet. As a result, the lactobacillus that are good for your health often die.  In addition, the fiber-fermenting bacteria are often absent, thereby eliminating the healthful effects of a high fiber diet.  Probiotics and prebiotics are often helpful in restoring healthy gut bacteria.  You can see Vince’s 2012 blog entry Gut microbiota, probiotics, prebiotics and synbiotics – keys to health and longevity.

13.   Reducing amyloid beta aggregates – One of the hallmarks of Alzheimer’s disease is the accumulation of misfolded, aggregates of a protein called amyloid beta. Fortunately, there are two natural compounds that if taken in large quantities can reduce amyloid-beta plaques in the brain.  They are Ashwagandha and curcumin.  Both of these are effective in reducing amyloid beta plaques.  The MEND program recommend doses of 500 mg for Ashwagandha and 400 mg for curcumin.  Because curcumin is so poorly absorbed, it is better to take a liposomal or nanoparticle form of the curcumin, like Bio-curcumin 95. Curcumin can be taking as a pill, but it may be absorbed much better in curry that has coconut oil, since the coconut oil creates an emultion and micelles which can be absorbed by the lymphatic system and thereby “bypass” the liver and the “first pass effect”.   Ashwagandha is much better absorbed and does not have as much of a problem. It can be taken as a pill, but also can be taken as a tea.   My friend Dr. Vince Giuliano has made a liposomal form of these two compounds together with two complementary anti-inflammatory herbal extracts which he believes get into the blood stream in concentrations that are 8-10 times higher than by pill form.  He has written about these and other phytosubstances a number of times, e.g.(ref) (ref) (ref) (ref) (ref).

14.   Cognitive enhancement – This category was probably added to the MEND program for supplements that could not be categorized elsewhere. They specifically recommend the natural product called Bacopa monniera and Magnesium. Bacopa monnieri is also called “water hyssop”, “herb of grace”, “Indian pennywort” and Withania somnifera.  Bacopa monniera has been shown to reduce amyloid plaque and prevent synaptic decline in mouse models of AD.  One possible mechanism by which Bacopa monnieri works is to enhance LDL receptor-related protein, which is the “amyloid exporter” in the brain.  There are many studies that show a benefit from Bacopa monniera In humans. A meta-analysis of 6 high quality clinical trials of Bacopa monniera showed that 9 out of 17 tests showed improved performance in the domain of “memory free recall”. In a study on Okadaic acid induced memory impaired rats, the effect of standardized extract of Bacopa monnieri and Melatonin on the Nrf2 pathway was investigated.  “OKA caused a significant memory deficit with oxidative stress, neuroinflammation, and neuronal loss which was concomitant with attenuated expression of Nrf2, HO1, and GCLC. Treatment with BM and Melatonin significantly improved memory dysfunction in OKA rats as shown by decreased latency time and path length. The treatments also restored Nrf2, HO1, and GCLC expressions and decreased oxidative stress, neuroinflammation, and neuronal loss. Thus strengthening the endogenous defense through Nrf2 modulation plays a key role in the protective effect of BM and Melatonin in OKA induced memory impairment in rats.” There is a special form of magnesium which is much better incorporated into the cell called Magnesium-L-threonate, aka MgT.  Both can be taken as a capsule.  The dose Bacopa monniera they recommend is 250 mg/day. However, most of the clinical trials recommend dosages of 300-450 mg/day.

15.  Vitamin D3 –Vitamin D3 seems to be quite different than the other vitamins for a variety of reasons. The most important difference is that Vitamin D levels should be checked and individuals need to adjust their dose based on their serum vitamin D3 levels. To prevent AD, the levels of Vitamin D3 need to be > 50 nmol/L.  The strongest evidence for this comes from two recent studies from 2014.  One was a 5 year study in 1,658 elderly patients who started the study with no dementia. During the 5 years, 171 of the 1,658 developed dementia (10% risk over 5 years).  This study looked at “all cause dementia”, of which 90% is Alzheimer’s dementia (AD) and Vascular dementia (VD).  The risk of developing dementia when serum Vitamin D3 levels were > 50 nmol/L was very low.  However, those with Vit D3  levels between 25 and 50 nmol/L had a 1.53 fold higher risk of developing dementia of any type.  Those with levels below 25 nmol/L had a 2.25 nmol higher risk of developing dementia of any type.  The 2^nd study reported in 2014 was from Denmark and followed 10,186 individuals in the Danish population for 30 years.  They looked at the risk of specific kinds of dementia and the relationship to Vitamin D3.  For Alzheimer’s disease (AD), the risk of AD type dementia was 1.25-1.29 fold higher in those with serum Vit D3 levels below 25 nmol/L.  For Vascular Dementia (VD), the risk of VD type dementia was 1.22 fold higher in those with serum Vit D3 levels below 25 nmol/L.  In conclusion, low Vitamin D3 levels is one of the largest risk factors for dementia and the easiest to prevent.  Most people do not get their Vitamin D3 levels checked.  Do you know what yours is?

16.   Increasing Nerve Growth Factor (NGF) – Hericium erinaceus and ALCAR — Although there are many growth factors that make nerve cells grow, the most important one is probably Nerve Growth Factor (NGF).  NGF is a growth factor made and secreted by astrocytes in the brain and spinal cord.  NGF enhances neuronal stem cell regeneration of the brain.  Exercise is a potent stimulator of NGF secretion. There are several natural compounds that stimulate nerve growth factor secretion.  They include extracts from the mushroom, Hericium erinaceus. Although there are other edible mushrooms that are good for you, of the 4 edible mushrooms that were studied for their effect on NGF secretion, only Hericium erinaceus induced the secretion of NGF from human astrocytes in the Hippocampus of the brain.  Another compound that stimulates the secretion of NGF is Acetyl-L-carnitine, aka ALCAR.  Acetyl-L-carnitine also helps with neuropathic pain.   In rodent models of Alzheimer’s disease, 150 mg/kg/day of ALCAR induced NGF secretion and increased choline acetyltransferase activity, which increasea acetylcholine levels in the hippocampus.

17.   Provide the substrates for synaptic formation – uridine, choline, citocolin, DHA, EPA, and herring roe — The ability to form synaptic connections between neurons is a key part of forming memory. Several key molecules are needed to create these synapses and dendritic spines that are not made by the human body (e.g. DHA) or are made in inadequate amounts (e.g. citicoline).   The omega-3 fatty acid called docosahexaenoic acid (DHA) is probably the “rate-limiting substrate” in the formation of presynaptic and postsynaptic proteins.  DHA alone will increase the formation of synapses and increase cognitive performance in humans and experimental animals, but the addition of two other circulating precursors for phosphatidylcholine also enhance memory formation.  These two other precursors are uridine (which gives rise to brain UTP and CTP) and choline (which gives rise to phosphocholine).   Phosphatidylcholine (PC) is the major phosphatide found in human neuronal connections. The other two major synaptic ingredients are uridine and DHA.  Studies have shown that the aministration of choline, uridine, and DHA together have a greater effect than the sum of the individual effects (i.e. they have a synergistic effect on generating synapses and dendritic spines). DHA alone increased the synthesis of hippocampal phospholipids by 8-75%, with the greatest percentage being in the synthesis if PC (phosphatidylcholine).  There are still controversies as to how much DHA a person should take per day.

The MEND program recommends 320 mg of DHA/day, but other experts recommend as much as 2,000 mg/day of DHA.  Herring roe, the eggs from the Herring forage fish, is another good source of n-3 polyunsaturated fatty acids that have a high phospholipid content.  MOPL 30 is a supplement product made by Artic Nutrition that includes a lot of phospholipids and a 3:1 ratio of DHA:EPA.  The MOPL 30 proprietary supplement not only increased neuronal generation, it also decreased plasma triacylglycerol and non-esterified fatty acids as well as increased HDL-cholesterol.  Although fasting glucose did not change, the glucose measurement on OGTT decreased at 10 minutes and 120 minutes into the test.   Instead of taking herring roe, uridine, or choline, the MEND program recommends citocoline (aka CDP-Choline) an intermediate compound in the generation of phosphatidylcholine from choline (i.e. already half made).  It is marketed under many names worldwide, including Ceraxon, Cognizin, NeurAxon, Somazina, Synapsine, etc. Studies have shown that citocoline increases dopamine receptor densities, prevents memory impairment, improve focus and mental energy.  Citocoline may also help treat attention deficit disorder (ADD).  The MEND program recommends a dose of 500 mg of Citocoline twice a day, 320 mg of DHA per day, and 180 mg of EPA per day.

18.   Optimize antioxidants – mixed tocopherols, tocotrienols, Selium, blueberries, NAC, Vit C, a-lipoic acid.  Although the free radical theory of aging has largely been proven to be incorrect as the “cause of aging”, there is no question that the “effect of aging” includes free radical damage to proteins, lipids, and nucleic acids that make up a cell.  To try to mitigate these “downstream effects” of aging, many believe that the judicious use of antioxidants still plays a useful role in treating neurodegeneration.  In this blog we have questioned that viewpoint and have pointed out that “antioxidants” like those mentioned often have powerful epigenetic impacts that better explain their actions(ref)(ref).

19.  Optimize Zn:fCu ratio – Alzheimer’s disease may be caused (in part) by copper toxicity — The fact that Alzheimer’s disease was rare prior to 1900, yet now being very common has led many experts to look for environmental “causes” of AD. One of the leading “suspects” in a long list of environmental risks for AD is inorganic copper, which comes from drinking water and supplement pills. There is clear evidence from human subjects that serum free copper is elevated with AD and that the level of free copper in the serum correlates with cognition and predicts cognition loss.  Animal studies have replicated these findings and have shown that as little as 0.12 ppm of coper in distilled drinking water in cholesterol-fed rabbits greatly enhanced the formation of AD.

A 2^nd feature of AD is that those affected also have Zinc deficiency.  A small clinical trial published in 2014 showed that in patients over the age of 70, Zinc supplementation protected against cognitive loss and also reduced serum free copper levels in AD patients.  For these reasons, it is unclear if the efficacy of Zinc therapy is on restoring normal Zn levels or if it is due to reducing Cu levels.

The following Table lists the remaining interventions in Dale Bredesen’s list.  These are fairly clear and we will not expand on them here.

Neuroinflammation “causes” all of the neurodegeneratove diseases

Although we will save most of our discussion on the science of AD to the coming Part 2 blog entry in this series, we comment here a bit more on the the science behind most of the above interventions – their neuroinflammatory nature.

In all neurodegeneratiave diseases (both familial and sporadic cases), there is evidence of a chronic, low grade brain inflammation that does not go away.  Histologically, this is called “gliosis”, a term that describes what is seen under the microscope. As mentioned above, microglial cells are increased in number and they appear “angry” (i.e. they are activated) likely due to the presence of Aβ_1-42.  It is likely that these microglial cells are secreting pro-inflammatory factors which are causing the inflammation, although the picture is actually much more complex.  Vince has written about this in 2011 and before in the blog entries Key roles of glia and microglia in age-related neurodegenerative diseases, New views of Alzheimer’s disease and new approaches to treating it, and Alzheimer’s Disease Update. We surface some additional insights here and in Part 2..

This illustration portrays some of the inflammatory processes that go on when microglia and astrocytes are activated:

Image may be NSFW.
Clik here to view.

Image and legend  source The 2014 publication Inflammasomes in neuroinflammation and changes in brain function: a focused review  “Cytokines hypothesis of neuroinflammation: Implications in comorbidity of systemic illnesses with psychiatric disorders. Pro-inflammatory cytokines can migrate between systemic circulation and brain in both directions which could explain the comorbidity of systemic illnesses with psychiatric disorders. There are three pathways for the transport of pro-inflammatory cytokines from systemic circulation to brain as described by Capuron and Miller (2011): Cellular, Humoral, and Neural. Moreover, PAMPs and DAMPs from trauma, infection, and metabolic waste can prime glial cells to express pro-inflammatory cytokines TNF-α, IL-1β, and IL-6. When expressed, these cytokines activates granulocytes, monocytes/macrophages, Natural Killer, and T cells and together contribute to the pathophysiology of neuroinflammation. Chronic neuroinflammation could result in neurodegeneration and associated psychiatric disorders. These pro-inflammatory cytokines also stimulate production and expression of anti-inflammatory cytokine by glial cells that function as negative feedback to reduce the expression of pro-inflammatory cytokines, subsiding the neuroinflammation. MCP-1, Monocyte chemoattractant protein-1; CP, Choroid plexus; CVO, Circumventricular organ.”

The chronic inflammation viewpoint of Alzheimer’s disease is related to but somewhat different than the Beta Amloid viewpoint, the viewpoint covered in my recent blog entry The Amyloid Beta face of Alzheimer’s Disease.

The situation is described in a 2014 publication by Landry and Liu-Ambrose: “An alternative to the classic amyloid centric view of AD suggests that late-onset AD results from age-related alterations in innate immunity and chronic systemic inflammation (for review see Krstic and Knuesel, 2013).

So, a basic strategy for preventing or delaying the onset of neurodegenerative diseases is to mount a multifront war on systematic inflammation.  The 25 Bredesen interventions described above are initiatives in that war.

By James P Watson with comments and editorial assistance by Vince Giuliano

Introduction by Vince Giuliano:

This is the third blog entry in the series NAD+ an emerging framework for health and life extension.  The first two entries were   Part 1: The NAD World, and Part 2: Deeper into the NAD World, hopeful interventions  We invite our readers to review these for background. 

Ever since David Sinclair’s seminal 2013 publication relating insufficiency of NAD+ to mitochondrial dysfunctionality(ref) , and and for some time before that, many research scientists have thought that insufficient nuclear levels of NAD+ are a major cause of diseases and aging processes including mitochondrial dysfunctionality,  associated deleterious metabolic consequences, and insufficient DNA repair.  Based on much research, this point seems to be solid and incontrovertible.  Having higher levels of NAD+ would doubtlesly be a good thing, especially for older people whose levels decline with age.  The major though not the only avenues of benefit are realized via sirtuins, particularly SIRT1. As is well-recognized and pointed out in many of out earlier blog entries, the sirtuins are essential for multiple key biological processes such as DNA repair and healthy mitochondrial metabolism.  Further, with aging and disease they tend to be in declining supply.  When there is not enough SIRT1. serious consequences can ensue .

In the growing excitement about NAD science during last two years, some researchers and entrepreneurs have further thought that human NAD+ levels can likely be enhanced by ongoing supplementation with NAD precursors such as nicotinamide riboside (NR) or  with  nicotinamide mononucleotide (NMN), The thinking has been that such supplementation might make a significant difference in human health and longevity.  However direct evidence  that NAD levels can be non-transiently enhanced in humans, either intracellular or extra-cellular, is thin to nonexistent.  No direct research shows this.  Likewise, evidence that human health or longevity benefits will result from continuous NAD precursor supplementats  is equally thin,  Enhancement of SIRT1 levels in mice via NMN supplementation and associated health benefits has been observed in only short term trials with mice, ones that lasted only 7 or 10 days (ref)(ref). There appears to be no clear evidence however, either human or animal, that continuing to take a NAD precursor supplement over a long term can lead to higher continuing levels of NAD+ or the many health benefits hypothesized to ensue.

This has led Jim Watson to investigate just what determines human NAD levels and what determines SIRT1 levels.  He found at least 50 such factors  In this current blog entry reports on what he believes are the most important 30 of these. 

This is another Chapter in the extended NAD story, but not the final one.  We expect soon to publish a Part 4 blog entry in this NAD World series.  That blog entry will look with more detail into several additional areas including NAD/Sirtuins and inflammation, and NAD and the much-discussed Warburg effect.  Despite a now-popular perception that scarcity of NAD+ is the main cause of the Warburg effect, Jim Watson points out that the effect  has multiple other causes including three long non-coding RNAs that promote it happening as a key effect in aging and cancer processes. Related to these causes,Jim discusses some possible intervention strategies that go beyond those normally discussed in the longevity literature.

1.  Subcellular compartment levels of NAD regulate aging 

The content of NAD in the nucleus as well as total cellular NAD levels declines with aging.

A.  Animal studies that showed how total cellular NAD decline with aging – DNA damage seems to be the most significant biomarker that correlates with the change in cellular NAD levels

Several papers in the 1990s showed that PARP activity increased with aging in both animal and human cells, but the evidence that NAD declined with aging was not discovered until the past 10 years.  In fact, a 1983 paper by Chapman, Zaun, and Gracy suggested the opposite effect – that NAD levels increased with aging.   The first paper to show that NAD depletion occurred was actually a heart failure study published in 2005.  Pillai and colleagues showed that PARP1 activation resulted in NAD depletion, reduced SIRT2 activity, and myocyte cell death. The next report was in 2008 by Parihar and colleagues, and was a study of rat hippocampal neurons.  In this in vitro study, they showed that a 50% decline in NAD(H) levels occurred in the aged neurons. (see reference below).  The 3rd report was a study in Wistar Rats, which showed a 4-7 fold decline in NAD+ levels with aging.  ThIs paper by Braidy and colleagues at the U of New South Wales in Sidney, Australia was the first study that specifically was designed to look at declining levels of NAD with aging. (2011).  What was remarkable in this study was that the DNA damage biomarker, pH2AX, seemed to correlate with the decline in NAD levels, even more than other biomarkers for oxidative stress.

B,  Human Studies showing that total cellular NAD levels decline with aging - DNA damage seems to be the most significant biomarker that correlates with the change in cellular NAD levels

Until recently, it was not known if human cells also underwent a decline in NAD content with aging.  The first paper to show this effect in humans was also from Sidney, Australia.  In 2012, Massudi and colleagues showed a precipitous drop in NAD levels within human skin cells, harvested from areas of skin with no sun exposure from infancy to old age.   Although measures of oxidative stress changed with aging (TBARS, MDA, F2-isoprostanes, etc.), the most significant change that correlated with aging was the DNA damage marker, pH2AX, which showed a significant increase with age in both males and females (p < 0.003). This suggests that the most important feature of cellular aging is DNA damage, not lipid oxidation or oxidative stress, per se.   Along with this increase in DNA damage was a dramatic increase in PARP activity (10-fold) in males.  PARP enzymes are NAD-consuming enzymes that sense and help repair single stranded DNA breaks via the base-excision repair (BER) pathway.  Paradoxically, the increase in PARP activity  was not statistically significant in the female cohort.  Even more surprising was the fact that SIRT1 activity did now show a statistically significant change with aging in either males or females, however.  Again, this is puzzling, since NAD levels declined so much.

References: 

• ·      Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats
• ·      Age-associated changes in oxidative stress and NAD+ metabolism in human tissue

C,  Studies that showed how nuclear levels of NAD decline with aging – Studies of Wallerian degeneration in the brain and mitochondrial biogenesis in muscles linked NAD deficiency in the nuclear subcellular compartment to the occurrence of Wallerian degeneration, and the “mitochondrial failure” seen with aging. 

Long ago, there was circumstantial evidence that NAD levels may be regulated on a subcellular compartmental level, the significance of this was unknown, since no one had measured NAD levels in various sub cellular compartments.  For instance, it was well-known that three isoforms of NMNAT existed [NMNAT-1 in nuclear compartment, NMNAT-2   in the Golgi complex, and NMNAT-3 in the mitochondria], the significance of this was not understood.  NMNAT is a key enzyme since it is the only enzyme shared by both pathways for NAD synthesis, the NAD biosynthesis de novo pathway  and the NAD Salvage cycle pathway.

One clue to this significance was the discovery of a mutant mouse model of  Wallerian degeneration called the “Wlds mouse“,  which was protected from developing Wallerian degeneration following axonal injury.  It was discovered that the Wlds mouse  had an 3 extra copies of a 85 kb tandem triplication on the end of chromosome 4 that contained two protein-coding genes for UCH-L1 (UPS gene) and NMNAT-1 (NAD synthesis  gene for the nuclear isoform of NMNAT).  These extra copies of the NMNAT-1 gene in the Wlds mouse resulted in a 3-fold higher NMNAT activity in the brain, but whole brain NAD+ levels were not increased.  This Wlds mouse study was published by Wang and He from Boston in 2009, but they did not show that nuclear NAD levels were actually reduced with aging or Wallerian degeneration.

More recently, in a December 2013 paper Gomes and colleagues from Sinclair’s lab in Boston showed that in mice, declining NAD levels in the nucleus of  muscle cells induced a pseudohypoxic state, disrupting nuclear-to-mitochondrial communication with aging.  In 3 separate experiments, he “knocked down” NMNAT-1, NMNAT-2   and NMNAT-3.  Only NMNAT-1 knock-down mimicked aging and the mitochondrial dysfunction seen with aging.  Although not published, Sinclair has measured NAD levels in the nuclear subcellular compartment and has showed that nuclear NAD levels decline with aging in mice.  In his key 2013 paper, he did publish evidence that IP supplementation with 400 mg/kg of NMN for one week reversed this “pseudohypoxic state” by promoting Tfam-dependent transcription of mitochondrially-encoded OXPHOS genes.

In summary, the indirect evidence about nuclear NAD levels from Wang and He’s work with the Wlds mice with 3 extra copies of the nuclear-specific isoform of NMNAT (NMNAT-1) as well as the more direct work on NMNAT-1 knockdown by Gomes and colleagues in Sinclair’s laboratory have provided tantalizing clues to a major cause of a “Universal phenotype of Aging:”   that of the decline in mitochondrial-encoded OXOPHOS genes, which includes increased ROS generation, Warbug-type metabolism, oxidative stress, NAD depletion, and eventually   ATP depletion in cells.  By turning on the Tfam gene via a SIRT1-mediated mechanism, induced by intraperitoneal high-dose NMN supplementation, Gomes and colleagues   increased expression of mitochondrial DNA encoded genes, thereby increasing the expression of mitochondrially-encoded OXPHOS genes.

Caveat:  The above three paragraphs make a strong case that one major cause of aging is a decrease in the nuclear levels of NAD co-enzyme, required for so many nuclear proteins involving DNA repair, epigenetic gene regulation, and apoptosis.  Unfortunately, no one has yet pin-pointed the cause of this “nuclear NAD decline” or demonstrated  that it can be reversed for more than one week.  The rest of this blog entry goes over 29 possible reasons why nuclear levels of NAD decline with aging.  Until someone demonstrates that they can permanently restore nuclear NAD levels, the exact cause of this aging phenomena must still be considered speculative.  Koch’s postulates must be proven.

2.  NAD/NADH ratio regulates aging independently of NAD content

-The longevity gene, NQO1, regulates aging by altering the NAD/NADH ratio in cells.   NQO1 does this by oxidizing NADH to NAD.   Beta-lapachone increases NQO1 enzyme activity and quercetin increases Nrf2-mediated gene expression of NQO1. 

Not only is the NAD content in the nucleus important for delaying/preventing aging, the redox ratio of NAD/NADH is also very important for delaying/preventing aging independently of the total NAD found in the cell.  Many genetic studies in model organisms have searched for “longevity genes” that regulate lifespan.  One of the curious findings from these studies is the gene that codes for the protein “NADH-quinone oxidoreductase 1″, or NQO1.  NQO1 oxidizes NADH to NAD, thereby increasing the NAD/NADH ratio.   Interestingly, Lee and colleagues from Korea recently showed that feeding animals beta-lapachone (aka Beta-L), an exogenous NQO1 co-substrate, prevented the age-dependent decline of motor and cognitive function in aged mice.  Beta-lapchone is a compound originally obtained from the Lapcho tree and has been used for medical purposes for many years.  Beta-L fed mice did not alter their food intake or locomotor activity, but did increase their energy expenditure as measured by VO2max and by heat generation.  The Beta-L fed mice developed changes in gene expression that mimicked 30% caloric restricted diets.  Another molecular effect of beta-Lapachone is that it induces apoptosis in breast and prostate cancer cells.

Gene polymorphisms in the NQO1 gene are strong prognostic indicators for breast cancer.  For instance, the NQO1 2 genotype (P187S) predicts poor survival from breast cancer(ref).  The relative risk for breast cancer in this with the P187S genotype is 6.15, when compared to control groups.  The P187S genotype does not affect local recurrence, but affects survival.

Interestingly, benzene poisoning is associated with the mutation of the NQO1 gene at codon 187, which creates the 609C-T mutation of the NQO1 gene.  This results in complete loss of the enzymatic activity of NQO1 protein.  By this mechanism, benzene produces a NOA1 “loss-of-function” mutation and induces hematological malignancies. This appears to be a major mechanism for chemotherapy-induced secondary malignancies, which are called “therapy-related malignancies”.  Two diseases that benzene induces are “therapy-related  leukemia” and “therapy-related myelodysplastic syndrome“.  The NQO1 gene can also be transcriptionally unregulated by a polyphenol called quercetin.  Specifically, quercetin increases gene expression of the NQO1 gene via an Nrf2 transcription factor mediated pathway(ref)(ref)(ref).  Specfically, quercetin enhances the binding of Nrf2 to the NRF-ARE binding site on the NQO1 gene promoter.   Quercetin also increases Nrf2-mediated transcriptional activity by up regulation gut e expression of Nrf2 mRNA and Nrf2 protein.  Quercetin also reduces the level of Keap1 protein, the binding partner of Nrf2, which prevents Nrf2 nuclear translocation.  Quercetin reduces Keap-1in a post-translational mechanism, thereby reducing Nrf2 ubiquitination and proteasomal degradation.  Another unusual up regulator of NQO1 is the toxin, dioxin.

The NAD World Part 4 blog enry contains additional discussion related to NQ01 and how it is egulated, beta lapachone and other topics mentioned in this item

References:

• LongevityMap Gene – Gene details HGNC symbol NQO1 
• Nrf2 mediates cancer protection but not prolongevity induced by caloric restriction
• The Protein Level of PGC-1α, a Key Metabolic Regulator, Is Controlled by NADH-NQO1
• In Vivo Role of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) in the Regulation of Intracellular Redox State and Accumulation of Abdominal Adipose Tissue
• Pharmacological activation of NQO1 increases NAD⁺ levels and attenuates cisplatin-mediated acute kidney injury in mice
• Enhanced activation of NAD(P)H: quinone oxidoreductase 1 attenuates spontaneous hypertension by improvement of endothelial nitric oxide synthase coupling via tumor suppressor kinase liver kinase B1/adenosine 5′-monophosphate-activated protein kinase-mediated guanosine 5′-triphosphate cyclohydrolase 1 preservation
• β-Lapachone ameliorates murine cisplatin nephrotoxicity: NAD+, NQO1, and SIRT1 at the crossroads of metabolism, injury, and inflammation

3.  Clock/BMAL1

CLOCK and BMAL1 regulate the circadian expression of the SIRT1 gene.  Day/night cycles are thus the #1 factor that determines SIRT1 expression.SIRT1 regulates circadian gene expression by deacetylating PER2

With long lists like those in this blog entry, it is easy to throw up your hands and say “its too complicated!”  Well it isn’t!  If you want to skip the list and just ask “What is the most important regulator of SIRT1 expression,” I do not think anyone would argue with the statement that CLOCK/BMAL1 bind to the gene promoter for SIRT1 and regulate the diurnal change in SIRT1 gene expression. (see references).

For example, it has been well-documented that liver insulin sensitivity correlates with the two circadian transcription factors CLOCK and BMAL1.  BMAL1, CLOCK, and SIRT1 all must work together to “turn on” and “turn off” 15% of the genome in human cells every day.  Unless the expression of these three proteins is coordinated, hepatic insulin resistance develops.   Constant darkness “dysregulates” the coordinated timing of BMAL1 and SIRT1.  As a result, BMAL1 and SIRT1 expression decreases with constant darkness and hepatic insulin resistance is induced.  Interestingly resveratrol can dramatically reverse the “dysregulation” of SIRT1-dependent circadian genes by increasing SIRT1 activity.  SIRT1 regulates circadian gene expression by PER2 deacetylation.

References:

• CLOCK/BMAL1 regulates circadian change of mouse hepatic insulin sensitivity by SIRT1
• The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control
• SIRT1 regulates circadian clock gene expression through PER2 deacetylation
• Interaction of circadian clock proteins PER2 and CRY with BMAL1 and CLOCK

4.  Long non coding RNAs 

The long non-coding RNA transcribed from the anti-sense strand of the SIRT1 gene regulates SIRT1 gene expression via a “trans-regulatory” mechaism.   Long non-coding RNAs also appear to regulate cellular senescence, metabolism and many other cellular functions.  

Introduction/Background

Recently, there has been dramatic shifts in the thinking of geneticists about the role of “junk DNA” in human disease, aging, and normal development.  Specifically, over 56,000 unique RNA transcripts have been identified via next generation sequencing (also called “deep sequencing” or RNAseq).  Of these, over 9,000 long non-coding RNA “genes” have been identified that make many more than 9,000 different RNA transcripts.  Although there are many other types of non coding RNA besides long non-coding RNA, these RNA sequences that are > 200 base pairs in length have garnered the most attention since they seem to have such powerful regulatory functions over and above even epigenetic gene regulatory mechanisms.

Some recent papers have suggested that of the 6,000+ gene polymorphisms (SNPs) that have been linked to disease by GWAS studies, as many as 93% of these SNPs are not due to protein-coding regions of the human genome, but instead are in regulatory areas.  Another surprising finding was that at least 5,000 (of the more than 9,000 DNA sequences that encode for long non-coding RNA) are not evolutionarily conserved.  What does this mean?  It means that these long non-coding RNA may be the “youngest” forms of gene regulation and may account for the unique characteristics of homo sapiens, such as our ability to make tools, form language, develop written communication, music, art, religion, socialization, etc.  In short, long non-coding RNA may be why we are “human”, rather than looking and acting more like our nearest cousins, the chimpanzees and bonabos.

SIRT1 gene-specific lncRNA

It is not surprising then that a long non-coding RNA has been discovered that regulates SIRT1 gene expression.  (see reference below).  This long non-coding RNA is transcribed from the anti-sense strand of the SIRT1 gene.  This lncRNA is called “SIRT1 antisense long non coding RNA” or SIRT1AS lncRNA for short.  Wang and colleagues from Shanghai, China discovered this and published their finding in April, 2014.  They were able to show that SIRT1 AS lncRNA expression results in an increase in expression of the SIRT1 gene.  They isolated SIRT1 AS lncRNA from differentiating myotubes from developing skeletal muscle, as well as from the spleen.  It appears that the SIRT1 AS lncRNA expression is both temporal and tissue-specific.  For instance, higher levels of SIRT1 AS lncRNA were expressed in undifferentiated, younger tissues,   Likewise, SIRT1 AS lncRNA was expressed in a tissue-specific fashion.  In the spleen, SIRT1 AS lncRNA levels were higher than in skeletal muscle.

SIRT1 AS lncRNA may counteract microRNA that inhibit SIRT1

miRNA that down regulate SIRT1 gene expression appear to be blocked or their effect attenuated by the SIRT1 AS lncRNA.  Thus, the function of SIRT1 AS lncRNA may be to counteract the effects of miR-34a, miR-217, miR-181a, and the other microRNA that increase SIRT1 mRNA degradation and thereby reduce SIRT1 protein expression.

Long non coding RNAs and cellular senescence

Gene-specific lncRNAs can regulated only one specific gene, like the SIRT1 AS lncRNA described above.  However, many more lncRNAs regulate many genes rather than one specific gene.  Gene-specific lncRNAs typically regulate nearby genes, which is a method called “regulation in-cis”.   Since SIRT1 plays a role in cellular senescence, it is likely that the “Anti-sense” senescence-associated lncRNAs play an role in regulating genes that SIRT1also regulates.  A list of the top 12 up-regulated Antisense lncRNAs with cellular senescence along with a list of the top 15 Anti-sense  lncRNAs that are down regulated with cellular senescence are listed below.

In addition to the Anti-sense lncRNAs described above, a number of lncRNAs have been found that originate in pseudogenes, genes that no longer code for proteins, but are still transcribed from their sense or anti-sense strand.   lncRNAs can regulate a single gene or regulate hundreds of genes as well. lncRNAs that regulate many genes typically do so via a method called “regulation in-trans”.  These lncRNAs are not located near by the genes that they regulate, but instead, are typically located long distances away, even on other chromosomes.  Many of these long non-coding RNAs are found in areas with no nearby protein-coding genes.  For this reason, they are called “Long intergenic non coding RNAs” or lincRNAs.

Several lincRNAs have been discovered that regulate cellular senescence via “trans regulatory” methods.  These lncRNAs appear to be “novel” and not within or associated with a particular protein-coding gene or a pseudogene.

Long non coding RNAs and metabolism

Since lncRNAs have been discovered that regulate cellular senescence, it is no surprise to find out that lncRNAs regulate metabolism as well.  There is strong evidence that insulin resistance may be mediated in part by lncRNAs.  This includes tissue-specifc lncRNAs and generalized lncRNAs.  For instance, the long non coding RNA called “H19″ may be involved in the intergenerational transmission of diabetes mellitus.  In a large next-generation RNA sequencing study of pancreatic RNA transcripts, over 1,000 lncRNAs were discovered in pancreatic islet beta cells.  Of these, 40% were long intergenic non coding RNAs (lincRNAs) and 55% were Anti-sense strand long non-coding RNAs (AS lncRNAs).  Interestingly, the non-coding RNA sequences found via this next generation sequencing study were more “tissue-specific” than the mRNA for protein-coding genes in the pancreatic tissue examined.  In other words, non-protein coding RNAs had a more “tissue specific signature” than the protein-coding mRNAs that were sequenced.  This is quite remarkable.

References:

• 2014 Identification, stability and expression of Sirt1 antisense long non-coding RNA
• 2013 Senescence-associated lncRNAs: senescence-associated long noncoding RNAs
•  2014 Regulation of metabolism by long, non-coding RNAs
• 2012 Transgenerational glucose intolerance with Igf2/H19 epigenetic alterations in mouse islet induced by intrauterine hyperglycemia

Antisence Long Non-coding RNAs that are Differentially  Regulated with the induction of Cellular Senescence

Image may be NSFW.
Clik here to view.

Summary:  It is likely that much of the regulation of protein-coding genes and the unique aspects of humans, human aging, and human disease may be due to the “invisible human genome”, which is the large number of non-coding RNAs.  This field is exploding, with new RNA sequences being discovered every day.  Already, an Antisense strand long noncoding RNA has been discovered at the SIRT1 gene, which increases SIRT1 gene expression by a yet unknown mechanism.  One possibility is that this lncRNA prevents or inhibits the functions of miRNAs which increase SIRT! mRNA degradation.   Other lncRNAs have been found that regulate cellular senescence, metabolism, etc. via “trans regulatory” or “cis regulatory” methods.

5.  Nicotinamide (Nam)

Nicotinamide is a direct inhibitor of both the SIRT enzymes and the PARP enzymes.

The accumulation of excess nicotinamide in cells is probably a major cause of aging.  Whereas we typically associate “NAD deficiency” with aging, “Nam excess” may have a similar effect.  To no one’s surprise, the levels of the two compounds are inversely related in aging.

Nam plays a role in both aging and in disease.   In hypertension and in aging individuals with normal blood pressure, Nam inhibits the methylation-mediated degradation of catecholamines.  Thus Nam excess plays a role in hypertension (see references below).

Nicotinamide also has an epigenetic effect. When SIRT1 is inhibited, cells age and cancer oncogenes are re-activated.  SIRT1 silences these genes  by histone deacetylation of H3K9 and H4K16 residues on the histones of these oncogenes.

A recent article showed that in rats, nicotinamide supplementation during pregnancy causes global DNA hypomethylation in rat fetuses.  Nicotinamide has detrimental effects in development, detrimental metabolic effects, and detrimental epigenetic effects when given to young rats.  Low dose nicotinamide increased weight gain in developing rats.  High dose nicotinamide did not, however.  The livers of nicotinamide-fed young rats had more DNA damage (8oxoG), impaired glucose tolerance, and increased insulin resistance.  Nicotinamide increased the levels of N-methylnicotinamide in the blood and decreased betaine levels in the blood.  This resulted in a global hypomethylation of DNA in the rat genome.  Nicotinamide also had “gene-specific effects” on CpG islands within the promoters of the following genes:

1. NNMT gene – this was down-regulated
2. DNMT genes – these were down-regulated
3. Homocysteine metabolism genes – these were down-regulated
4. Antioxidant genes and oxidative stress protection genes – these were down-regulated

Since niacin is converted into nicotinamide in human tissues, high dose niacin probably produces all of the above effects.  A recent paper called niacin and nicotinic acid “methyl consumers” and strongly suggested that high niacin/nicotinic acid intake is bad.

Excess nicotinamide has also been shown to increase plasma serotonin and histamine levels in humans, due to disrupting the metabolism of these neurotransmitters.  This is probably due to the fact that methyl donors and methylation enzymes are needed for serotonin/histamine metabolism.   Most importantly, nicotinamide is a direct inhibitor of the Sirtuin enzymes (SIRT1-7) and the PARP enzymes (all 17 of the PARPs).

The molecular mechanism by which Nam works is very straightforward. Nam acts as a direct inhibitor of the SIRT1 enzyme pocket where NAD binds.  Thus Nam is a “competitive inhibitor” of NAD and is “bad” when it comes to most cancers, aging, and most diseases.

On the other hand, inhibition of SIRT1 by Nicotinamide may be a “good thing” in the brain, where it may prevent NAD+ depletion and thereby protect neurons against excitotoxicity and neuronal cell death induced by PARP1.

As the cell consumes NAD (by SIRT1-7, PARP1, PARP2, Tankyrases, CD38, CD157, ARTs, and other enzymes), the NAD is consumed, leaving the by-product, Nam.   There are two primary ways  of “disposing of Nam”.  They are methylation/excretion or recycling of Nam into NMN (and subsequently NAD) via the “NAD salvage cycle”.  

Here are the problems with both of these methods of reducing Nam levels in the cell.  (see #2 and #3 below).

References:

• 2002 Inhibition of Silencing and Accelerated Aging by Nicotinamide, a Putative Negative Regulator of Yeast Sir2 and Human SIRT1
• 2006 Inhibition of SIRT1 Reactivates Silenced Cancer Genes without Loss of Promoter DNA Hypermethylation
• 2009 Nicotinamide Prevents NAD+ Depletion and Protects Neurons Against Excitotoxicity and Cerebral Ischemia: NAD+ Consumption by SIRT1 may Endanger Energetically Compromised Neurons
• Nicotinamide supplementation induces detrimental metabolic and epigenetic changes in developing rats
•  2013 Association of Nicotinamide-N-Methyltransferase Gene rs694539 Variant with Patients with Nonalcoholic Steatohepatitis
• 2013 Excess nicotinamide increases plasma serotonin and histamine levels
• 2013 Excessive nicotinic acid increases methyl consumption and hydrogen peroxide generation in rats
• 2014 Metabolism: Targeting a fat-accumulation gene

Conclusion:  It is now clear that high concentrations nicotinamide are harmful to health.  HIgh doses of dietary niacin probably produce the same effects, despite the many benefits of high dose niacin.  With aging, nicotinamide levels already go up.  Adding more nicotinamide is probably not going to “cure” aging.  Adding a methyl donor to eliminate nicotinamide (such as betaine) may be a good thing.

6.  NAMPT

-NAMPT is the rate-limiting step in the NAD Salvage Cycle, and is regulated in a circadian fashion.

The enzyme that converts Nam back into NMN in the “NAD salvage cycle” is Nicotinamide phosphoribosyl transferase, or NAMPT.  Unfortunately, NAMPT is regulated by circadian rhythms and is primarily up-regulated at night.  It is inhibited by not sleeping, however.  It is also down-regulated by eating and sedentary lifestyle.  Fasting and exercise dramatically up-regulate NAMPT.   I really do not see how NMN or NR will really change the gene expression of NAMPT.  If anything, since NMN is the “product” of the NAMPT enzyme, high levels of NMN may actually have a “feedback inhibition effect” on NAMPT, just like Nictoinamide has a “feedback inhibition effect” on SIRTs and PARPs.

Paradoxically, in a recent study in zebrafish, resveratrol actually DECREASES the expression of NAMPT.  This may be via a “feedback inhibition effect”, since SIRT1 “auto regulates” its own gene expession (see references).   Another interesting “twist” is that Angiotensin II receptor blockers (ATR type 1 blockers) actually increase NAMPT gene expression.  This may be the molecular mechanism behind the longevity effects of ATR1 blocker medications like Telmisartan.

References:

•  2009 Circadian Clock Feedback Cycle Through NAMPT-Mediated NAD+Biosynthesis
• 2008 Glucose Restriction Inhibits Skeletal Myoblast Differentiation by Activating SIRT1 through AMPK-Mediated Regulation of Nampt

• 2009 Circadian Control of the NAD+ Salvage Pathway by CLOCK-SIRT1
• 2012 Modulatory effect of resveratrol onSIRT1, SIRT3, SIRT4, PGC1α andNAMPT gene expression profiles in wild-type adult zebrafish liver
• 2009 Disruption of the Ang II type 1 receptor promotes longevity in mice
• Metabolism: Targeting a fat-accumulation gene

 7.  NNMT – Methylation of Nicotinamide – Nam)

Methylnicotinamide is a “mitohormetic compound” that regulates longevity

Of all of the unusual aspects of Sirtuins and NAD metabolism, N-methylnicotinamide is probably the hardest one to understand.  Recent evidence has shown that for the longevity effects of NAD metabolism to occur in nematodes (C. elegans), NAM must be methylated and then used by the gene, GAD-3, to produce low levels of hydrogen peroxide, thereby acting as a “mitohormetic compound”.  This low level of hydrogen peroxide induces mitochondrial biogenesis and is necessary for nematode lifespan extension.

Reference:  2013 Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide

This article came out last year and one of the co-authors is David Sinclair.

However, that is not the entire story.  There is much more to the story of the methylation of nicotinamide.  Most of this has to do with stopping the toxic effects of nicotinamide from occurring, due to the inhibitory effects of Nam on both the Sirtuin enzymes (SIRT1-7) and the PARPs (1-17).

Over 85% of transmethylation reactions occur in the liver, including the methylation of nicotinamide.  With the oral intake of nicotinamide, the liver could either methylate the nicotinamide or convert it to NAD.  From many studies, it is clear that the liver preferentially methylates nicotinamide, rather than converting it to NAD.   The reason why the liver methylates nicotinamide is that high concentrations of nicotinamide cause cell injury and cell death, since nicotinamide inhibits all 7 of the Sirtuins and also inhibits the PARP enzymes (PARP1,PARP2, etc).

For this reason, with oral intake of Nam, serum levels of N-methylnicotinamide increase.  Only 15% of nicotinamide methylation occurs outside of the liver.  Nicotinamide methylation by NNMT also requires a methyl donor, such as SAMe or trimethylglycine, which is also called betaine.  When nicotinamide is methylated, it is then excreted, thereby reducing Nam levels in the body and preventing SIRT/PARP inhibition.

GWAS studies of polymorphisms in the NNMT gene have revealed an amazing association with Non-alcoholic steatohepatitis (NASH).  The NNMT gene SNP, rs694539, is a SNP found in the regulatory portion of NNMT gene.  The “GG” genotype protects against NASH, with an OR of 0.58.  The “AA” genotype increases the risk of NASH with an OR of 7.3.   This suggests that methylation of nicotinamide is an important factor in preventing NASH (see references below).   This same polymorphism (rs694539) has been linked to bipolar disorder recently.  The association was “female gender-specific” and did not influence male bipolar risk.

A recent novel theory discussing how “too much NNMT” and “too little NNMT” may both play a role in aging and disease has been proposed. This theory suggests that excess dietary nicotinic acid consumption results in molecular/cellular  “methyl consumption” and plays a role in disease. This new theory has been published in Nature and is being taken seriously.  The main hypothesis is the increase in “methyl consuming compounds” in our diet contributes to metabolic syndrome and many other “man-made diseases”.  Another aspect of this new theory is that NNMT is a “fat accumulation gene”  (see references below), since the expression of NNMT is unregulated with increased dietary intake of food, especially foods that are rich in niacin and nicotinic acid.   Specifically, the expression of the NNMT gene correlates with the percentage of fat in 20 different mouse strains.  The main cause of the up regulation of the NNMT gene is overeating/overfeeding.   In fact, Kraus and colleagues recently showed that the administration of methylnicotinamide inhibited NNMT and this increased NAD levels and SAM-dependent gene expression.

References:

• 2012 Nicotinamide, NAD(P)(H), and Methyl-Group Homeostasis Evolved and Became a Determinant of Ageing Diseases: Hypotheses and Lessons from Pellagra
• 2013 Excessive nicotinic acid increases methyl consumption and hydrogen peroxide generation in rats 2011 Dietary methyl-consuming compounds and metabolic syndrome
• 2013  Excessive nicotinic acid increases methyl consumption and hydrogen peroxide generation in rats
• 2014 Targeting a fat-accumulation gene

This theory has been proposed because of recent studies which show a paradoxical effect occurs when NNMT is over-expressed.  When this occurs, the NNMT over-expression results in diet-induced obesity.  This has been shown in humans and in animal models.  For instance, the NNMT gene is unregulated in fat cells with obesity and T2DM. When the NNMT gene is “knocked down”, it protects against diet-induced obesity.

Other references for nicotinamide methylation:

• 1973 Nicotinamide methyltransferase and S-adenosylmethionine:5′-methylthioadenosine hydrolase. Control of transfer ribonucleic acid methylation
• http://www.nature.com/nchembio/journal/v9/n11/full/nchembio.1352.html  2013 Role of sirtuins in lifespan regulation is linked to methylation of nicotinamide
• 1994 Nicotinamide methylation in patients with cirrhosis
• 1984 Nicotinamide methylation tissue distribution, developmental and neoplastic changes
• 1984 Nicotinamide methylation and its relation to NAD synthesis in rat liver tissue culture: Biochemical basis fro the physiological activities of 1-methylnicotinamide
• 1965 Inhibition of the methylation of nicotinamide by chlorpromazine
• 1973 Nicotinamide methyltransferase and S-adenosylmethionine:5′-methylthioadenosine hydrolase. Control of transfer ribonucleic acid methylation
• 2012 Toxic effects of nicotinamide methylation on mouse brain striatum neuronal cells and its relation to manganese
• 2013  Association of Nicotinamide-N-Methyltransferase Gene rs694539 Variant with Patients with Nonalcoholic Steatohepatiti
• 2015  Association of nicotinamide-N-methyltransferase mRNA expression in human adipose tissue and the plasma concentration of its product, 1-methylnicotinamide, with insulin resistance2013 Association of nicotinamide-N-methyltransferase (NNMT) gene rs694539 variant with bipolar disorder
• 2014 Metabolism: Targeting a fat-accumulation gene

Here is the recent (2014)article on NNMT knock-down and the protection of diet-induced obesity: Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity

8.   PARP-1 and PARP-2

PARPs are the #1 Intracellular “NAD consumers” .  PARP-2 is also a direct inhibitor of SIRT1.

• The Poly-ADP-ribose polymerases (PARPs) are a large family of enzymes involved with DNA damage detection, DNA damage repair, and also cellular apoptosis.  They are voracious “NAD consumers” and use as much as 100-150 molecules of NAD when activated by one DNA break.   As a result, cells can become NAD depleted in the nucleus, where the PARPs reside.  They are also voracious “ATP consumers” and use up as much as 100-150 molecules of ATP when activated by one DNA break.  As a result, cells can become ATP depleted, which then induces cellular death.  Thus PARPs may be an important way of killing a cell by “suicide,” if there is too much DNA damage.
• PARP-1 is not a direct inhibitor of SIRT1, but because it consumes so much more NAD than Sirtuins do, PARP1 inhibitors or PARP-1 “knock down” have been shown to increase SIRT1activity in cells.
• PARP-2, on the other hand, is a direct negative regulator of SIRT1, independent of NAD activity. When PARP-2 activity increases, which it does with aging (by 10-fold), SIRT1 is inhibited, regardless of NAD levels.  However, the deletion of the PARP-2 gene results in hepatic cholesterol accumulation and decreased HDL lipoproteins.

References:

• 2011 PARP-1 Inhibition Increases Mitochondrial Metabolism through SIRT1 Activation
• 2013 PARP-2 knockdown protects cardiomyocytes from hypertrophy via activation of SIRT
• 2011 PARP-2 Regulates SIRT1 Expression and Whole-Body Energy Expenditure
• 2014 Pharmacological Inhibition of Poly(ADP-Ribose) Polymerases Improves Fitness and Mitochondrial Function in Skeletal Muscle
• 2011 Interference between PARPs and SIRT1: a novel approach to healthy ageing?
• 3013 PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosylation
• 2014 MicroRNA-149 Inhibits PARP-2 and Promotes Mitochondrial Biogenesis via SIRT-1/PGC-1α Network in Skeletal Muscle
• 2014 Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels
• ·       2014 Antagonistic crosstalk between SIRT1, PARP-1, and -2 in the regulation of chronic inflammation associated with aging and metabolic diseases

Conclusion: There is probably an “antagonistic cross talk” between SIRT1, PARP-1, and PARP-2 due to their mutual demand/need for NAD.  With aging, there is a “sterile inflammation” that occurs, often referred to as “inflammaging”.  Inflammaging appears to be directly under the control of the NF-kB transcription factor with antagonistic crosstalk between SIRT1, PARP1, and PARP2 signaling pathways.  There may be a role for a PARP inhibitor for health.  Pharmacological inhibition of PARPs has already been shown to improve skeletal muscle fitness and mitochondrial function in rodent models. In Part 4 of this NAD World series, we will discuss inflammationaging and the roles of NF-kB, NAD and SIRT1 in much further detail

9.  CD38 

CD38 is probably the #1 Extracellular “NAD consumer” (whereas PARPs are the #1 intracellular consumer)

CD38 is one of several “ectoenzymes” found outside the cell that are “NAD consumers”.  (The others are CD157, ART1, ART2, ART3, and ART4).  CD38 is a multifunctional membrane-bound extracellular enzyme that plays a key role in immunity, autoimmunity, and calcium signaling.  CD38 consumes NAD and makes cyclic-ADP ribose (cADPR).  Unfortunately, CD38 is a very inefficient enzyme and consumes as many as 100 NADs for every one cyclic-ADP-ribose that it makes.  For this reason, some experts on CD38 feel that the #1 function of CD38 is to regulate cellular NAD levels.  A strong argument for this theory is the recent discovery that CD38 is found inside the cell as well, bound to membranes on the inner portion of the cell nucleus.  Here it could deplete nuclear NAD.  Interestingly, the apple skin-derived flavanoid, apigenin, is a powerful inhibitor of CD38.  Treatment of cell cultures with apigenin increased NAD levels in the cells, reduced global acetylation of proteins, and reduced the acetylation of p53 and RelA-p65 subunits of NF-kB.

Reference: 2012 Flavonoid apigenin is an inhibitor of the NAD+ ase CD38: implications for cellular NAD+ metabolism, protein acetylation, and treatment of metabolic syndrome.

The classic description of CD38 has not been that of an “intracellular NAD level regulator”, but part of a signaling system involving cyclic ADP-ribose and calcium. Cyclic-ADP-ribose (cADPR) functions as a second messenger in the cell to trigger calcium release from the sarcoplasmic reticulum. CD38 is a very important membrane-bound enzyme found on the surface of many cells, but the highest density of CD38 are on the surface of immune cells such as monocytes/macrophages.

In the brain, CD38 is very important for the secretion of oxytocin from oxytocin-producing cells in the hypothalamus. Oxytocin has traditionally been thought of as the “maternal milk/nurturing hormone”, but in the brain it functions as a neurotransmitter.  In the brain, there are many locations for oxytocin type 1 (OTR1) and oxytocin type 2 (OTR2) receptors, located mostly in the telencephalon.  Oxytocin appears to be the “Peptide that binds our hearts in love”.  It has clearly been linked to maternal love, brotherly love, spousal/opposite sex affection, and community spirit.  It may be the peptide that is secreted in response to religious experiences of love for God as well.

References:

• 2009 CD38 as a regulator of cellular NAD: a novel potential pharmacological target for metabolic conditions
•  2002 CD38 expression is an important prognostic marker in chronic lymphocytic leukaemia
• CD38 expressed on human monocytes: A coaccessory molecule in the superantigen-induced proliferation
• 2004 CD38 expression on CD8 T lymphocytes as a marker of residual virus replication in chronically HIV-infected patients receiving antiretroviral therapy
• 2005 CD38 expression levels in chronic lymphocytic leukemia B cells are associated with activation marker expression and differential responses to interferon stimulation

Here is a diagram that illustrates the possible way that CD induces NAD depletion and therefore causes metabolic syndrome:

Image may be NSFW.
Clik here to view.

Ref for diagram:  CD38 as a regulator of cellular NAD: a novel potential pharmacological target for metabolic conditions

” Possible mechanism of regulation of SIRT1 and AMPK pathway by CD38 inhibition.

10.  MicroRNAs

16 microRNA have been found to regulate SIRT1 expression.  These miRNA are mostly negative regulators of SIRT1 expression by their binding to the 3′ UTR of SIRT1 mRNA, increasing SIRT1 mRNA degradation before they can be transcribed.  Different microRNA are expressed by different triggers, such as EtOH, p53, and Diabetes type II.

16 different micrRNAs have been found that regulate SIRT1 expression (see table below).   Different triggers induce the expression of each miRNA.   These microRNA all down-regulate SIRT1 expression.  This occurs because microRNA bind to a region in the 3′ untranslated region (3′ UTR) in the SIRT1 mRNA,  thereby increasing the degradation of SIRT1 mRNA.  Thus microRNA are a “post-transcriptional regulators” of SIRT1 gene expression.  This miR-mediated effect is likely the mechanism by which cancer increases the expression of SIRT1 and also the mechanism by which aging decreases the expression of SIRT1.

miR-217 - an miR that is activated by drinking EtOH

Ethanol also activates miR-217 gene expression and is one of the primary mechanisms responsible for alcoholic fatty liver disease and NASH (which is the clinically symptomatic form of alcoholic fatty liver disease).  NASH can lead to alcoholic cirrhosis, liver failure, ascites, and death.  miR-217 is also the mechanism by which HIV infections shorten lifespan, since tat-activated LTR expression induces miR–217.

miR-34a – an miR that is activated by p53

miR-34a has been the most studied microRNA when it comes to SIRT1 expression. It is involved in pancreatic cancer, colorectal cancer, prostate cancer, brain cancer, liver cancer, normal neural differentiation, liver metabolism, endothelial cell senescence, and endothelial progenitor cell senescence.

Ectopic miR-34a reduces SIRT1 expression. The gene for miR-34a is “turned on” by p53 protein.   miR-34a has been shown to induce cancer cell apoptosis in colon cancer cells.  miR-34a also promotes endothelial cell senescence in atherosclerosis.

miR-181a – an miR that is increased in diabetes type II

miR-181a binds to a region in the 3′ untranslated region (3′ UTR) in the SIRT1 mRNA, thereby increasing the degradation of SIRT1 mRNA.  Thus miR-181a is a “post-transcriptional regulator” of SIRT1 gene expression.   Over-expression of miR-181a results in insulin resistance.  Studies of the serum (plasma) of diabetics has shown that miR-181a is increased in the serum.  Studies of diabetics has also shown that miR-181a is increased in hepatocytes.  There is hope that inhibiting miR-181a may be a strategy for treating diabetes type II.

There are many other miRNAs that regulate SIRT1, listed below.

Image may be NSFW.
Clik here to view.

Table reference

References:

• 2012 MicroRNA Regulation of SIRT1
• 2009 MicroRNA 217 Modulates Endothelial Cell Senescence via Silent Information Regulator 1
• 2008 miR-34a repression of SIRT1 regulates apoptosis
• 2009 Cellular Regulation of SIRT1
• 2012 MiR-217 is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation by down-regulation of SIRT
• 2012 MicroRNA-217 Promotes Ethanol-induced Fat Accumulation in Hepatocytes by Down-regulating SIRT1
• 2010 MicroRNA-34a regulation of endothelial senescence 
• 2012 Downregulation of miR-181a upregulates sirtuin-1 (SIRT1) and improves hepatic insulin sensitivity
• 2012  MicroRNA Regulation of SIRT1

11.  p53

There are two binding sites for p53 on the promoter of SIRT1. They are located at positions 168 and 178 upstream from the TSS.  p53 binding to the SIRT1 promoter prevents gene expression of SIRT1, except with starvation. With starvation stress, p53 dissociates from the SIRT1 promoter and FoxO3a can “knock p53 off these two binding sites (168 and 178), thereby removing the repressive effects of p53 from the SIRT1 promoter, thereby resulting in SIRT1 gene expression.

References:

• 2010 SIRT1 and p53, effect on cancer, senescence and beyond
• 2014 SIRT1 phosphorylation by AMP-activated protein kinase regulates p53 acetylation

12.  FoxO3a and Eating (especially sugar):

Glucose induced Insulin signaling activates the Insulin/IR/IRS-1/PIP3K/Akt pathway which prevents Foxo3a from migrating into the cell nucleus and activating the SIRT1 gene. Fasting does the opposite. FoxO3a also enters the nucleus with fasting and “bumps” the two p53s off the two binding sites on the SIRT1 promoter, thereby abolishing the repression of SIRT1 by p53.

Reference:   2004 Stress-Dependent Regulation of FOXO Transcription Factors by the SIRT1 Deacetylase

13.  E2F1

The cell cycle and apoptosis regulator.

In actively growing cells, E2F1 is the transcription factor which controls cell-cycle fluctuations in SIRT1 levels. E2F1 induces SIRT1 gene expression in response to other factors also, such as cellular oxidative stress from exercise, low dose ETOH, chemo, XRT, and maybe your “oxidative stress water.” If this water would work as a “controllable timeable ROS dose,” this is the molecular mechanism by which it would work. OBTW, SIRT1 also has a negative feedback loop, inhibiting E2F1.

References:

• 2012 SIRT1: new avenues of discovery for disorders of oxidative stress
• 2011 Transcriptional and Nontranscriptional Functions of E2F1 in Response to DNA Damage
• 2014 AMP-activated Protein Kinase α2 and E2F1 Transcription Factor Mediate Doxorubicin-induced Cytotoxicity by Forming a Positive Signal Loop in Mouse Embryonic Fibroblasts and Non-carcinoma Cells
• Targeting cardiovascular disease with novel SIRT1 pathways
• 2014 Sirt1 is a tumor promoter in lung adenocarcinoma

14.  HIC1:CtBP co-repressor complex

The SIRT1 promoter has a binding site for a co-repressor complex called “HIC:CtBP”, which decreases SIRT1 gene expression. With CR and fasting or “fasting Mimetics like 2-DG”, the affinity of CtBP for HIC goes down, thereby increasing SIRT1 gene expression several fold. This is one of the primary molecular mechanisms of fasting.

References:

• 2012 Molecular dissection of the interaction between HIC1 and SIRT1
• 2012 Hypermethylation of HIC1 Promoter and Aberrant Expression of HIC1/SIRT1 Might Contribute to the Carcinogenesis of Pancreatic Cancer
•  2013 Identification of p21 (CIP1/WAF1) as a direct target gene of HIC1 (Hypermethylated In Cancer 1)
• 2013  Signification of Hypermethylated in Cancer 1 (HIC1) as Tumor Suppressor Gene in Tumor Progression

15.  ChREBP

 The carbohydrate response element binding protein.

ChREBP, is up-regulated with the dietary intake of carbohydrates. ChREBP is the “molecular link” between carbohydrate ingestion and high triglycerides in the blood.  (i.e. the glucose-induction of triglyceride synthesis.)   How does this occur?  Well, ChREBP represses the transcription of SIRT1.

ChREBP may play a major role in alcoholism and alcohol-induced fatty liver disease.  In a mouse model of binge drinking, ChREBP acetylation was increased dramatically and was recruited to gene promoters in mice.  The acetylation of ChREBP was dependent on alcohol metabolism rate.  In mice with mutant forms of ChREBP that could not be acetylated, the ChREBP-dependent genes could not be “turned on”.  ChREBP silencing in mice that were fed alcohol prevented the increase in triglycerides that normally occurs with binge drinking.  In addition, SIRT1 was down-regulated in these mouse models of EtOH binge drinking, due to the direct inhibitory effect of ChREBP on SIRT1 gene expression.

References:

• 2011 CREB and ChREBP oppositely regulate SIRT1 expression in response to energy availability
• 2015 Novel role for ChREBP in the control of ethanol metabolism and susceptibility to binge drinking
• 2015 Novel role for ChREBP in the control of ethanol metabolism and susceptibility to binge drinking
• 2013 Novel insights into ChREBP regulation and function
• 2012 The lipogenic transcription factor ChREBP dissociates hepatic steatosis from insulin resistance in mice and humans
• 2014 Liquid fructose downregulates Sirt1 expression and activity and impairs the oxidation of fatty acids in rat and human liver cells
• 2012 Glucose sensing by ChREBP/MondoA–Mlx transcription factors

Conclusions:  ChREBP is a “glucose sensor”.   ChREBP is the direct molecular link between high fructose or high glucose intake and the hepatic formation of triglycerides.  ChREBP is also a major inhibitor of SIRT1 gene transcription.  More importantly, ChREBP is the “binge drinking gene.”

16.  CREB

The cyclic-AMP response element binding protein,

CREB, activates SIRT1 gene expression. This is activated by low glucose levels, which happens with fasting.  CREB may also be the transcription factor that explains some of the paradoxical effects of SIRT1 over-expression.  In mice that over-express SIRT1, an atherogenic diet does not worsen glucose metabolism.  Instead, SIRT1 over-expression protects these mice fed an atherogenic diet from glucose dysregulation (i.e. insulin resistance).

However, in these SIRT1 over-expressed mice on an atherogenic diet, their atherosclerotic lesions actually get much worse than controls.   The reason for this is that SIRT1 deacetylates CREB, preventing its cyclic-AMP phosphorylation.  Thus SIRT1 inhibits CREB from activating gluconeogenic genes and inhibits CREB from activating hepatic lipid metabolism and excretion. In summary, CREB activates SIRT1 gene expression, and SIRT1 has a negative feedback effect on CREB function.  This explains how SIRT1-mediated CREB deacetylation regulates the balance between glucose and lipid metabolism.

ReferenceS:

• 2011 CREB and ChREBP oppositely regulate SIRT1 expression in response to energy availability
• 2011 Proatherogenic Abnormalities of Lipid Metabolism in SirT1 Transgenic Mice Are Mediated through Creb Deacetylation
• 2011 Sirt1 Mediates Neuroprotection from Mutant Huntingtin by Activation of TORC1 and CREB Transcriptional Pathway
• 2011 Sirt1 Mediates Neuroprotection from Mutant Huntingtin by Activation of TORC1 and CREB Transcriptional Pathway 

Conclusion:  CREB is a “glucose and lipid sensor” with reciprocal interactions with SIRT1.  CREB activates SIRT1, which is the opposite of ChREBP.  Fasting activates CREB, whereas fasting inhibits ChREBP.  Moreover, when there is too much fat, but SIRT1 is over-expressed, CREB cannot prevent atherosclerosis and atherosclerosis worsens.

17.  TLX

TLX is one of the “orphan nuclear receptors”.

TLX binds to a TLX-response element in the SIRT1 promoter.  TLX is short for “Tailess.”

TLX is a very important transcriptional repressor in the brain, especially in neural stem cells and is vital to normal growth and development. As of now, no endogenous ligand for these ligand-dependent transcription factors has been identified.  It is a “druggable” target, however.

So far, only three compounds have been found out of a 20,000 compound high-throughput screen that bind to TLX (ccrp1, ccrp2, and ccrp3).  Although many functions for TLX have been discovered, the main cellular function of TLX appears to be keeping neural stem cells in their undifferentiated, proliferative state.  TLX regulates the expression of another nuclear receptor, the “retinoic acid receptor” or RAR.  Thus TLX is an important receptor. TLX is an oncogene-induced senescence suppressor inside and outside of the brain.  It has been shown to be effective in the prosate.

TLX  co-regulates the cyclin-D kinase inhibitor, CDKN1A (aka p21WAF/CIP1) with SIRT.  Very little is known about this orphan nuclear receptor other than the fact that it activates SIRT1 gene expression.

References:

• 2015 Orphan nuclear receptor TLX functions as a potent suppressor of oncogene-induced senescence in prostate cancer via its transcriptional co-regulation of the CDKN1A (p21WAF1/CIP1) and SIRT1 genes
• 2015 TLX: A master regulator for neural stem cell maintenance and neurogenesis
• 2013 Insulin induces neurite outgrowth via SIRT1 in SH-SY5Y cells
• 2014 SIRT1 is required for oncogenic transformation of neural stem cells and for the survival of “cancer cells with neural stemness” in a p53-dependent manner
• 2008 Pathological aggression in “fierce” mice corrected by human nuclear receptor 2E1
• 2014 The Human Orphan Nuclear Receptor Tailless (TLX, NR2E1) Is Druggable
• 2004 Expression and function of orphan nuclear receptor TLX in adult neural stem cells

18.  C/EBP-alpha and beta

C/EBP-alpha and C/EBP-beta – the two hepatic CCAAT/enhancer binding proteins with opposite effects on SIRT1/

In the liver, there are two opposing transcription factors that play a major role in liver biology, glucose metabolism, and fat metabolism: CCAAT/enhancer binding proteins alpha and beta.  These two transcription factors are also expressed elsewhere, outside of the liver, but the general role of the two appear to be similar – they have opposing roles on gene expression.

•  The CCAAT/enhancer-binding protein alpha (C/EBP-alpha) is a transcription factor that represses many genes and activates many genes.  The most important 3 are SIRT1, p53, and PGC-1a.
• There is also a C/EBP-beta that has the opposite effects on these genes.

For instance C/EBP-alpha represses the hTERT gene, thereby preventing cancer induction of telomerase.   C/EBP-beta, on the other hand increases the expression of the hTERT gene, thereby increasing telomerase enzymes.

C/EBP-alpha activates SIRT1 gene expression via binding to the promoter region of SIRT1.  The opposite is true about C/EBP-beta.  It represses SIRT1 gene expression.

In old age, SIRT1 cannot be up-regulated very well due to the repressor effects of the C/EBP-beta/HDAC1 complex, which both work together to suppress SIRT1 gene expression.

Interestingly, pomegranate seed oil has 3 ingredients in it (xanthigen, fucoxanthin, and punicic acid) that appear to down regulate C/EBP-beta and thus prevent fat accumulation.  Xanthigen up regulates SIRT1 and AMPK signaling in differentiated fat cells also.

References:

• 2012 CEBP factors regulate telomerase reverse transcriptase promoter activity in whey acidic protein-T mice during mammary carcinogenesis
• 2014  Terminating hepatocyte proliferation during liver regeneration: The roles of two members of the same family (CCAAT-enhancer-binding protein alpha and beta) with opposing actions
• 2012 Transcription factor CCAAT/enhancer-binding protein alpha and critical circadian clock downstream target gene PER2 are highly deregulated in diffuse large B-cell lymphoma 2012 Xanthigen Suppresses Preadipocyte Differentiation and Adipogenesis through Down-regulation of PPARγ and C/EBPs and Modulation of SIRT-1, AMPK, and FoxO Pathways
• 2013 Transcriptional and Translational Regulation of C/EBPβ-HDAC1 Protein Complexes Controls Different Levels of p53, SIRT1, and PGC1α Proteins at the Early and Late Stages of Liver Cancer
• 2011 The reduction of SIRT1 in livers of old mice leads to impaired body homeostasis and to inhibition of liver proliferation†

Conclusion: The two CCAAT/enhancer-binding proteins alpha and beta have important opposing effects on tissue regeneration, glucose metabolism and fat metabolism.  In general, C/EBP-alpha prevents fat accumulation and promotes liver regeneration, whereas C/EBP-beta has the opposite effect.  C/EBP-alpha increases SIRT1 gene expression, whereas C/EBP-beta and HDAC-1 combine to repress the promoter of SIRT1.

19.  BRCA1

BRCA1 increases the expression of NAMPT, PARP1 and SIRT1, whereas BRCA1 mutation, promoter methylation, or knockdown decreases NAMPT, PARP1, and SIRT1 gene expression, but paradoxically increases NAD levels, which then increase SIRT1 activity (but not SIRT1 gene expression).  Thus BRCA1 may be a “balancer” between SIRT1 gene expression and SIRT1 protein activity

Two decades ago, the first breast cancer susceptibility gene was discovered and called “BRCA1″.  Now we know the BRCA1 protein (which is involved in double stranded DNA repair), is mutated in hereditary forms of breast and ovarian cancer.  Inherited BRCA1 mutations can lead to cancers of the breast, ovary, and many other organs.  For breast cancer, the risk due to the mutation is increased to 56-80%.  For ovarian cancer, the risk is increased to 15-60%.   BRCA1 regulates the expression of 7% of the mRNA in cancer cells.  This is probably why it is so important and should therefore be called a “transcriptional regulator”.

Although a lot of research was being done about BRCA1 and cancer, no one had linked BRCA1 to Sirtuins or NAD until very recently.  Recently, the BRCA1 protein was found to control NAMPT-mediated NAD synthesis, which was a surprise.  Another surprise was that NAD levels could have a “feedback activation” of BRCA1 expression.  Moreover, it has been shown that BRCA1 is a positive regulator of PARP1 levels and NAD-dependent PARP1 activity.  Most recently, BRCA1 was found to also positively regulate SIRT1 expresión. BRCA1 binds to the SIRT1 promoter and activates SIRT1 gene expression. SIRT1 then inhibits the expression of Survin by deacetylating the H3 histones of the Survivin gene. BRCA1 also inhibits Survivin. Without SIRT1 or BRCA1, Survivin levels increase and Cancer develops.

BRCA1 may also play a role in preventing hypertension.  One of the major pathways in spontaneous hypertension is the Angiotensin II pathway and the activation of the ATR1 and ATR2 receptor by Angiotensin II.  ATR1 receptor activation induces NAD(P)H oxidase-induced free radical formation and the vasoconstriction of blood vessels due to reduced NO production.  A recent article showed that BRCA1 limits Angiotensin II ATR1-mediated redox signaling, thereby improving vascular reactivity and reduces blood pressure in spontaneously hypertensive mice.

References:

• 2015 Linking BRCA1 to NAD World
• 2014 A novel crosstalk between BRCA1 and sirtuin 1 in ovarian cancer
• 2012 497 BRCA1 Limits AT1-Mediated Redox Signaling, Improves Vascular Reactivity and Reduces Blood Pressure in Spontaneously Hypertensive Rats

Image may be NSFW.
Clik here to view.Illustration Reference:  2015 Linking BRCA1 to NAD World  “Figure 1. Proposed model of crosstalk among BRCA1, SIRT1 and PARP1. A, BRCA1 inactivation may regulate SIRT1 and PARP1 levels, and induce an increase in NAD-mediated SIRT1 and PARP1 activity. B, the model shows a significant effect of BRCA1 in the maintenance of SIRT1-related biological processes. C, a proposed model to maintain stable BRCA1 and PARP1-related DNA repair ability.”

20.  SNPs in the SIRT1 promoter

Evolution and the “Feast or Famine” SNP in the SIRT1 promoter region in Northern India. rs12778366 is a single nucleotide polymorphism (SNP) found 1.46 kb upstream from the TSS in the SIRT1 promoter that predisposes N Indians to type II diabetes. > 80% of N. Indians have this SNP and have a 6-9 fold higher risk of T2DM. This SNP was probably “selected by famines” that often occurred in N India for 1,000s of years. Now it is a liability, since famines no longer occur

21.  EGR1 

Mechanical stretching of muscles:

The SIRT1 gene is upregulated by the stretching of muscle fibers via the transcription factor “Early Growth Response Factor 1″, or EGR1. Mechanical stretching of muscles increases mRNA for SIRT1 by 2.2 fold and SIRT1 protein by 100%! This is why stretching before and after exercise is so important, since SIRT1 expression causes FoxO3a deacetylation (and thereby induction of the mitochondrial SOD gene) as well as Nrf2 Deacetylation (and thereby induction of many antioxidant genes).

22.  DBC1 (aka CCAR2)

“Deleted in Breast Cancer 1″ is probably a bad name for this protein, since it may not even be deleted in most breast cancers.

For this reason, the new name for DBC1 is “Cell cycle activator and apoptosis regulator 2″, or CCAR2. DBC1 directly interacts with SIRT1 by forming a stable complex with DBC1, thereby preventing the activity of SIRT1 in vitro and in vivo.  Interestingly, SIRT1 and DBC1 protein levels are higher in breast cancer tissues, compared to age-matched controls, but not at the transcriptional level.  At the transcriptional level, there does not appear to be up regulation of SIRT1 and DBC1.  This up regulation of SIRT1 and DBC1 is therefore at the “postranscriptional level” (i.e. via microRNA or microRNA sinks).  In gastric adenocarcinoma, overexpression of SIRT1 and DBC1 are actually associated with a better prognosis.

Reference:

• 2013 hMOF Acetylation of DBC1/CCAR2 Prevents Binding and Inhibition of SirT1
• 2008 DBC1 is a negative regulator of SIRT1
• 2008 Negative regulation of the deacetylase SIRT1 by DBC1
• 2010 Balance between SIRT1 and DBC1 expression is lost in breast cancer
• 2009 Interactions between DBC1 and SIRT1 are deregulated in breast cancer cells
• 2012 Expression of SIRT1 and DBC1 in Gastric Adenocarcinoma
• 2008  DBC1 is a negative regulator of SIRT1

23.  c-Myc

There is now evidence that the SIRT1 gene expression is “downstream” from the oncogene, c-Myc.  In cancer, there is a “positive feedback loop” that occurs, which results in contributes to the development of cancer.  Specifically, c-Myc activates SIRT1, which in turn promotes SIRT1 function.   Likewise, SIRT1 promotes c-Myc function.  Here is the article on this.  This “mutual positive feedback” between SIRT1 and c-Myc is why there is so much confusion as to whether SIRT1 is a tumor suppressor or a tumor enhancer.

Reference:  2011 The c-MYC oncoprotein, the NAMPT enzyme, the SIRT1-inhibitor DBC1, and the SIRT1 deacetylase form a positive feedback loop

“Silent information regulator 1 (SIRT1) represents an NAD⁺-dependent deacetylase that inhibits proapoptotic factors including p53. Here we determined whether SIRT1 is downstream of the prototypic c-MYConcogene, which is activated in the majority of tumors. Elevated expression of c-MYC in human colorectal cancer correlated with increased SIRT1 protein levels. Activation of a conditional c-MYC allele induced increased levels of SIRT1 protein, NAD⁺, and nicotinamide-phosphoribosyltransferase (NAMPT) mRNA in several cell types. This increase in SIRT1 required the induction of the NAMPT gene by c-MYC. NAMPT is the rate-limiting enzyme of the NAD⁺ salvage pathway and enhances SIRT1 activity by increasing the amount of NAD⁺. c-MYC also contributed to SIRT1 activation by sequestering the SIRT1 inhibitor deleted in breast cancer 1 (DBC1) from the SIRT1 protein. In primary human fibroblasts previously immortalized by introduction of c-MYC, down-regulation of SIRT1 induced senescence and apoptosis. In various cell lines inactivation of SIRT1 by RNA interference, chemical inhibitors, or ectopic DBC1 enhanced c-MYC-induced apoptosis. Furthermore, SIRT1 directly bound to and deacetylated c-MYC. Enforced SIRT1 expression increased and depletion/inhibition of SIRT1 reduced c-MYC stability. Depletion/inhibition of SIRT1 correlated with reduced lysine 63-linked polyubiquitination of c-Myc, which presumably destabilizes c-MYC by supporting degradative lysine 48-linked polyubiquitination. Moreover, SIRT1 enhanced the transcriptional activity of c-MYC. Taken together, these results show that c-MYC activates SIRT1, which in turn promotes c-MYC function. Furthermore, SIRT1 suppressed cellular senescence in cells with deregulated c-MYC expression and also inhibited c-MYC–induced apoptosis. Constitutive activation of this positive feedback loop may contribute to the development and maintenance of tumors in the context of deregulated c-MYC.

24.  AROS

Active Regulator of SIRT1

AROS is an endogenous activator of SIRT1.  AROS may bind to the site where resveratrol and other STAC activators bind to SIRT1, but this is still unclear.  What is clear is that AROS increases SIRT1 activity and works with SIRT1 to suppress p53 activity.  Specifically AROS works with SIRT1 to deacetylate p53, thereby reducing p53-mediated transcriptional activity (gene expression of genes dependent on p53).  Thus SIRT1 and AROS are negative feedback regulators of p53.

Reference: 2007 Active Regulator of SIRT1 Cooperates with SIRT1 and Facilitates Suppression of p53 Activity

25.  HuR

The Hu protein called HuR is an RNA binding protein that stabilizes the SIRT1 mRNA, preventing its degradation

Many RNA binding proteins have been recently discovered that degrade or stabilize messenger RNA (mRNA).  This includes the mRNA degrading RNA-binding proteins, AUF1, BRF1, TTP, and KSRP. The mRNA-stabilizing RNA binding proteins include the elav/Hu proteins, of which HuR is one.  HuR is probably the most well-known RNA-binding protein that reduces mRNA degradation.  It binds to the SIRT1 mRNA in the cytoplasm to prevent the SIRT1 mRNA from being degraded. Whereas the 16 different miRNA that bind to SIRT1 mRNA all promote its degradation, HuR binds to the same 3′ UTR region on the SIRT1 mRNA, thereby preventing the miRNA-mediated degradation of the SIRT1 mRNA.  The net result of HuR is that there is more SIRT1 protein as a result of the same level of SIRT1 gene transcription.

Reference: 2007 Phosphorylation of HuR by Chk2 Regulates SIRT1 Expression

26.  JNK2

The c-Jun Kinase, JNK2 phosphorylates the SIRT1 protein, thereby stabilizes the SIRT1 protein 

The SIRT1 protein has several phosphorylation sites on Serine amino acid side chains.  Ser27 is one of these sites that gets phosphorylated indirectly by JNK2 activation.  When the Ser27 site on SIRT1 is phosphorylated, the SIRT1 protein becomes much more resistant to proteasome-mediated degradation.  Thus it increases the half life of the SIRT1 protein from < 2 hrs to > 9 hours.  This is a very important part of maintaining SIRT1 protein levels within the cell.

Reference: 2008 JNK2-dependent regulation of SIRT1 protein stability

27.  Resveratrol, SRT1720, SRT2104, EX527

Resveratrol is a natural STAC activator.  Synthetic STAC activators such as SRT1720 have also been synthesize

The initial excitement about Sirtuins was primarily directed towards the natural compound, reseveratrol, found in red grape skins, Japanese knotweed, and many other plants.   Resveratrol and other STAC activators only activate SIRT1 and not SIRT2-7 or the PARP enzymes.  For this reason, resveratrol may hold certain advantages over NAD therapy or NAD precursor therapy such as NR or NMN.  The STAC-activating site on SIRT1 is near amino acid E320.  This site is not present on the other 6 isoforms of mammalian SIRT (SIRT2-7).

References:

• Sirtuin-activating compound
•  SIRT1 Activators: The Evidence STACks up  
• Evidence for a common mechanism of SIRT1 regulation by allosteric activators
• New findings on mechanism of activation of sirtuins may vindicate Sirtris founders

28.  Lamin A

Lamin A is part of the nuclear cytoskeleton and may bind to the C-terminus of SIRT1.   Resveratrol may activate SIRT1 in a Lamin A-dependent manner.

There is a link between accelerated aging and the Laminopathies.  The most well-known laminopathies is Hutchinson-Gilford Progeria Syndrome, or HGPS.  In HGPS, a mutation in the Lamin A gene produces a mutant protein called “progerin”, which results in a breakdown of the cytoskeleton of the nuclear matrix.  This results in premature aging and usually death due to an MI or stroke during teenage years.  Recently, Gosh and colleagues from China have linked Lamin A and SIRT1.  According to their work, the C-terminal tail of SIRT1 binds to Lamin-A.  Thus lamina A may serve as a “SIRT1 anchor” in the cell nucleus.

Gosh and colleagues also showed that reseveratrol activates SIRT1 in a Lamin A-dependent manner.  Specifically, they showed that resveratrol increased the binding of SIRT1 to Lamin A and also down-regulated FoxO3 acetylation (SIRT1 is a known FoxO3 deacetylator).  Not all experts agree with this mechanism of action.  It has not been verified by a 2nd research laboratory.   However, this is very intriguing that reseveratrol may be a compound that could help treat HGPS.

Reference: 2013 Resveratrol activates SIRT1 in a Lamin A-dependent manner

Aging 

Aging lowers SIRT1 activity by multiple mechanisms.   The primary mechanism was once thought to be due to decreased gene expression of the SIRT1 gene, but this may not be true in the majority of cases.  Instead, age-induced reduction in SIRT1 activity is probably due to declining levels of NAD, increasing levels of Nicotinamide, and increasing levels of DBC1.

Reference: 2011 Age Related Changes in NAD+ Metabolism Oxidative Stress and Sirt1 Activity in Wistar Rats

Coming”  Part 4 of the NAD world (comment by Vince Giuliano)

By James P Watson with contributions and editorial assistance by Vince Giuliano

This Part 4 blog entry goes even further into the extended NAD story, examining important subtopics:

1. The ratio NAD+/NADH in cells and the body, which may be as or more important than absolute levels of NAD+ for driving health.
2. The NQ01 gene which drives the NAD+/NADH ratio, and factors related to its activation and expression: BET proteins, the 20S proteasome, BET inhibitors, NQ01 regulation of PGV-1alpha, Nrf2 regulation, etc
3. The Warburg effect, changes in cell metabolism characteristic of cancers and aging, its causes, effects and how NAD+ level is only one of several factors affecting it. Why the view that “The Warburg effect is caused by a nuclear state of pseudohypoxia which is caused by insufficient NAD+” is incomplete.
4. Reversing Warburg metabolism – known approaches and the possible use of phytosubstances; possible limits of reversal.
5. SIRT1 and inflammation, and why control of inflammation has such paramount importance
6. In the course of these discussions, a review of possible intervention approaches. ones not commonly discussed in the longevity litterture.

Image may be NSFW.
Clik here to view.NAD+ kinase Image source

The  Part 1 blog entry in the NAD World series provided an overview treatment of the NAD World and its nuances: to identify the major molecular entities involved, their roles, health and longevity ramifications, the reasons for the current excitement, and to begin to clarify what is actually known and what the remaining uncertainties are. 

The Part 2 blog entry in the NAD World series concentrates on the reasons for focusing on NAD+, particularly with respect to interventions that are seriously likely to lead to longer healthier lives.  It discusses molecular processes in the NAD salvage cycle that are responsible for the health-inducing and life-extending properties of calorie restriction, and further discuss the key roles of Sirtuins, SIRT1, SIRT6 and SIRT7 in particular.

The Part 3 blog entry in the NAD World series identifies 30 Major Factors that Control SIRT1 Expression, SIRT1 Activity, and SIRT1-mediated Aging.I.  The NAD+/NADH ratio and what affects it. The NQ01 gene.

   I.  The NAD+/NADH ratio and what affects it. The NQ01 gene

 What is of prime importance for health and longevity may not be the actual concentration of NAD+ in cells or cell nucli, but rather the NAD+/NAD ratio which may not be affected by NAD precursor supplements and rather be driven by other matters such as expression of the NQ01 gene,

In the Part 1 blog entry, we characterized the NAD Salvage Cycle by which NAD+ and NADH (the oxidized and reduced forms of NAD) are cycled back into each other and the de-novo pathway  via which new NAD is introduced into the body and cells.  And. in Parts 1,2 and 3 we discussed how various factors affect the cycling process such as circadian clock gene control.  In Part 3 we introduced and initially discussed the NQO1 gene which is a point of departure for the current discussion. “The longevity gene, NQO1, regulates aging by altering the NAD/NADH ratio in cells.   NQO1 does this by oxidizing NADH to NAD.   Beta-lapachone increases NQO1 enzyme activity and quercetin increases Nrf2-mediated gene expression of NQO1.”

I am now confidence that supplementation with a NAD+ precursor like NMN or NR transiently increased the ratio of NAD+  to NADH (NAD/NADH), but the ratio returns to normal in the course of continued supplementation.  This is because there is an enzyme that regulates the NAD/NADH ratio.  The ratio is NOT determined by dietary intake or IV intake of any compound.  It is enzymatically controlled. That enzyme is called  “NAD(P)H dehydrogenase, quinone 1″, or NQO1 for short.  NQO1 is an unusual gene in that it requires NADH as a co-factor but does not convert the NADH into nicotinamide, like the Sirtuins or PARPs.  Instead, it converts NADH to NAD+.  (i.e. it only oxidizes NADH to NAD+).

The oxidation of NADH increases NAD+ levels  within the cell and at the same time, decreases NADH levels within the cell.  As a result, the NAD+/NADH ratio  increases (or the NADH/NAD+ ratio decreases).

Why is NQO1 such an important gene?  Well here are the “Top 10 Reasons”.

1,  NQO1 regulates the intracellular redox state of the cell and thus, the ratio of NAD/NADH in the cell.

This is the main reason why I now think that NQO1 is so important.  Mice that are homozygous negative (both genes knocked out) for NQO1 have an increase in the NADH/NAD ratio (and an increase in the NADPH/NADP ratio).  NQO1 knockout mice strangely enough have lower blood glucose levels, less abdominal fat.  However, they have higher levels of triglycerides, beta-hydroxybutyrate, pyruvate, and lactate.   They also have higher levels of glucagon.   More importantly, the NQO1 “knock out” mice have lowered rates of pyridine nucleotide synthesis,  reduced glucose metabolism, and reduced fatty acid metabolism.  This is not surprising since NQO1 is the controller of the 20S PC mediated degradation of PGC-1alpha.

Unfortunately, cancer cells have also discovered this wonderful property of NQO1.  Many cancers unregulate NQO1 either via Nrf2 pathways or by other methods, such as the loss of miR suppression of mRNA for NQO1.  A recent study shows that higher levels of the NQO1 protein predict poor prognosis in non-small cell lung cancer.  This is a sobering thought – cancer cells up-regulate antioxidant genes!  This does not mean that we should avoid phytosubstances that up-regulate NQO1, it just means that “cancer cells are smart”.

Introducing:  BET proteins

The opposite problem occurs in aging cells.  Aging cells have lower levels of expression of the gene NQO1.  This is not just due to a “lack of broccoli” or a “lack of exercise”.   Instead, the gene NQO1 is down-regulated by proteins called “epigenetic readers”.  The two “epigenetic readers” that suppress the Nrf2-induction of the NQO1 gene are called “Bromodomain and Extraterminal Proteins” (or BET proteins).  Specifically, Brd2 and Brd4 proteins “sit on top” of the histone protein acetylated lysines at the promoters of the Nrf2-dependent genes.  As a consequence, Nrf2 and the other transcription factors that “turn on” NQO1 gene cannot turn the gene on.  (This is why BET inhibitors like JQ1 are so exciting).

Summary:  NQO1 regulates the ratio of NAD/NADH and the ratio of NADP/NADPH by oxidizing NADH to NAD+.   Warburg-type metabolism ensures that most of the NAD(H) within the cell is in the reduced form (NADH).  NQO1 is one of the few genes that oxidizes NADH to NAD+.  When both genes for NQO1 are  “knocked out”, there is even more NADH in the reduced state.  This results in a lower NAD/NADH ratio (or higher NADH/NAD ratio).  Thus, NQO1 is the “anti-Warburg gene”.   This is why NQO1 is so important.

References:

2001 In Vivo Role of NAD(P)H:Quinone Oxidoreductase 1 (NQO1) in the Regulation of Intracellular Redox State and Accumulation of Abdominal Adipose Tissue

2015 NQO1 protein expression predicts poor prognosis of non-small cell lung cancers

2013 Bromodomain and extra-terminal (BET) proteins regulate antioxidant gene expression

2.  NQO1 regulates the level of the co-activator, PGC-1a,within the cell by controlling degradation rate via the 20S proteasome.

PGC-1a is my “favorite co-activator” because it is so important in mitochondrial biogenesis.  I always thought that exercise activated PGC-1a gene via the “exercise kinase” called AMPK. (See he blog enry PGC-1-alpha and exercise).   This is why I was shocked to find out that NQO1 actually regulates PGC-1a, not by the increase in expression of the NQO1 gene, but the the rate that PGC-1a is degraded.  Expression of NQ01 keeps PGC-1a from being degraded.  What I found out is that the level of PGC-1a protein in a cell is primarily determined by its degradation rate, not its synthesis rate.

Like many regulatory factors, PGC-1a has an extremely short half life.  All of these extremely short-lived proteins are regulated by degradation rates, not synthesis rates.  In the past, it was thought that PGC-1a degradation was only regulated by the ubiquitin-proteasome system (UPS).  The UPS method involves a “protein tagger” that goes around putting a ubiquitin “tag” on the protein to be degraded.

Introduce: the 20S proteasome

However, recently a new process of proteasomal degradation has been discovered that does NOT involve any ubiquitination.  Specifically, this proteasome does NOT require ubiquitination of the protein and this proteasome system is called the “20S proteasome catalytic particle”  (aka 20S PC).  Unlike the ubiquitin-dependent, 26S proteasome system (UPS), the 20S proteasome does not require protein unfolding to degrade the protein.  (i.e. it can degrade proteins even without unfolding them).  Moreover, the 20S proteasome can handle oxidized proteins much better than the UPS 26S proteasome.  As a consequence, the 20S proteasome is the “oxidized protein degrader in stressed cells”.  For instance, it takes 4 times as much hydrogen peroxide to inhibit the 20S proteasome as it does to inhibit the 26S proteasome of the UPS.

Introducing: intrinsically disordered proteins (IDPs)

Not all proteins are degraded by the 20S proteasome, however. The main type of proteins degraded by the 20S PC system are called “intrinsically disordered proteins” (or IDPs).  Interestingly, the 20S proteasome system seems to be regulated by oxidative stress, via the glutathionylation of cysteine residues in the alph-rings of the 20S proteasome.

In conclusion, PGC-1a is a “intrinsically disordered protein” (IDP) that is regulated by its degradation rate.  When PGC-1a is damaged by oxidation or when the cell is under oxidative stress (like with aging), the 20S proteasome controls its degradation rate and thus the levels of PGC-1a within the cell.   Other IDPs besides PGC-1a include p53, c-fos, C/EBPa, p63, p33, p73a, and ornithine decarboxylase (ODC).

Interestingly, the 20S PC system has a “gate keeper” that inhibits the IDPs from being degraded.  Guess who the “gatekeeper” is for 20S PC? Yes, it is NQO1. That is how NQO1 expression keeps PGC-1a around.

 Summary:

There is strong evidence now that the levels of PGC-1a in cells is regulated primarily by the degradation rate of PGC-1a, and only secondarily by the gene expression of the PGC-1a gene.  There are two degradation pathways for PGC-1a.  The two pathways are the Ubiquitin Proteasome system (UPS) and the ubiquitin-independent proteasome system called 20S PC..

Under conditions of no oxidative stress, the UPS system may regulate PGC-1a levels within the cell.  However when the cell is under cellular stress and the PGC-1a protein is damaged by ROS-induced oxidation, the 20S proteasome controls the degradation rate of PGC-1a. NQO1 is the “gate-keeper” for this 20S PC system that prevents PGC-1a from being degraded during periods of cellular oxidative stress.  Thus with aging, the 20S PC system is more important than the26S proteasome  (i.e. the UPS) and thus the 20S proteasome degrades PGC-1a in the cell, unless NQO1 protects it from degradation. Thus it appears that under conditions of oxidative stress, such as with aging, NQO1 may be a major factor that controls the concentration of PGC-1a in the cell. 

References:

 2013 The Protein Level of PGC-1α, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

2001 Degradation of oxidized proteins by the 20S proteasome

2014 Regulating the 20S Proteasome Ubiquitin-Independent Degradation Pathway

1998 Comparative resistance of the 20S and 26S proteasome to oxidative stress

2013 The Protein Level of PGC-1α, a Key Metabolic Regulator, Is Controlled by NADH-NQO1

2006 20S proteasomes and protein degradation “by default”

1996 Degradation of oxidized proteins in K562 human hematopoietic cells by proteasome

2013 Redox regulation of the proteasome via S-glutathionylation

3.  Lower levels of NQO1 leads to increased sensitivity to chemical-induced skin carcinogens.

A very interesting study was done looking at skin-induced cancer from carcinogens.  This study looked at NQO1-null mice and found that their levels of NADH was higher (as expected).

Normally, when skin is exposed to chemical carcinogens, p53 is rapidly unregulated.  In the NQO1-null mice, exposure to chemical carcinogens did not induce p53 and as a result, the cells did not undergo apoptosis.  Instead, they underwent transformation to cancer cells.  This is an amazing and very important finding.  One  feature of p53 induction in the skin is an increase in the appearance of melanin in the skin.  This is normally called a “tan” by most people, but on a molecular level, a “tan” is actually p53 induction.  This is why people with dark skin have a lower incidence of skin cancer (it is not all to do with the sun block effect of melanin.  It is all about p53).

In a study of various mutations in the NQO1 gene in human basal cell carcinomas, 3.1% of 457 cases were found to have “loss-of-function” mutations in the NQO1 gene.  Those “NQO1 loss-of-function” individuals were found to have more skin cancers than those with other mutations, but this was not statistically significant.  The authors concluded that NQO1 mutations were clearly associated with skin cancer risk, but that these mutations only accounted for a minority of skin cancers.

Summary:  NQO1 stabilizes p53 and prevents its degradation. p53 levels in the cell is tightly regulated by two separate degradation pathways – a ubiquitin-dependent pathway that is dependent on the p53 binding partner, Mdm2;  or the ubiquitin-independent pathway that is dependent on NQO1.   It appears that the NQO1-dependent (ubiquitin-independent) pathway is the most important pathway for regulating p53 levels within the cell.

In the experiment above, NQO1-null mice did not induce p53 in response to carcinogens and the damaged skin cells would not undergo apoptosis, as they should to prevent cancer formation.  As a result, the NQO1-null mouse skin cells developed cancer.  These effects were thought to be directly due to the lack of binding of NQO1 to p53, which would preven the 20S PC degradation of p53 in the cell.  This means that p53 induction and cell apoptosis is dependent on NQO1-mediated stabilization of p53, preventing the 20S PC degradation of p53.  This is another reason why HQO1 is so important.   However in humans, loss-of-function mutations in the NQO1 gene only account for 3.1% of human skin cancers.

References:

 2005  Lower Induction of p53 and Decreased Apoptosis in NQO1-Null Mice Lead to Increased Sensitivity to Chemical-Induced Skin Carcinogenesis

2003 p53 hot-spot mutants are resistant to ubiquitin-independent degradation by increased binding to NAD(P)H:quinone oxidoreductase 1

1999 Association of NAD(P)H:quinone oxidoreductase (NQO1) null with numbers of basal cell carcinomas: use of a multivariate model to rank the relative importance of this polymorphism and those at other relevant loci

4.  NQO1 regulates blood pressureby eNOS, ACE, and an LKB1/AMPK-mediated preservation in GTPCH-1

Another fascinating study showed that activation of NQO1 ameliorates spontaneous hypertension in a rat model.  As you may know, spontaneous hypertension does not normally occur in rodents.  But in certain strains of inbred rats, bred to develop spontaneous hypertension, high BP does occur and is thought to be mediated by a decline in nitric oxide production by endothelial cells.  In this rat model of spontaneous hypertension, activation of NQO1 by beta-lapachone relieved the hypertension in these rats. The positive effects of beta-lapachone were thought to be mediated by NQO1-induction of endothelial nitric oxide (eNOS).

Another study showed that the effect of NQO1 was to regulate the acetylation of eNOS.  When an eNOS inhibitor was used, the positive effects of beta-lapachone was completely blocked.

In a separate study, beta-lapachone was used to study the effects of the shedding of the enzyme angiotensin converting enzyme (ACE), which converts Angiotensin I to Angiotensin II in the blood stream.  This study showed that beta-lapachone increased NQO1 activity which resulted in reduced cleavage and secretion of ACE into the extracellular space surrounding the cells that synthesized ACE.

In the most recent study, further elucidation of the eNOS mediated mechanism was analyzed and figured out. In this study, they showed that the increase in NAD+ levels in the aortic endothelial cells resulted in an increase in LKBA deacetylation, and AMPK phosphorylation.  This was followed by an increase in GTP-cyclohydrolase-1 preservation and tetrahydrobiopterin/dihydrobiopterin ratio.  This explained the rest of the story on how beta-lapachone reduced blood pressure.

Both beta-lapachone and the polyphenol, epicatechin, have the effect of reducing blood pressure.  Beta-lapachone does this via the direct activation of NQO1, whereas epicatechin does this by activating Nrf2.   Nrf2 is the transcription factor that turns on the NQO1 gene.

Summary:  NQO1 elevated the ratio of NAD/NADH in the endothelial cells and increases eNOS activity via an AMPK-dependent mechanism.  The increase in AMPK phosphorylation resulted in a preservation of the GTP cyclohydrolase-1 (GTPCH-1), which resulted in a lowering of blood pressure. The elevation in the NAD/NADH ratio also results in a reduced cleavage and secretion of ACE into the bloodstream, thereby reducing Angiotensin II formation.  As a result of the eNOS-mediated method and the ACE-reduction mediated molecular mechanism, the hypertension in rats resolved.   As a result of all this research, NQO1 activation has been recently proposed as a strategy for controlling hypertension (see lst reference below).

References:

2011  Activation of NAD(P)H:quinone oxidoreductase ameliorates spontaneous hypertension in an animal model via modulation of eNOS activity

2013 NQO1 Activation Reduces Blood Pressure via Regulation of eNOS Acetylation in Spontaneously Hypertensive Rats

2013 NQO1 Activation Regulates Angiotensin–Converting Enzyme Shedding in Spontaneously Hypertensive Rats

2014 Enhanced activation of NAD(P)H: quinone oxidoreductase 1 attenuates spontaneous hypertension by improvement of endothelial nitric oxide synthase coupling via tumor suppressor kinase liver kinase B1/adenosine 5′-monophosphate-activated protein kinase-mediated guanosine 5′-triphosphate cyclohydrolase 1 preservation

2012 Epicatechin lowers blood pressure, restores endothelial function, and decreases oxidative stress and endothelin-1 and NADPH oxidase activity in DOCA-salt hypertension

2014 NQO1 activation: a novel antihypertensive treatment strategy?

5.  “Loss of function” Polymorphisms in the NQO1 gene are associated with carotid artery atherosclerosis/plaques and stroke risk

This was the most amazing study.  There is a well-known polymorphism in the NQO1 gene called the “C609T variant.”  The C609T polymorphism results in a complete loss of enzymatic activity of NQO1 due to protein instability.  The C609T SNP is very common in Asia and has been well-described in Japanese, Korean, and Chinese ethnic groups.  Individuals with the “C allele” have a lower risk of carotid plaque disease, whereas individuals with the “T allele” have a higher risk of carotid atherosclerotic disease (OR = 1.65).  In Korea, 42% of the population have one “T allele” and 1% of the population have two copies of the “T allele”.  16% of Caucasians have one or two copies of the C609T variant, whereas 49% of Chinese have one or two copies of this SNP.

Another common polymorphism in the NQO1 gene is the C465T mutation.  This SNP results in a reduction in enzyme activity, but not a complete loss of function like the C609T SNP.   The incidence of this SNP is very low in all populations, varying from 0-5% (see reference below).

References:

2009  The C609T variant of NQO1 is associated with carotid artery plaques in patients with type 2 diabetes

2009 An Association between 609 C → T Polymorphism in NAD(P)H: Quinone Oxidoreductase 1 (NQO1) Gene and Blood Glucose Levels in Korean Population

6.   Bromodomain and Extraterminal Proteins (BET) supppress Nrf2-mediated gene expression (including NQO1)

Introducing:  BET protein inhibitors

We mentioned Bromodomain proteins in Item 1 above.  As you may recall, these are “epigenetic readers” that “read” the post-translational modifications of histone proteins.  The BET proteins (Brd2, Brd3, Brd4, and BrdT) bind to acetylated lysine residues on histone and non-histone proteins.  As a result, they either INCREASE or DECREASE the transcription of the genes associated with these acetylated lysine histones.  As it turns out, the Nrf2 genes are regulated by Brd2 and Brd4 proteins.  These complex with the acetylated lysine residues on histones located at the promoters of Nrf2-regulated genes.  As a result, Nrf2 either cannot increase or decrease the expression of these target genes.  When they gave the cells a BET protein inhibitor called JQ1, it increased the expression of Nrf2-genes including HO-1, NQO1, and GCLC, all of which are important in anti-oxidant defense and regulation of intracellular redox state. JQ1 administration resulted in a 10-fold increase in HO-1messenger RNA and a 3-fold increase in HO-1 protein levels.  JQ1 administration resulted in a 3-fold increase in NQO1 mRNA expression and a 3-fold change in NQO1 protein expression.  JQ1 also increased mRNA expression for GCLC by 2-4 fold and GCLC protein expression by 3-4 fold.

Interestingly, JQ1 also inhibited the expression of the ROS-producing protein, Nox.  As a result of this down-regulation of Nox, there was less free radicals in the cell (less H2O2).  This resulted in less oxidative stress in the cell due to both a reduced ROS production and increased anti-oxidant enzymes.

This page lists 14 other BET inhibutors,  And this 2011 paper discusses the discovery and characterization of small-molecule BET inhibitors

Conclusion: Bromodomain “epigenetic readers” can “shut off” the expression of Nrf2 genes and increase the expression of free-radical producing genes (Nox).  Inhibition of BET proteins with JQ1 has the effect of increasing the expression of Nrf2 genes and decreasing the expression of free radical-producing genes (Nox).  This may be a key discovery as one of the major causes of oxidative stress-induced aging may be BET proteins.)  And may also be why eating Broccoli and exercising have failed to lengthen life span.  JQ1, the most well-studied BET inhibitor, suffers from poor pharmacokinetics with a high clearance and low oral biovailabillity in animal studies.  This is why a lot of work is going into developing better BET inhibitors.

References:

2014 Bromodomain and Extra-Terminal (BET) proteins suppress nuclear factor E2-related factor 2 (Nrf2) -mediated antioxidant gene expression

2014  Bromodomain and Extraterminal Proteins Suppress NF-E2–Related Factor 2–Mediated Antioxidant Gene Expression

2014 Bromodomain and Extra-Terminal (BET) proteins suppress nuclear factor E2-related factor 2 (Nrf2) -mediated antioxidant gene expression

2013 Bromodomain and extra-terminal (BET) proteins regulate antioxidant gene expression

2014  Bromodomain and Extraterminal Proteins Suppress NF-E2–Related Factor 2–Mediated Antioxidant Gene Expression

2014 Bumping into BET inhibitors

2014 New benzazepine BET-inhibitors with improved oral bioavailability 

7.  The NQO1 gene is regulated by other factors besides Nrf2 – c-Jun, Nrf1, Jun-B, Jun-D, etc.

Quercetin “turns on” the NQO1 gene via Nrf2.   Dioxin “turns on” the NQO1 gene via both AhR, Arnt, and Nrf2.  Luteolin inhibits expression of NQO1 and drug-metabolizing enzymes via AhR and Nrf2 pathways.

As you may know, the levels of Nrf2 and the location of Nrf2 proteins within the cell is primarily regulated by the binding partner of Nrf2, aka Keap1.  There is a new name for Keap1, called INNrf2, but this new name is having trouble getting any attention in the scientific literature  This Important transcription factor and its binding protein havr been often discussed in this blog.  A comprehensive  series of entries on Nrf2 was published in this blog in 2012: Part1, Part2 and Part3.

An older article from 2000 showed that the NQO1 gene is regulated by several factors other than Nrf2 binding to the ARE segment of the NQO1 promoter.  They showed the transcription factor, c-Jun, can also bind to the ARE promoter sites on Nrf2-dependent genes.  So do the transcription factors Nrf1, Jun-B, and Jun-D.  Polyphenols can “turn on” NQO1 via Nrf2.  Toxins like dioxin can also ‘turn on” Nrf2, but require the assistance of the aryl hydrocarbon receptor, AhR, and the the aryl hydrocarbon receptor nuclear translocator, Arnt.

References:

2000 Regulation of genes encoding NAD(P)H:quinone oxidoreductases

2001 Induction of human NAD(P)H:quinone oxidoreductase (NQO1) gene expression by the flavonol quercetin

1991 Human NAD(P)H:quinone oxidoreductase (NQO1) gene structure and induction by dioxin

2000 NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms

2015 Constitutive expression of the AHR signaling pathway in a bovine mammary epithelial cell line and modulation by dioxin-like PCB and other AHR ligands

  2014 Luteolin modulates expression of drug-metabolizing enzymes through the AhR and Nrf2 pathways in hepatic cells

8.  The Asian vegetable, “pak choi,” reduces colon inflammation and colon cancer even better than broccoli sulforaphanes

In Asia, there is a very popular type of green, leafy vegetable called “pak choi”.  As a child, I ate this frequently as part of our regular diet in Thailand.  It is not a particularly “tasty” vegetable and reminds me of a cross between spinach and cabbage, but when boiled or steamed, it is a common vegetable eaten with rice.

A recent study showed that pak choi and brassica vegetables both activated cytoprotective genes, but the sets of genes that were activated were different.  Specifically, pak choi, broccoli, brussel sprouts, and other brassica vegetables all activated the typical Nrf2-target genes (NQO1, GSTM1, SRXN1, GPX2), whereas pak choi alone activated the AhR target gene,CYP1A1.  The relevance in the current context is that NQO1 belongs to a group of the aryl hydrocarbon receptor (AhR) battery of drug-metabolizing enzymes that are characteristically induced by both AhR agonists and Nrf2 activators(ref).               r

More importantly, in the studied mouse models of colitis and colon cancer, the glucosinolate-rich pak choi drastically reduced colitis and colon tumor number, whereas the broccoli-diet did not reduce colitis or colon tumor number in mice.

Conclusion:  The presence of glucosinolates (sulphoraphanes, etc.) in food does not necessarily reduce colon inflammation,  and colon cancer.  It appears that certain foods may have more stable or different glucosinolates that are more effective than others at preventing cancer and inflammation.  The Asian vegetable, pak choi,  appears to be more effective than broccoli and other brassica vegetables in down-regulating inflammation and preventing colon cancer.  (This study was done in Germany, by the way, and was not sponsored by the pak choi industry).

Reference:  2014  Glucosinolates from pak choi and broccoli induce enzymes and inhibit inflammation and colon cancer differently

9.  Physical methods and many readily available drugs and phytosubstances increase NQO1 expression or increase NQO1 activity

Including hyperthermia, heat shock, photodynamic herapy, sulindac, dimethylfumarate, taxifolin, sulforaphane,  resveratrol, and cisplatin.

A recent discovery that an old, common, generic NSAID that is still available at the drug store also activates the NQO1 gene.  Sulindac, a long neglected compound used to treat arthritis, activates the NQO1 gene.  Also, previous work has shown that several other compounds up-regulate the NQO1 gene or increase the activity of NQO1.   This includes cisplatin, resveratrol, dimethyl fumarate, taxifolin, sulforaphane, and the glucosinolates in pak choi.  Since the cancer-killing effects of beta-lapachone are dependent on the levels of NQO1, all of the above compounds work synergistically to kill cancer with beta-lapachone.

Several physical methods have been shown to increase NQO1 gene or protein activity.  This includes hyperthermia, heat shock, and photodynamic therapy.  This may be how photodynamic therapy works in cancer cells.  Interestingly, beta-lapachone works synergistically with these physical methods as well.

References:

2014 Sulindac Compounds Facilitate the Cytotoxicity of β-Lapachone by Up-Regulation of NAD(P)H Quinone Oxidoreductase in Human Lung Cancer Cells

2015 NQO1 protein expression predicts poor prognosis of non-small cell lung cancers

2014 The Chemotherapeutic Effects of Lapacho Tree Extract: β-Lapachone

2013 Preventive Effects of NSAIDs, NO-NSAIDs, and NSAIDs Plus Difluoromethylornithine in a Chemically Induced Urinary Bladder Cancer Model

  2015 Effect of glycosylation patterns of Chinese eggplant anthocyanins and other derivatives on antioxidant effectiveness in human colon cell lines

10.  Beta-lapachone, a compound found in the bark of the South American Lapacho tree, is a potent activator of the NQO1 protein and produces ROS in cancer cells, but reduces ROS in non-cancer cells.  It also inhibits pathological retinal neovascularization, but does not inhibit physiological neovascularization. 

The most exciting thing about NQO1 is that there is a natural, cheap, compound found in the tree bark of a South American tree.  The compound is called beta-Lapachone and is a NQO1 activator.  Specifically, NQO1 is a “two-electron transfer” enzyme that can extinguish free radicals in normal cells, but produces free radicals in cancer cells.  It has been shown to be a very effective compound for treating lung cancer.  Here is how it works:

• Beta-lapachone undergoes a redox cycle by NQO1, which reduces beta-lapachone to an unstable semiquinone.  The semiquinone then rapidly undergoes a two-step oxidation back to the parent stable compound, beta-lapachone.  This produces what is called a “perpetuating futile redox cycle”.  This results in an unbalance of intracellular reactive oxygen species in cancer cells, resulting in the cell death of the cancer cells.  This “perpetual futile redox cycle” is totally dependent on the concentration of NQO1 within cells. Here is a diagram of the reaction:

Image may be NSFW.
Clik here to view.

Illustration reference:   2014 The Chemotherapeutic Effects of Lapacho Tree Extract: β-Lapachone

The downstream effects of perpetual futile redox cycling include 4 apoptotic pathways and one necroptotic pathway:

1. Mitochondrial-induced apoptosis – The induction of ROS in mitochondria opens the MPTP pores and results in PARP activation and caspace activation.  This induces apoptosis.  
2. ER-induced apoptosis - The induction of ER stress induces sarcoplasmic release of calcium which induces high levels of cytoplasmic Ca++.  This also induces apoptosis via the ER.
3. DNA-damage mediated apoptosis – beta-lapachone also induces Topoisomerase I and II.  The activation of topoisomerases  induces DNA breaks, which induces PARPs.  This PARP hyper-activation induces apoptosis independently from mitochondrial ROS or ER stress.
4. Cell cycle arrest-induced apoptosis - The futile redox cycling of beta-lapachone also induces cycle cycle arrest via the activation of p21, p27, and the phosphorylation of JNK, PI3K, and Akt.  This induces cancer cell apoptosis as well.
5. Calpain-induced cell necrosis – Unlike the 4 pathways above, futile redox cycling also induces calcium influx into the cells independently of ER stress.  This calcium influx into the cell activates Calpain, which induces cell death by the necrosis pathway, not the apoptosis pathway.

Conclusion:  beta-lapachone induces cancer cell death by five different pathways, all dependent on perpetual futile redox cycling which is dependent on NQO1 expression.   Here is a diagram that illustrates these 5 pathways:

Image may be NSFW.
Clik here to view.