AGINGSCIENCES™ – Anti-Aging Firewalls™

Perhaps the two most comprehensive theories explaining aging in my treatise are Programmed Epigenomic Changes and Stem Cell Supply Chain Breakdown.  Recent research related to the epigenetics of stem cells deals with the profound underlying relationships between those two theories.  The research relates to questions such as “What causes a stem cell to proliferate (e.g. reproduction through mitosis making more stem cells of the same kind), and what causes a stem cell to differentiate (e.g. generate more specific progenitor or somatic cells)?  The subject has been called Epigenetic alchemy for cell fate conversion.   I review some of that current research here. The key players I am going to focus on here are DNA methyltransferases and their key regulatory roles.

Background on DNA methylation

I have discussed DNA methylation and its role in aging in a number of my earlier blog entries.  See for example Epigenetics, epigenomics and aging, DNA methylation, personalized medicine and longevity and Histone acetylase and deacetylase inhibitors, DNA demethylation – a new way of coming at cancers,  and Epigenetics going mainstream.

“DNA methylation, particularly when applied to CG-rich promoter sequences, has been shown to silence gene expression in a heritable manner. DNA methylation is therefore a form of cellular memory. Because DNA methylation is not encoded in the DNA sequence itself, it is called an epigenetic modification (“epi”, Greek origin: “above” or “upon”). The transcriptional silencing associated with 5-methylcytosine is required for fundamental biological processes such as embryonic development, protection against intragenomic parasites, X-inactivation(ref).”,

It has long been known that “DNA methylation is impacted by aging and impacts on aging(ref).  Methylation in the promoter region of genes is thought generally to be associated with gene silencing.  Longevity-related and health-promoting genes may be turned off in the process of aging due to progressive methylation(ref).”  I remind my readers that the 13^th theory of aging  covered in my treatise, Programmed Epigenomic Changes, envisages aging as a systematically articulated set of epigenomic changes including  changes in DNA methylation in cells accumulated with aging. One researcher goes so far as to assert that DNA methylation is the cause of aging.  See my blog entry Homicide by DNA methylation.

Much of the new research relates to the life-and-death roles of DNA methyltransferases in adult stem cells and what causes stability in embryonic stem cells.  “– the DNA methyltransferase (DNA MTase) family of enzymes catalyze the transfer of a methyl group to DNA. DNA methylation serves a wide variety of biological functions.  All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor(ref).” As mentioned, a methyl group transferred to  a GpC site in the promoter region(ref) of a gene generally serves to silence that gene.  “CpG sites are regions of DNA where a cytosine nucleotide occurs next to a guanine nucleotide in the linear sequence of bases along its length. “CpG” is shorthand for “—C—phosphate—G—“, that is, cytosine and guanine separated by a phosphate(ref).”  

The new research relates to How DNA methyltransferases 1.  initiate and maintain methyl marks, 2. are involved in self-renewal of  embryonic stem (ES) cells, and 3. act in somatic (adult) stem cells including: hematopoietic, epithelial, neural and muscle cells.  It also relates to the molecular factors that keep embryonic stem cells from differentiating and the role of methyltransferases once those cells start differentiating.  I start out with research on adult stem cells. 

DNA methyltransferases and  adult stem cells

The April 2010 review publication DNA methylation in adult stem cells: New insights into self-renewal summarizes the important role of methyltransferases in preserving adult stem cell lineages. “Methylation of cytosine residues in the context of CpG dinucleotides within mammalian DNA is an epigenetic modification with profound effects on transcriptional regulation. A group of enzymes, the DNA methyltransferases (DNMTs) tightly regulate both the initiation and maintenance of these methyl marks. Loss of critical components of this enzymatic machinery results in growth, viability and differentiation defects in both mice and humans, supporting the notion that this epigenetic modification is essential for proper development. Beyond this, DNA methylation also provides a potent epigenetic mechanism for cellular memory needed to silence repetitive elements and preserve lineage specificity over repeated cell divisions throughout adulthood. Recent work highlighting the specialized roles of DNA methylation and methyltransferases in maintaining adult somatic stem cell function suggests that further dissection of these mechanisms will shed new light on the complex nature of self-renewal.” 

The 2010 study DNMT1 maintains progenitor function in self-renewing somatic tissue gets down to more specifics.  “Progenitor cells maintain self-renewing tissues throughout life by sustaining their capacity for proliferation while suppressing cell cycle exit and terminal differentiation. DNA methylation provides a potential epigenetic mechanism for the cellular memory needed to preserve the somatic progenitor state through repeated cell divisions. DNA methyltransferase 1 (DNMT1) maintains DNA methylation patterns after cellular replication. Although dispensable for embryonic stem cell maintenance, the role for DNMT1 in maintaining the progenitor state in constantly replenished somatic tissues, such as mammalian epidermis, is unclear. Here we show that DNMT1 is essential for epidermal progenitor cell function. DNMT1 protein was found enriched in undifferentiated cells, where it was required to retain proliferative stamina and suppress differentiation. In tissue, DNMT1 depletion led to exit from the progenitor cell compartment, premature differentiation and eventual tissue loss. Genome-wide analysis showed that a significant portion of epidermal differentiation gene promoters were methylated in self-renewing conditions but were subsequently demethylated during differentiation.  — These data demonstrate that proteins involved in the dynamic regulation of DNA methylation patterns are required for progenitor maintenance and self-renewal in mammalian somatic tissue.”

The 2009 publication DNA methyltransferase 1 is essential for and uniquely regulates hematopoietic stem and progenitor cells establishes a similar critical role for DNMT1  in hematopoietic stem and progenitor cells. “DNA methylation is essential for development and in diverse biological processes. The DNA methyltransferase Dnmt1 maintains parental cell methylation patterns on daughter DNA strands in mitotic cells; however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we conditionally disrupted Dnmt1 in the hematopoietic system. Defects were observed in Dnmt1-deficient HSC self-renewal, niche retention, and in the ability of Dnmt1-deficient HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had specific impact on myeloid progenitor cells, causing enhanced cell cycling and inappropriate expression of mature lineage genes. Dnmt1 regulates distinct patterns of methylation and expression of discrete gene families in long-term HSCs and multipotent and lineage-restricted progenitors, suggesting that Dnmt1 differentially controls these populations. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment.”

The methyltransferases are also important in maintaining genomic stability of neural stem cells. Then 2009 study Cellular epigenetic modifications of neural stem cell differentiation reports : “Emerging information indicates that epigenetic modification (i.e., histone code and DNA methylation) may be integral to the maintenance and differentiation of neural stem cells (NSCs), but their actual involvement has not yet been illustrated. In this study, we demonstrated the dynamic nature of epigenetic marks during the differentiation of quiescent adult rat NSCs in neurospheres. A subpopulation of OCT4(+) NSCs in the neurosphere contained histone marks, trimethylated histone 3 on lysine 27 (3me-H3K27), 2me-H3K4, and acetylated H4 (Ac-H4). A major decrease of these marks was found prior to or during differentiation, and was further diminished or reprogrammed in diverse subpopulations of migrated NSCs expressing nestin or beta-III-tubulin. –. Furthermore, we found an outward translocation of DNA methylation marker 5-MeC, DNMT1, DNMT3a, and MBD1 in NSCs as differentiation began and proceeded; 5-MeC from homogeneous nucleus to peripheral nucleus, and DMNT1a and 3a from nuclear to cytoplasm, indicating chromatin remodeling. —  These results indicate that chromatin is dynamically remodeled when NSCs transform from the quiescent state to active growth, and that DNA methylation modification is essential for neural stem cell differentiation.”

Embryonic and induced pluripotent stem cells and maintenance of pluripotency

The methyltransferases play somewhat of a different role when it comes to fully pluripotent cells – embryonic stem cells and, most likely, induced pluripotent stem cells.  Philosophically, I like the position taken in the 2008 paper Capturing pluripotency.  “In this Essay, we argue that pluripotent epiblast founder cells in the embryo and embryonic stem (ES) cells in culture represent the ground state for a mammalian cell, signified by freedom from developmental specification or epigenetic restriction and capacity for autonomous self-replication. We speculate that cell-to-cell variation may be integral to the ES cell condition, safe-guarding self-renewal while continually presenting opportunities for lineage specification.”

A key question is “When does a pluripotent stem cell like an Esc or iPSC stay pluripotent and when does it differentiate into a less-pluripotent state?  Addressing this question is the September 2009 publication Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. “Coordinated transcription factor networks have emerged as the master regulatory mechanisms of stem cell pluripotency and differentiation. Many stem cell-specific transcription factors, including the pluripotency transcription factors, OCT4, NANOG, and SOX2 function in combinatorial complexes to regulate the expression of loci, which are involved in embryonic stem (ES) cell pluripotency and cellular differentiation. This review will address how these pathways form a reciprocal regulatory circuit whereby the equilibrium between stem cell self-renewal, proliferation, and differentiation is in perpetual balance. We will discuss how distinct epigenetic repressive pathways involving polycomb complexes, DNA methylation, and microRNAs cooperate to reduce transcriptional noise and to prevent stochastic and aberrant induction of differentiation. We will provide a brief overview of how these networks cooperate to modulate differentiation along hematopoietic and neuronal lineages.”

Also addressing the same question is the 2008 publication Esrrb activates Oct4 transcription and sustains self-renewal and pluripotency in embryonic stem cells.  “The genetic program of embryonic stem (ES) cells is orchestrated by a core of transcription factors that has OCT4, SOX2, and NANOG as master regulators. Protein levels of these core factors are tightly controlled by autoregulatory and feed-forward transcriptional mechanisms in order to prevent early differentiation. Recent studies have shown that knockdown of Esrrb (estrogen-related-receptor beta), a member of the nuclear orphan receptor family, induces differentiation of mouse ES cells cultured in the presence of leukemia inhibitory factor. – Supporting all of these data, stable transfection of Esrrb in ES cell lines proved sufficient to sustain their characteristics in the absence of leukemia-inhibitory factor. In summary, our experiments help to understand how Esrrb coordinates with Nanog and Oct4 to activate the internal machinery of ES cells.”

These two studies suggest that, unlike the case for adult stem cells, other factors like Esrrb are important in maintaining the undifferentiated status of fully pluripotent stem cells.  This result was also observed in a 2006 mouse study which concluded “Our results indicate that ES cells can maintain stem cell properties and chromosomal stability in the absence of CpG methylation and CpG DNA.”  The 2010 publication Polycomb complexes act redundantly to repress genomic repeats and genes also suggest other mechanisms that inhibit differentiation in ESCs.

What causes embryonic stem cells to differentiate? The 2009 report Cdk2ap1 is required for epigenetic silencing of Oct4 during murine embryonic stem cell differentiation indicates “Here, we show that Cdk2ap1, a negative regulator of Cdk2 function and cell cycle, promotes Oct4 promoter methylation during murine embryonic stem cell differentiation to down-regulate Oct4 expression.”

When ESCs do differentiate, then promoter methylation comes into play as indicated in the April 2010 publication Targeting of de novo DNA methylation throughout the Oct-4 gene regulatory region in differentiating embryonic stem cells. “Differentiation of embryonic stem (ES) cells is accompanied by silencing of the Oct-4 gene and de novo DNA methylation of its regulatory region. Previous studies have focused on the requirements for promoter region methylation. We therefore undertook to analyze the progression of DNA methylation of the approximately 2000 base pair regulatory region of Oct-4 in ES cells that are wildtype or deficient for key proteins. We find that de novo methylation is initially seeded at two discrete sites, the proximal enhancer and distal promoter, spreading later to neighboring regions, including the remainder of the promoter. De novo methyltransferases Dnmt3a and Dnmt3b cooperate in the initial targeted stage of de novo methylation. Efficient completion of the pattern requires Dnmt3a and Dnmt1, but not Dnmt3b. Methylation of the Oct-4 promoter depends on the histone H3 lysine 9 methyltransferase G9a, as shown previously, but CpG methylation throughout most of the regulatory region accumulates even in the absence of G9a.”

Summarizing the situation:

·        The pluripotency and differentiation of ESCs and iPSCs are regulated by complex networks which maintain dominance of cell ground-state pluripotency transcription factors like OCT4, SOX2 and NANOG until differentiation is triggered.  Apparently, the methyltransferases do not play a dominant role in that process though they may be involved.

·        When ESCs start to differentiate, silencing of the OCT4 gene seems to take place via promoter methylation of this gene.  At that point methyltransferases become important for maintaining lineages of adult stem cells

.·        Adult stem cells, including neural progenitor cells and hematopoietic stem cells depend on DNA methylation for their survival in undifferentiated state.  This methylation in turn depends critically on the actions of DNA methyltransferases.  In plain language, the methyltransferases keep lineages of adult stem cells continuing in their niches throughout life instead of having all the adult cells differentiating early in life leaving no reserves of such cells.

So, DNA promoter regulation via methylation in stem cells is an important mechanism for the operation of the stem cell supply chain. 

There is also a growing number of publications on DNA methylation and the role of methyltransferases in cancer stem cells, and I will probably take that topic up in a later blog post.

DNA repair is a major defense against the second cause of aging described in my treatise Cell DNA Damage.  Such repair is absolutely necessary. Damage can be caused by oxidative processes, radiation exposure, and exposure to environmental toxins, cigarette smoke and some antibiotics, and anti-inflammatory drugs(ref). Even without extraordinary exposure, in the course of a normal good day a person may have a million or more events of DNA damage occur in his or her body.  Further, the kinds of DNA damage can be of multiple types(ref).  Failure to repair damage can lead to cell death, cancer, a number of diseases and premature aging.   

In response to this challenge, cells have evolved numerous repair strategies.  Some are very clever and still being discovered. I discussed one such line of defense against an important form of DNA damage, double-strand breaks, in my March 2010 blog entry DNA repair cleanup failure – a root cause for cancers?  I concluded that entry by saying “There is a lot more interesting research related to DNA repair beyond the thread covered here and I will probably come back to that topic again before too long.”  That time is now. I review several additional natural DNA repair strategies together with news of recent discoveries.   

Ku and making ends meet 

In the earlier blog entry and with respect to the substance Ku that I am concerned with here, the focus is on one particular important kind of breaks, double-strand breaks (DSBs), breaks that can occur naturally in cell differentiation or that are created by radiation and certain chemicals.    A double-strand break results in a broken chromosome, and this kind of DNA damage is particularly difficult to repair.  Because these breaks completely threaten genomic integrity, evolution has provided us with a number of sophisticated approaches for automatic DNA repair. Non-homologous DNA end-joining (NHEJ) is the main pathway for repairing double-stranded DNA breaks.  It functions throughout the cell cycle to repair such lesions. “NHEJ typically utilizes short homologous DNA sequences called microhomologies to guide repair. These microhomologies are often present in single-stranded overhangs on the ends of double-strand breaks. When the overhangs are perfectly compatible, NHEJ usually repairs the break accurately.^[1][2][3][4] Imprecise repair leading to loss of nucleotides can also occur, but is much more common when the overhangs are not compatible. Inappropriate NHEJ can lead to translocations and telomere fusion, hallmarks of tumor cells(ref).^[5] ” 

A protein called Ku has been known for some time to be involved in NHEJ(ref)(ref).  A colorful animation of the role of Ku in NHEJ can be found here.  Previously, it was thought that Ku worked simply by recognizing  broken ends and then recruiting other factors that cleaned up the ends and then joining them again.  The ends-cleanup processing is necessary because the strand breaks are often associated with nucleotide damage so that simply connecting ends would result in mutated chromosomes. The April 2010 online publication Ku is a 5′-dRP/AP lyase that excises nucleotide damage near broken ends says that Ku does the ends-cleanup job itself. “Ku had previously been presumed only to recognize ends and recruit other factors that process ends; our data support an unexpected direct role for Ku in end-processing steps as well.”  As reported in Science Daily quoting Dale Ramsden one of the authors and investigators, “Ku is a very exciting protein because it employs a unique mechanism to repair a particularly drastic form of DNA damage.  — Damage to DNA in the form of a broken chromosome, or double strand break, can be very difficult to repair — it is not a clean break and areas along the strand may be damaged at the level of the fundamental building blocks of DNA — called nucleotides — It has been assumed in the past that double strand breaks are the most difficult class of DNA damage to repair and it is often presumed that they simply cannot be repaired accurately.”  Part of the importance of this new research is showing that repairs of double-strand breaks can be more accurate than previously thought.  Ku-based healing is not only at the chromosome level but also at the nucleotide level. 

DNA unwrapping/wrapping in repairing single-strand breaks. 

My power boat ties up to the dock using heavy 3-strand polyvinyl rope.  If a single strand breaks I can fuse it together with heat from a small blowtorch.  But first I must unwrap the strands some to identify the break and make room for the repair.  It turns out that the DNA repair machinery does something very similar.  The 2009 publication DNA wrapping is required for DNA damage recognition in the Escherichia coli DNA nucleotide excision repair pathway reports on such unwrapping.  As explained simply in a Science Daily article: “They found that the proteins that initially recognize the damage amplify the distortion of the DNA around the damaged site by bending the DNA and separating the strands of the double helix. This makes it easier for the next protein to recognize and cut out the damaged portion of the DNA. The cells then patch up the empty space using the healthy half of the DNA as a model to repair the cell to its original state. — The study was conducted using a DNA repair system operated in E. coli, but the findings are applicable to other cells because they adopt similar systems.”  The following item relates to the repair process that takes place after the DNA strands unwind. 

A shuttlebus first-responder repair protein, SSB 

For some time, the protein SSB has been known to play a role in excision repair(ref) of DNA single-strand breaks.  The 2009 publication SSB protein diffusion on single-stranded DNA stimulates RecA filament formation lends insight into the ways in which SSB works. “Single-stranded DNA generated in the cell during DNA metabolism is stabilized and protected by binding of ssDNA-binding (SSB) proteins. Escherichia coli SSB, a representative homotetrameric SSB, binds to ssDNA by wrapping the DNA using its four subunits. However, such a tightly wrapped, high-affinity protein–DNA complex still needs to be removed or repositioned quickly for unhindered action of other proteins. — tetrameric SSB can spontaneously migrate along ssDNA. Diffusional migration of SSB helps in the local displacement of SSB by an elongating RecA filament. SSB diffusion also melts short DNA hairpins transiently and stimulates RecA filament elongation on DNA with secondary structure. This observation of diffusional movement of a protein on ssDNA introduces a new model for how an SSB protein can be redistributed, while remaining tightly bound to ssDNA during recombination and repair processes.”   

A press release from the University of Illinois  explains the actions of SSB in simpler terms.  “– a single-stranded DNA-binding protein (SSB), once thought to be a static player among the many molecules that interact with DNA, actually moves back and forth along single-stranded DNA, gradually allowing other proteins to repair, recombine or replicate the strands.”  SSB is a first responder. Think of it as a crew first sent out on a small railway shuttlebus car when there is trouble with the tracks.  The crew includes the supervisor who will oversee emergency measures and the repairs.  “Whenever the double helix of DNA unravels, exposing each strand to the harsh environment of the cell, SSB is usually first on the scene, said University of Illinois physics professor and Howard Hughes Medical Institute investigator Taekjip Ha, who led the study. — Although DNA unwinding is necessary for replication or recombination, it is normally a transient process, he said. Exposed single-stranded DNA (ssDNA) can be damaged or degraded by enzymes in the cell. Damaged DNA may also come unwound, and ssDNA can bond to itself, forming hairpin loops and other problematic structures. — “If you have lots of single-stranded DNA in the cell, basically it’s a sign of trouble,” Ha said. “SSB needs to come and bind to it to protect it from degradation and to control what kind of proteins have access to the single-stranded DNA.” Although other proteins are known to travel along double-stranded DNA, this is the first study to find a protein that migrates back and forth randomly on single-stranded DNA, Ha said.” 

“– the researchers showed that SSB diffuses randomly back and forth along single-stranded DNA, and that this movement is independent of the sequence of nucleotides that make up the DNA. They also found that an important DNA repair protein in E. coli, RecA, grows along the ssDNA in tandem with the movement of SSB. As the RecA protein extends along the DNA strand it prevents the backward movement of the SSB. — The researchers also found that SSB can “melt” small hairpin loops that appear in single-stranded DNA, straightening them so that the RecA protein can bind to and repair them. In this way SSB modulates the activity of RecA and other proteins that are involved in DNA repair, recombination and replication. — “SSB may be a master coordinator of all these important processes,” Ha said(ref).” 

The role of HMGB1 

No, HMGB1 is not an agency in the Russian secret service.  HMGB1 stands for high mobility group box protein 1.   It is a protein that pays an important role in DNA repair, though what to do about it is controversial. HMGB1 is an Alarmin. “Alarmins are a newly described and still emerging group of structurally diverse, but functionally related, molecules that include defensins, cathelicidins, eosinophil-derived neurotoxin, and HMGB1 — All are released in response to infection and tissue damage, and mediate innate immunity and tissue repair(ref).”  Let’s start with the bad reputation for HMGB1, which is that it causes inflammation and plays a role in creating epileptic episodes and is implicit in the progress of many cancers.  For example the medical news report Salute: epilessia? è colpa della molecola HMGB1 translate into Health:  epilepsy?  It the fault of the molecule HMGB1.  Many publications suggest that targeting HMGB1 could provide effective cancer therapies(ref).  The 2003 publication Dealing with death: HMGB1 as a novel target for cancer therapy suggests the development of anti-cancer drugs that work by inhibiting HMG1. HMGB1 is thought to play a key role in chronic inflammatory autoimmune disease and as well as in severe, acute systemic inflammatory disease(ref). ”Because HMGB1 plays a key role in inflammation, it’s also being targeted in drugs under development for rheumatoid arthritis and sepsis. “   A quite different view of HMGB1 is suggested in the 2008 medical news report Suspect protein HMGB1 found to promote DNA repair, prevent cancer.  “An abundant chromosomal protein that binds to damaged DNA prevents cancer development by enhancing DNA repair”–  “Long known to attach to sites of damaged DNA, the protein was suspected of preventing repair. “That did not make sense to us, because HMGB1 is a chromosomal protein that’s so abundant that it would be hard to imagine cell repair happening at all if that were the case,” Vasquez (senior researcher in the study) said.  In a series of experiments — Vasquez and first author Sabine Lange, — tracked the protein’s impact on all three steps of DNA restoration: access to damage, repair and repackaging of the original structure, a combination of DNA and histone proteins called chromatin. — First, they knocked out the gene mouse embryonic cells and then exposed cells to two types of DNA-damaging agents. One was UV light, the other a chemotherapy called psoralen that’s activated by exposure to darker, low frequency light known as UVA. In both cases, the cells survived at a steeply lower rate after DNA damage than did normal cells. — Next they exposed HMGB1 knockout cells and normal cells to psoralen and assessed the rate of genetic mutation. The knockout cells had a mutation frequency more than double that of normal cells, however, there was no effect on the types of mutation that occurred. –Knock out and normal cells were then exposed to UV light and suffered the same amount of damage. However, those with HMGB1 had two to three times the repair as those without. Evidence suggests that HMGB1 works by summoning other DNA repair factors to the damaged site, Vasquez said.”

Going back to the issue discussed above of DNA unwrapping/wrapping in repairing single-strand breaks, HMGB1 apparently plays an important role in that process.  “Lange and Vasquez hypothesize that HMGB1 normally binds to the entrance and exit of DNA nucleosomes, so is nearby when DNA damage occurs. It then binds to and bends the damaged site at a 90-degree angle, a distortion that may help DNA repair factors recognize and repair the damage. After repair it facilitates restructuring of the chromatin(ref).”

“Pinpointing HMGB1’s role in repair raises a fundamental question about drugs under development to block the protein, Vasquez said.” “Our findings suggest that depleting this protein may leave patients more vulnerable to developing cancer.”  I wonder if this view is giving any pause to those who are out  pushing the development of HMGB1 inhibitors as drug candidates?  I think it should.

I expect I will be coming back to DNA repair yet-again before too long.

Up until a couple of months ago, the answer seemed very clear to me.  Resveratrol offers a number of powerful health-promoting effects.  Also, it turns on the SIRT1 gene activating the same evolutionary-conserved pathway that is known to confer longevity in case of calorie restriction.  But a pair of recent publications cast doubt on this picture.  This blog entry reviews research on the beneficial health effects of resveratrol and whether or not it indeed activates the SIRT1 gene.  I also touch on other substances being developed to turn on the SIRT1 gene and how certain big pharma and biotechcompanies appear to be lining up taking conflicting viewpoints.  Large stakes are involved.   

Resveratrol  (trans-resveratrol, 3,4′,5-trihydroxystilbene) is a substance occurring naturally in several plants in response to stress, attack by pathogens such as bacteria or fungi, or ultraviolet  radiation.  Discovered to be in red wine in 1992, the substance has been extensively studied for its medicinal and possible anti-aging properties.  The resveratrol site of the Linus Pauling Institute, is a good source of information on all aspects of the substance with 109 literature citations but appears not to be up-to-date since no citations subsequent to 2006 are listed. The Wikipedia article on resveratrol is also a good general information source but appears also not to be completely current. 

Bioavailability of resveratrol is low because it is rapidly metabolized and eliminated.  “–much of the basic research on resveratrol has been conducted in cultured cells exposed to unmetabolized resveratrol at concentrations that are often 10-100 times greater than peak concentrations observed in human plasma after oral consumption (ref)(ref)(ref).”   Therefore, conclusions of such studies may not be applicable even for people who take large amounts of supplementary resveratrol. New micronized forms of resveratrol and sublingual tablets may increase bioavailability(ref)(ref).  Resveratrol appears to exercise few side effects even at large dose levels(ref)(ref).  Since effects may vary widely by individual depending on genetic makeup, however, it is appropriate to exercise caution with large doses. 

Example health benefits of resveratrol 

·        Studies have included looking at resveratrol’s actions in leukemia and in colon, stomach, pancreatic, esophageal, intestinal, and breast cancers, both from preventative and therapeutic viewpoints.  “Although resveratrol can inhibit the growth of cancer cells in culture and in some animal models, it is not known whether high intakes of resveratrol can prevent cancer in humans(ref).” See the discussion and citations here. Multiple forms of biological activity against cancers can be involved. For example, resveratrol might prevent cancer by inhibiting  the expression of certain cytochrome p450 enzymes(ref).   To find recent and current publications related to resveratrol and cancer, I suggest doing a search on “cancer resveratrol” in PubMed.org.

·        “Resveratrol has been found to exert a number of potentially cardioprotective effects in vitro, including inhibition of platelet aggregation (47, 48, 68), promotion of vasodilation by enhancing the production of NO (46, 69) and inhibition of inflammatory enzymes (34, 70, 71). However, the concentrations of resveratrol required to produce these effects are often higher than those that have been measured in human plasma after oral consumption of resveratrol (7)(ref).”  There is an impressive number of current and recent publications related to resveratrol and cardiovascular issues as can be found by doing a search on “cardiovascular resveratrol” in PubMed.org. 

·        Numerous studies indicate that resveratrol might be be useful in control of obesity and diabetes.  A March 2010 publication Resveratrol, obesity and diabetes states “It is well established that resveratrol exerts beneficial effects in rodents fed a high-calorie diet. In some studies, resveratrol was reported to reduce body weight and adiposity in obese animals. The action of this compound involves favourable changes in gene expressions and in enzyme activities. The accumulating evidence also indicates the benefits of resveratrol in diabetes and diabetic complications. It is known that resveratrol affects insulin secretion and blood insulin concentration. In animals with hyperinsulinemia, resveratrol was found to reduce blood insulin. Moreover, numerous data indicate that in diabetic rats, resveratrol is able to reduce hyperglycemia.”

·        “In November 2008, researchers at the Weill Medical College of Cornell University reported that dietary supplementation with resveratrol significantly reduced plaque formation in animal brains, a component of Alzheimer and other Neurodegenerative diseases.^[23] In mice, oral resveratrol produced large reductions in brain plaque in the hypothalamus (-90%), striatum (-89%), and medial cortex (-48%) sections of the brain. In humans it is theorized that oral doses of resveratrol may reduce beta amyloid plaque associated with aging changes in the brain(ref).”

·        Re. resveratrol and bone loss see these articles.  Re. resveratrol and prostate function see these articles.  Re. resveratrol and stroke see these articles.  Re. resveratrol and cataracts see these articles.  Re resveratrol and its anti-viral activities see these articles. 

The main messages up to this point are: 

·        Resveratrol appears to offer a wide range of potential health benefits based on biochemical, cell-level and in some cases, small-animal studies.  Except for bioavailability, it appears to be an amazing supplement.

·        It is unclear as to whether taking the usual doses of commercial resveratrol supplements lead to any of these benefits, however, first because of absence of any clinical studies on humans, and second because of resveratrol’s low bioavailability profile. 

Resveratrol activating SIRT1 

SIRT1, you will recall is the evolutionary-conserved gene activated by calorie restriction that, when activated, provides health and life extension in a number of lower species.  The SIRT1 protein is the most interesting member of the sirtuins, protein deacetylases. I have discussed aspects of SIRT1 in several blog posts and cited this current review of 10 years of research in sirtuins by Leonard Guarante and a colleague. 

In the course of these discussions, I have in keeping with the literature repeatedly asserted that resveratrol activates SIRT1.  I have also provided multiple literature citations to back up this assertion.   The blog post SIRT1, mTOR, NF-kappaB and resveratrol and this 2010 publication, for example, indicates how resveratrol inhibits mTOR signaling while activating SIRT1 showing a linkup between the mTOR “shortivity” pathway and the SIRT1 “longevity” pathway.  This article suggests a linkup between TXNIP, another “shortivity” pathway,  and the SIRT1 “longevity” pathway.   

I repeat a couple of paragraphs here from my blog post Visit with Leonard Guarante which are directly relevant to the present discussion.  “The work of Guarante and his colleagues has led to the identification of  resveratrol as an activator of SIRT1 and later to the establishment of Sirtris Pharmaceuticals, a company devoted to the discovery of small-molecule activators of sirtuins that could address diseases of aging.   Reported in the Sirtris web site, “A long-term study of middle-aged mice shows resveratrol improves health and mimics some benefits of dietary restriction(ref).”

“David Sinclair, a key player in sirtuins research at Harvard and founder of Sirtris, originally came from Australia to work at MIT in Guarante’s lab.   Leonard is on the Board of Sirtris which has been acquired by GlaxoSmith Kline (GSK).  The 2007 publication Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes by Sinclair and other Sirtris-affiliated  authors is one of a number of publications relating SIRT1 to type 2 diabetes.  “Resveratrol, a polyphenolic SIRT1 activator, mimics the anti-ageing effects of calorie restriction in lower organisms and in mice fed a high-fat diet ameliorates insulin resistance, increases mitochondrial content, and prolongs survival^10–14   Here we describe the identification and characterization of small molecule activators of SIRT1 that are structurally unrelated to, and 1,000-fold more potent than, resveratrol. These compounds bind to the SIRT1 enzyme—peptide substrate complex at an allosteric site amino-terminal to the catalytic domain and lower the Michaelis constant for acetylated substrates. In diet-induced obese and genetically obese mice, these compounds improve insulin sensitivity, lower plasma glucose, and increase mitochondrial capacity. In Zucker fa/fa rats, hyperinsulinaemic-euglycaemic clamp studies demonstrate that SIRT1 activators improve whole-body glucose homeostasis and insulin sensitivity in adipose tissue, skeletal muscle and liver. Thus, SIRT1 activation is a promising new therapeutic approach for treating diseases of ageing such as type 2 diabetes(ref).” 

Sirtris currently has four SIRT1 activator substances in Phase IIa clinical trials(ref).  Phase I safety and dose trials of SRT2104 are complete.  The substances are identified as SRT2104, SRT2379 and SRT501.  Trials relate to metabolic disease (Type 2 Diabetes), inflammation, cardiovascular disease and oncology.”

The April 2009 paper Sirtuin activators summarized the most-accepted view of the situation at the time of its publication “CONCLUSIONS: To date, resveratrol is the most potent natural compound able to activate SIRT1, mimicking the positive effect of calorie restriction. Resveratrol might help in the treatment or prevention of obesity and in preventing the aging-related decline in heart function and neuronal loss. As resveratrol has low bioavailability and interacts with multiple molecular targets, the development of new molecules with better bioavailability and targeting sirtuin at lower concentrations is a promising field of the medicinal chemistry. New SIRT1 activators that are up to 1000 times more effective than resveratrol have recently been identified. These improve the response to insulin and increase the number and activity of mitochondria in obese mice. Human trials with a formulation of resveratrol with improved bioavailability and with a synthetic SIRT1 activator are in progress.”  The new “1000 times more effective” SIRT1 activators referred to here are the molecules developed by and being tested by Sirtris.As written in a February 2010 Special Report in Gen Sirtuins: Antiaging Medicines or Marketing? – “A large body of basic research does indeed support the nomination of SIRT1 activators as antiaging drugs. SIRT1 activation has profound metabolic effects: It regulates glucose or lipid metabolism through its deacetylase activity for over two dozen known substrates and has a positive role in the metabolic pathway through its direct or indirect involvement in insulin signaling. It also stimulates glucose-dependent insulin secretion from pancreatic β cells and directly stimulates insulin-signaling pathways in insulin-sensitive organs. SIRT1 also reportedly influences adiponectin secretion, inflammatory responses, gluconeogenesis, and levels of reactive oxygen species, which together contribute to the development of insulin resistance.” 

The dissenting publications 

Two dissenting publications appeared recently, ones that challenge whether resveratrol or any of the Sirtris small-molecule activators actually activate SIR1 Also indirectly challenged is whether Sirtris has a sound platform of drug candidates with which to succeed.   

The October 2009 paper Resveratrol is Not a Direct Activator of SIRT1 Enzyme Activity in the journal Chemical Biology and Drug Design  is by a group of authors associated with Amgen Inc.  The researchers claimed that the activation of SIRT1 observed by Sinclair and his colleagues and reported in the publication Small molecule activators of SIRT1 as therapeutics for the treatment of type 2 diabetes was not real but was an artifact of the use of a high-throughput in vitro fluorescence polarization assay.  The Amgen authors write “Here, we show that: (i) the Fluor de Lys-SIRT1 peptide is an artificial SIRT1 substrate because in the absence of the covalently linked fluorophore the peptide itself is not a substrate of the enzyme, (ii) resveratrol does not activate SIRT1 in vitro in the presence of either a p53-derived peptide substrate or acetylated PGC-1alpha isolated from cells, and (iii) although SIRT1 deacetylates PGC-1alpha in both in vitro and cell-based assays, resveratrol did not activate SIRT1 under these conditions.” 

The second dissenting paper SRT1720, SRT2183, SRT1460, AND RESVERATROL ARE NOT DIRECT ACTIVATORS OF SIRT1 was published in January 2010 in the Journal of Biological Chemistry.  Several of the co-authors are associated with Pfizer.  SRT1720, SRT2183 and SRT1460 were earlier reported by Sirtris to be SIRT1 activators.  This is a highly technical paper where the researchers developed their own assay approach.  “These data demonstrate that neither the Sirtris series nor resveratrol would serve as useful  pharmacological tools due to their highly promiscuous profiles. – Our results show that the Sirtris series of compounds and resveratrol have little or no effect on SIRT1 activity even with these two full length protein substrates. — In summary, our detailed assessment of the Sirtris series and resveratrol involving several biochemical assays with native substrates and biophysical studies employing NMR, SPR, and ITC demonstrate that these compounds are not direct SIRT1 activators. We also demonstrated that SRT1720 does not show beneficial effects in a rodent diabetes model, which is in contrast to that previously reported (26). The broad selectivity assessment against over 100 targets including receptors, enzymes, ion channels, and transporters show that the Sirtris series and resveratrol are highly promiscuous and would not serve as useful pharmacological tools for studying SIRT1 pathways. In the literature, resveratrol has been widely referred to as a “SIRT1 activator” (For selected recent references, see 40-44) and routinely used to “activate” SIRT1 in variouscellular assays, with only a few questioning the original study that reported its ability to activate SIRT1 in an artificial substrate-based fluorescent assay (28,29,39,45). Likewise, the Sirtris compounds have been referred to as “SIRT1 activators” in recent publications (46-48). Our present data are significant for the field as we provided strong evidence that neither the Sirtris series nor resveratrol are direct SIRT1 activators.” 

Where am I in all this? 

First of all, I am skeptical of the skeptics.  This paper by the Pfizer people appears to condemn resveratrol along with the Sirtris small-molecule compounds for two reasons: 1.  It does not really directly activate SIRT1, and 2.  It is biochemically highly “promiscuous,”  making it a kind of biochemical whore.  Even if point 1. Were correct, what about the very large number of publications (hundreds) from researchers around the world written over nearly two decades documenting the positive health benefits of resveratrol quite independently of whether it has any SIRT1 action?  Did all of those researchers miss seeing or overlook the promiscuity?  Were they carried away by resveratrol’s sexiness?  Perhaps, but I have trouble believing that.   And, if resveratrol were indeed so promiscuous, why does it seem to have so few negative side effects and so many positive effects?   

And as far as resveratrol and SIRT1 are concerned, I would ask the question “If resveratrol does not activate SIRT1, then why does it seem to provide so many of the same health benefits that would be expected from activating SIRT1?” 

Second, the stakes involved are big.  If the Sirtris small molecules turn out to be worthless, then just what will have GSK gotten for the $720 million they paid for Sirtris?  If the original studies done at MIT and Harvard and Sirtris on SIRT1 activation are based on faulty science, what will be the result on the reputations of the key scientists and laboratories involved?  

Third, the results of the Sirtris clinical trials should tell a lot.  If one of the substances turns out to be a blockbuster, this should send the SIRT1 activator skeptics scampering.  If all the Sirtris substances fizzle, then perhaps the rival skeptics are in part right.  But even if all the proprietary Sirtris SIRT1 activators fizzle as drugs, that still won’t explain all the positive research results on health benefits of good old resveratrol. 

Finally, being just an individual, I have no personal desire to get anywhere near the middle of a knife fight between pharmaceutical and biotech giants like Pfizer, GSK and Amgen and research institutions like Harvard and MIT. 

Please see the medical disclaimer for this blog.

Astragalus-based dietary supplements that are known to activate the expression of telomerase have been on the market for several years now.  However, there appears to be a significant difference between what these supplements are widely publicized to do and what published scientific research says they actually do.  Specifically, the promotion and press coverage often implies that such supplements will extend the lengths of telomeres in people who take them and thus confer longevity benefits.  However, there appears to be virtually no clinical research evidence to support such claims.  On the other hand, research does suggest that at least one of the supplements can provide several important health benefits.  In this blog post I seek to penetrate through the thick layers of commercial and PR fog about such supplements and get down to what is actually known about their actions. 

History 

During the 1990s and early 2000s, Geron, a small biotech company, was a leader in research relating to telomeres and telomerase.  Few scientists and no other significant biotech or pharmaceutical company paid much attention to telomeres or telomerase back then.  Based on its research, Geron applied for 279 patents related to telomeres or telomerase.  One of the major areas of research concern to Geron then was telomere activation as an approach to disease prevention and longevity.  The company discovered that certain extracts of the astragalus plant had a capability to activate the expression of telomerase in certain cell types, at least under test-tube conditions.   

From the Geron web site: “Geron, in collaboration with the Biotechnology Research Institute (BRC), a company established by the Hong Kong University of Science and Technology (HKUST), began screening for telomerase activators in early 2000. The source of material for the screen was natural product extracts from traditional Chinese medicines. In the course of the screening, several extracts were discovered that reproducibly up-regulated the low, basal level of telomerase in human skin cells. With analysis of the extract and further testing, one compound in the extracts was identified as a key telomerase activator. It was capable of activating telomerase in other human cells types (e.g., lymphocyte immune cells) at very low concentrations. Another compound, a derivative of the first, was also present in the extract but at lower concentrations and was also found to possess similar telomerase activating properties. These molecules are currently under development for the treatment of degenerative diseases. Other small molecule activators discovered during the course of the research may also be developed for certain disease indications.”

The research resulted in Geron applying for a patent on telomerase activators which was finally issued just a few months ago.  Filed 06/23/2004, the patent is called Compositions and Methods for Increasing Telomerase Activity.  Since publication 05/15/2008, the patent application can be read by anyone and the descriptions found there still provide much  of the scientific rationale for people taking astragalus-based telomerase-activator supplements.  The patent application introduction states “The present invention relates to methods and compositions for increasing telomerase activity in cells. Such compositions include pharmaceutical, including topical, and nutraceutical formulations. The methods and compositions are useful for treating diseases subject to treatment by an increase in telomerase activity in cells or tissue of a patient, such as, for example, HIV infection, various degenerative diseases, and acute or chronic skin aliments. They are also useful for enhancing replicative capacity of cells in culture, as in ex vivo cell therapy and proliferation of stem cells.” 

Geron subsequently shifted its focus to other areas of research including embryonic stem cell therapies and developing drugs that turn telomerase off in cancer cells.  As far as I can tell, Geron is currently pursuing telomere activation mainly via a subsidiary and marketing licensing agreements.  Geron is the majority owner of TA Therapeutics, a Hong Kong subsidiary which is focusing on telomerase activation for organ renewal and prolonging the lives of AIDS patients.  A US company, TA Sciences, has licensed one telomerase-activator extract from Geron called TA-65 in 2002, a nutraceutical it has been marketing it to the public for over three years now. 

Of the Geron-researched telomerase-activating products, two in particular have received the most attention: TA-65 being marketed to the public by TA Sciences and TAT2 under investigation as part of drug development by TA Therapeutics.  Both formulations are carefully guarded proprietary secrets of the companies involved.  I suspect the two substances are either highly related or identical.  There has been much speculation as to what TA-65 consists of, particularly in online longevity-related forums(ref)(ref).  Based on reading the Geron patent, it appears that a number of astragalus membranaceus extracts exhibit varying degrees of capability to promote the expression of telomerase(ref).  One extract mentioned in the patent is astragaloside IV, and another extract with roughly ten times the activation potency is cycloastragenol, and there are others as well.  Based on careful reading of the patent and the dosage originally suggested by TA sciences the best informed guess is that TA-65 and TAT2 are cycloastragenol, but this is only a guess.   

TA Sciences is an active marketing company and any search on Google related to telomerase will often produce prominent advertising related to TA-65 and its health and longevity benefits.  TA-65 does not come cheap.  When the company first started marketing it, it was available only as part of a “Patton Protocol” package with cost of $25,000 for the first year.  (Noel Patton is the founder of TA Sciences.)  Now, the Patton Protocol is offered in either an a-la-cart mode or as a full package.  The cost of six months of the protocol including the TA-65, some other supplements, a visit to a doctor and a number of diagnostic tests is $6,725.  Cost of a six-month supply of TA-65 alone is $4,000.  It is interesting that when it was originally marketed the daily dosage of TA-65 was 5 mg and the daily dosage has been increased now to 100mg, by a factor of 20.  This has led to speculation that the substance may not be pure cycloastragenol which is very expensive to produce. 

Besides TA-65 available from TA sciences, based on the information in the Geron patent other companies have started to market both astragaloside IV and cycloastragenol as telomerase activator supplements(ref)(rev).  These supplements are being sold considerably cheaper than TA-65 with cost of a 30-day supply typically running up to $80.  One such company, Revgenetics, decided to discontinue its cycloastragenol product line when the Geron patent was finally issued and sell of its existing stock at discount, charging $25 for a bottle which contains 30 5mg pills. 

The concept of telomerase activation 

A responsible formulation of the telomerase activation hypothesis is that through systemic intermittent activation of telomerase, specifically in stem and progenitor cells, it may be possible to delay shortening of telomeres and therefore delay the onset of multiple disease and degenerative processes associated with cell senescence.  A very informative 2007 PowerPoint Presentation by Joseph M. Raffaele MD (an affiliate of TA Sciences) states the scientific rationale for telomerase activation and lays out results of a small clinical trial of TA-65.  There is general consensus that too-short telomeres lead to cell senescence leading to the diseases and symptoms of aging.  However, it must be pointed out that many factors affect telomere length, that many complex factors both known and yet-unknown promote or delay the onset of cell senescence, and that telomerase activation does more than affect the lengths of telomeres.  For example, the protein TAp63 strongly affects senescence of stem cells(ref).  I return to this important point later. 

Research on telomerase activators 

So, what research exists on the effects of telomerase activators beyond that which went into the patent?  I will review research here that involves any of the four activator substances mentioned (TA-65, TAT2, Astragaloside IV, Cycloastragenol) recognizing that what is true for one activator may not be valid for another.  With one exception, I will confine myself to publications in established journals or reputable online research publishers and will avoid ungrounded assertions in press releases or opinions stated in blogs.  

·        A small human trial was conducted in 2005 of TA-41, a precursor of TA-65, I believe sponsored by TA Sciences.  This trial is described in a page on the TA Sciences web site and in the aforementioned PowerPoint Presentation.  The trial was a 24-week double-blind, placebo-controlled study involving 36 male subjects between 60 and 85 years of age, a relatively short trial with scale far smaller than typical Phase III FDA-approved trials.  TA-65 is the presumed major metabolite of TA-41. “ — subjects consumed 2 or 4 tablets daily of a placebo control substance (placebo groups) for 12 weeks or 2 or 4 tablets daily of a TA-65 precursor molecule (TA-41) for 12 weeks (product groups). The product tablets each contained 10 mg of TA-41 (an Astragalus extract) along with other botanical extracts and excipients. — The 12 week placebo or product use period was followed by a further 12 week follow-up period.”  My impression is that the experimental design of the study and the treatment of statistical measures were handled quite responsibly.  Nonetheless, because of the small sample size, statistical significance of the results is relatively crude.  The study treated .2 as the p-value for statistical significance though in larger studies .05 or even .01 are typical values.  The major benefits observed among those taking the products were “1.  Apparent improvement in certain immune system measures, 2. Apparent improvement in eye sight, 3.  Apparent improvement in certain sexual function measures, and 4. Apparent improvement in certain skin properties(ref).”  No significant adverse events were identified.  Detailed discussion and diagrams of results can be found on the TA Sciences web page for the study and in the PowerPoint presentation.  To my knowledge the results of this 2005 study have never been published in an established scientific journal.  Nonetheless the study seems to have been well done and I tend to take it seriously.  

·        Dr. Raffaele reports in his 2007 PowerPoint Presentation that “preliminary results of 16 patients o TA-65 for 3 months show an increase of mean lymphocyte telomere length.”  I have seen no further or subsequent details.

·        To my knowledge, there have been no further studies relating actual user experience of those taking TA-65 though by this time there should be considerable experience to report.  Those taking TA-65 as part of the Patton Protocol have had extensive measurements of aging-related biomarkers and their telomere lengths.  I would love to see the data derived from this user cohort laid out.

·        The 2008 study report Telomerase-based pharmacologic enhancement of antiviral function of human CD8+ T lymphocytes looked at exposing lymphocyte cells from HIV-infected donors to TAT2. “ — , during aging and chronic HIV-1 infection, there are high proportions of dysfunctional CD8(+) CTL with short telomeres, suggesting that telomerase is limiting. The present study shows that exposure of CD8(+) T lymphocytes from HIV-infected human donors to a small molecule telomerase activator (TAT2) modestly retards telomere shortening, increases proliferative potential, and, importantly, enhances cytokine/chemokine production and antiviral activity.  The enhanced antiviral effects were abrogated in the presence of a potent and specific telomerase inhibitor, suggesting that TAT2 acts primarily through telomerase activation.”  The study suggests a possible health benefit for HIV-infected individuals, individuals who experience an extraordinary high rate of telomere shortening in immune cells due to the disease.  This benefit would have to be verified in clinical tests.  This study says nothing about telomere lengthening.  This study was co-authored by Rita Effros, a leading researcher in the role of telomeres in HIV infections.  This study, by the way, referred to the experimental substance both as TAT2 and as cycloastragenol.

·        A 2005 study Telomerase Therapeutics for Degenerative Diseases describes possible benefits of telomerase activation but provides no experimental results.  There are numerous studies pointing to telomere shortening as an important process contributing to the advance of HIV and studies like this 2010 one looking at telomerase activity and replicative senescence in human CD8 T lymphocytes, but none of those studies are directly concerned with telomerase activation. 

·        The 2009 publication Cycloastragenol extends T cell proliferation by increasing telomerase activity covers another in-vitro study reporting “Naturally, there is a great deal of interest in finding inducers of telomerase that may help delay the onset of cellular aging. There are various nutraceuticals that claim to both increase the health of individuals and delay the onset of cellular aging. We tested the nutraceuticals resveratrol and cycloastragenol for their ability to enhance T cell functions in vitro. In this study we evaluated the effect of these compounds on cellular proliferative capacity, levels of telomerase activity, surface markers and cytokine secretion of human CD4 and CD8 T cells. Our results show that cycloastragenol moderately increase telomerase activity and proliferative capacity of both CD4 and CD8 T cells. These preliminary results suggest that nutraceuticals inhibit the onset of CD4 and CD8 cellular senescence.”  Like in the previously-discussed study the effect was moderate, outside the body, and the study said nothing about extending telomeres. 

Of the telomerase activators mentioned, perhaps the one best covered in the research literature is astragaloside IV.  As I state in my treatise; “Astragaloside IV has been systematically studied for its medicinal properties only recently, mostly in Chinese and European research centers. It is an antiinflammatory, antifibrotic and antioxidant. It is known to have vasodilation and cardioprotective properties. It is neuroprotective and can protect the myocardium against ischemia/reperfusion injury. There are no reported negative side effects. Yet, my impression is that much is yet to be learned about this substance. Specifically, there appears to be little if any research available in the public domain relating astragaloside IV’s medicinal properties to its ability to induce telomerase expression.”  

Research publications related to Astragaloside IV include the 2002 publication Effects of astragaloside IV on myocardial calcium transport and cardiac function in ischemic rats, the 2004 publication Astragaloside IV protects against ischemic brain injury in a murine model of transient focal ischemia, the 2009 publication Effects of Astragaloside IV on heart failure in rats, the 2009 publication Astragaloside IV attenuates cerebral ischemia–reperfusion-induced increase in permeability of the blood-brain barrier in rats, the 2008 report Astragaloside IV inhibits spontaneous synaptic transmission and synchronized Ca2+ oscillations on hippocampal neurons, the 2006 report Effects of astragaloside IV on pathogenesis of metabolic syndrome in vitro, and Effect of astragaloside IV on hepatic glucose-regulating enzymes in diabetic mice induced by a high-fat diet and streptozotocin, and the 2006 publication Astragaloside IV from Astragalus membranaceus Shows Cardioprotection during Myocardial Ischemia in vivo and in vitro.  While these and many other research publications relate to potentially beneficial effects of Astragaloside IV, none relate to or even mentions the substance’s role as a telomerase activator.  Of course, some or all of the reported benefits could ultimately be due to telomerase activation. 

Other than the study cited above, the only discussions of cycloastragenol health activities seem to be in longevity blogs chewing over the same material covered here.  It is a relatively unfamiliar substance.  As for astragaloside IV, cycloastragenol suppliers appear to be in China.  Purchasing either of these supplements from a US company, it is good to be on a lookout for independent laboratory verification of contents and purity.  

Observation 1: When it comes to telomerase activation, the contrast between what is reported as “research” in the general and commercial literature and what is reported in the filtered scientific research literature is singularly stark.  Pubmed.org is the definitive National Library of Medicine database of medical and related scientific research, containing millions of literature abstracts covering virtually every article in every research publications worldwide.  The following lists the number of items retrieved using Google and using Pubmed in response to the given query. 

Query                                      Found in Google     Found in Pubmed

TA-65 + telomerase                     21,100                7 (all irrelevant)

TAT2 + telomerase                          4,510                1 (cited here)         

astragaloside + telomerase          7,200                0

cycloastragenol + telomerase      1,820                1 (cited here) 

Observation 2: Published studies suggests that telomerase activation may have a positive effect on the immune function, though this conjecture based on lab cell-level studies must be confirmed via large-scale human studies.  How much affect using what activator and under what conditions are as yet not established.   There is also research strongly suggesting important potential health benefits from taking astragaloside IV in particular, and possibly also from taking TAT2 (likely to be the same thing).  How telomerase activation relates to the beneficial effects of these substances, however, remains mostly unstudied. 

Observation 3:  The case for specifically taking TA-65 is mainly based on propriety information provided by TA sciences and doctors offering TA-65 as a treatment, and by the original research done by Geron. The most compelling positive information is that derived from the 2005 human trial sponsored by TA Sciences.  While TA-65 has an immense standing in the popular literature I have had trouble finding any mention of it in the published scientific literature.  In fact, if a query about TA-65 is made in PubMed, part of the reply is “The following term was not found in PubMed: TA-65.”   

Observation 4:  I remind readers that there are research studies establishing that there are other interventions that result in longer telomeres besides taking the telomerase activators discussed here.  See the January 2010 blog entry Vitamins, supplements and telomerase – upregulation or downregulation?  And also see my blog entries Exercise, telomerase and telomeres, Timely telomerase tidbits, Breakthrough telomere research finding, and Telomere and telomerase writings. 

Observation 5: In the scientific literature I have found no published research whatsoever that establishes that any of the telomerase activators mentioned actually extends telomeres.  The closest the literature comes are statements like “moderately increases telomerase activity, ” “modestly retards telomere shortening,”  and “inhibit the onset of CD4 and CD8 cellular senescence.” And these statements are based on cell-level studies with results that may or may not be applicable in live humans.  It is interesting that the TA Sciences web site does not now make the claim that TA-65 actually extends telomeres.  Unfortunately, however, the claim keeps popping up in news stories and some blog postings about the activator substances. 

Observation 6:  What telomerase activators actually do in humans remains a mystery as far as the published scientific literature is concerned, and what the two proprietary activators consist of still remains a mystery as well. I keep awaiting more trustworthy published information. 

Back to the science of telomeres and telomerase 

For those familiar with the great complexities of telomere biology and pathways affecting telomere length management, it should not be surprising that is not so simple as “take a telomerase activator and get longer telomeres.”  Whether telomeres get longer or shorter or stay the same is determined not only by the presence of telomerase but also by interactions involving many signaling paths and activation cofactors.  “Telomere transcription is regulated by several mechanisms: developmental status, telomere length, cellular stress, tumour stage and chromatin structure(ref).”  Presence of a telomerase activator is only one factor in driving telomere lengths. The literature related to telomerase and telomeres is extremely extensive and I have barely touched on it here.  What is largely missing is literature specifically related to the astragalus-based telomerase activators.  

Further, telomerase has other activities besides telomere length maintenance.  Activating the TERT telomerase component may have other positive effects without making telomeres longer.  These positive effects can include promoting cellular and organismal survival(ref) and increasing the rate of differentiation of quiescent adult stem cells(ref).  So, in principle at least, a telomerase activator could convey health benefits independently of affecting telomere lengths.  We just don’t know the extent to which this happens in humans in response to the astragalus-based supplements.   

Personal experience 

My personal experience with telomerase activators may be untypical.  I started taking a large dose of astragalus extract in July 2007 with the intention of telomerase activation, switched to astragaloside IV 50mg a day in August 2008.  As of mid-December 2009, I switched to taking a 5mg cycloastragenol capsule together with a simple astragalus-extract pill which may possibly increase bioavailability.   On February 14 2010, I upped the daily cycloastragenol dose to 10mg. So I have been on some kind of telomerase activator or the other for close to three years now.  What is the effect?  I don’t know, especially because of all the other anti-aging supplements I have been taking and anti-aging lifestyle patterns I have been observing.  If I use the criteria mentioned in the TA Sciences trial, I can personally comment that now at the age of 80: 

1.      Immune system measures:  I seem to have as good or possibly better resistance to infections or viruses as ever.

2.     Eye sight: Distance vision acuity in both eyes is excellent though I require corrective lenses for reading and close-up vision.  My last eye exam showed no progression of druzen or signs of macular degeneration.

3.      Sexual function measures:   No decline in desire, perhaps even a bothersome increase.  Performance and satisfaction enhanced by sildenafil seems OK.

4.     Certain skin properties:  Quality and texture of skin for my age are excellent.

5.     Hair:  And I add one other thing I have been monitoring, which is hair.  About a year ago I wrote in my treatise “I have noticed a few small effects so far. The light patina of grey hairs on my mostly-bald scalp seems to me to be a bit thicker with a few black hairs as well. I have been nearly bald for over 30 years. It is known that in animal models at least, conditional telomerase induction causes proliferation of hair follicle stem cells (ref). It remains to be seen whether I will see more or darker hair as I continue with telomerase activation. Also there seems to be some increase in my sexual libido but this may be a subjective impression. I also do not know if my daily schedule of alternating taking the telomerase activator with the other supplements with a few hours of separation is effective or whether I would be better off alternating every other day or even every other week.”  Since then there are many more grey hairs but not black ones.  I am no longer absolutely bald.  The grey hairs seem to keep coming back but very slowly.   

I am planning to stay with the cycloastragenol supplements until my supply runs out in 3 months or so.  I am not sure what I am going to do for telomerase activation after then. I keep waiting for the “shoe to drop” with more definitive research results becoming available as I have been waiting for three years now.  I think it is ridiculous that we are still relying on 2005 test data from 36 people who were on an activator for only two weeks when now many people have been on such activators for three years or more and have been subjected to systematic age-biomarker and telomere length testing.

I have reported on Induced pluripotent stem cells (iPSCs) in my treatise and in numerous past blog entries(ref)(ref)(ref).  I have viewed these cells as probably providing  the golden keys to closing the loop on the stem cell supply chain allowing extension of human longevity(ref)(ref)(ref).  iPS cells can be created by forcing expression of certain genes in normal somatic (body) cells taken from an individual, like skin cells.  According to most of the literature until recently such cells are pluripotent  like human embryonic stem cells (hESCs), and can be induced to differentiate into any cell type.  They seemed to offer a better alternative for stem cell therapies than use of hESCs because they are autologous, i.e. derived from the same individual they are used on and therefore not subject to immune system rejection.  

As more research on iPSCs is being done, however, evidence has been building that the iPSCs that have been created so far are second-rate in a couple of key respects and so-far unfit for use in human therapies. 

The February 2010 study Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency looks at the differentiation of iPSCs into neural cells.   “For the promise of human induced pluripotent stem cells (iPSCs) to be realized, it is necessary to ask if and how efficiently they may be differentiated to functional cells of various lineages. Here, we have directly compared the neural-differentiation capacity of human iPSCs and embryonic stem cells (ESCs). We have shown that human iPSCs use the same transcriptional network to generate neuroepithelia and functionally appropriate neuronal types over the same developmental time course as hESCs in response to the same set of morphogens; however, they do it with significantly reduced efficiency and increased variability. These results were consistent across iPSC lines and independent of the set of reprogramming transgenes used to derive iPSCs as well as the presence or absence of reprogramming transgenes in iPSCs. These findings, which show a need for improving differentiation potency of iPSCs, suggest the possibility of employing human iPSCs in pathological studies, therapeutic screening, and autologous cell transplantation.”  The study was authored by Su-Chun Zhang and colleagues.  Su-Chun Zhang has published previous studies related to creating neural cells from stem cells such as the 2009 article Differentiation of spinal motor neurons from pluripotent human stem cells.   

I do not think these findings are very surprising since iPSCs are reverted from mature cells and may have short telomeres.  In other words, though newly-created iPSC cells can differentiate into any cell type like embryonic stem cells can, unlike embryonic stem cells the iPSCs made so far are mostly old and tired cells from the viewpoint of replicative potential.  I covered this point in the blog entry Telomeres and telomerase in Induced Pluripotent stem cells – not what we thought.  That blog entry cites the March 2010 publication Spontaneous reversal of the developmental aging of normal human cells following transcriptional reprogramming.  That new study contradicts what had been the prevalent assumption that reverting a cell to iPSC status restores the expression of telomerase and telomere length to equivalent embryonic stem cell length.  This study asserts that reverting a mature cell to iPSC status does not automatically restore its telomere lengths to those found in hESCs.  “Conclusion: Prematurely aged (shortened) telomeres appears to be a common feature of iPS cells created by current pluripotency protocols. However, the spontaneous appearance of lines that express sufficient telomerase activity to extend telomere length may allow the reversal of developmental aging in human cells for use in regenerative medicine.”   

Put differently, the March 2010 study says restoring a cell to pluripotency and providing the cell with youth are two quite different things and the former does not necessarily imply the latter.  Pluripotency of an iPSC implies that, like an embryonic stem cell, the iPSC can differentiate into any somatic cell type.  Absence of youth in this case means that the restored iPSC cell could probably not lead to many generations of descendents like an embryonic stem cell could,  because its telomere lengths are short like those in old cells.   In fact, iPSC cells can be near senescent.  The February 2010 Su-Chun Zhang article does not link the observed variability and inefficiency of iPSC differentiation for generating neural cells to short telomeres, but it seems to me that the association may be a key one.   

This question of telomere lengths of iPSCs, nonetheless, appears to be somewhat controversial.  The above-cited publication directly contradicts assertions in a number of earlier publications including the 2009 publication Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells.  That report asserted “We show here that telomeres are elongated in iPS cells compared to the parental differentiated cells both when using four (Oct3/4, Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming factors and both from young and aged individuals. We demonstrate genetically that, during reprogramming, telomere elongation is usually mediated by telomerase and that iPS telomeres acquire the epigenetic marks of ES cells, including a low density of trimethylated histones H3K9 and H4K20 and increased abundance of telomere transcripts.” 

It seems to me that it is essential to develop reliable techniques for creating iPSCs with long telomeres, perhaps by selecting mutant lines that naturally express telomerase.  That may go a long ways to correcting the problems of lack of differentiation capability and wide variability noted by Su-Chun Zhang and his colleagues.  Or, it might be necessary to address additional issues to get iPSCs to behave as well as hESCs.

At least three approaches to human longevity that have been discussed multiple times in this blog appear to be in trouble or the subject of controversy between groups of scientists: 1.  generation and use of effective and reliable induced pluripotent stem cells, 2. human telomere extension via telomerase activators, and 3. use of resveratrol and its homologs to activate SIRT1 and therefore provide health and longevity.  At best, each of these approaches faces unforeseen hurdles; at worst some of them may simply not be working out.  While I have liked these approaches and written about each from a positive viewpoint, there is a problematic or controversial side to each which I will cover in three different blog entries following this one.

How do we know if something we read about longevity is so?  This post is stimulated by the previous blog entry Another piece of DAF-16 research.  I list a few comments having to do with the creditability of longevity research reports in the general press and then comment on creditability of publications in the scientific literature: 

News reports in the popular press 

·        When the press reports discovery of a new “longevity gene,” don’t believe it until you check it out.  As in the case described in Another piece of DAF-16 research, what is being reported could just be a new study shedding incremental knowledge on a long-known and well-studied gene.

·        The same is true of other reports in the popular press relating to “longevity breakthroughs.”  In recent months, I have come across literally hundreds of articles with titles like “new fountain- of-youth substance discovered” that relate to telomerase.  Most report on minor incremental pieces of research.  Few point out that telomerase was discovered in 1985(ref) and has been intensely studied since. 

·        Recognize that most such reports are written by intelligent well-meaning writers who do not have in depth scientific backgrounds.  Typically, they will pick up on a press report issued by a university or a literature abstract that describes current research but provides no background. 

Reports in the published scientific literature 

·        Most basic discoveries relating to health or longevity are not one-time events but are reported in a developing stream of studies by different researchers located in different centers over a period of years.  Important results are invariably confirmed in multiple publications and the stream becomes a little river.

·        Once in a while, an important concept may not be picked up for years.  For example, the telomere shortening hypothesis and the possibility of telomerase were first proposed by Alexey Olovnikov in 1973(ref), but little attention was paid to it until the 1990s.  Eventually, telomerase was rediscovered and explained by Greider, Blackburn and Stoszak in 1985 and became part of a current rapidly-running river of research.

·        I tend not to trust therapies or health or longevity substances researched, developed, publicized and sold by a lone practitioner or single company, especially if they are based on a “scientific secret” or proprietary non-published data.

·        Once in a while some reported results are controversial with different researchers publishing contradictory results.  In such cases my commitment is to keep digging deeper. If I cannot come up with a conclusion, I will say so.

·        Scientific truth are not absolute, They are relative to the culture and knowledge of the time.  So, even streams of seemingly consistent reports can turn out to be incorrect.  For many decades, based on reputable published studies the medical establishment sternly warned people of the possible toxic consequences of taking more than 400IU of vitamin D a day.  Nowadays the medical establishment has finally acknowledged the important benefits of vitamin D and doses of 2,000IU per day and up are routinely recommended. 

·        In particular, care has to be paid to “what is so” when very large amounts of money are involved as in the case of blockbuster drugs.  Many lucrative money-maker drugs have remained on the market for years before being pulled off for safety or lack-of-efficacy reasons.  See the list here.  In the case of almost all drugs eventually pulled from the market, there were positive previously-published research results, in many cases written by consultant medical people quietly paid off by the drug companies involved(ref)(ref).  Reporting of research paid for by pharmaceutical companies tends to be systematically biased towards favoring the sponsor’s products(ref). “Conclusion Systematic bias favours products which are made by the company funding the research. Explanations include the selection of an inappropriate comparator to the product being investigated and publication bias.” 

It is often very difficult to spot biased research publications ghost-written by doctors paid off by pharmaceutical companies.  When I see a highly positive research review of a new drug, one of my initial reactions is to be a little suspicious.   In writing this blog I try to keep my reporting as unbiased as possible citing multiple sources over long time frames and avoiding reporting where there is a strong commercial interest involved coupled with scant knowledge.  But I am not sure I have always been successful.  For a period, for example, I was possibly too captivated by the idea of telomere extension via astragaloside IV or cycloastragenol as a key anti-aging technology.  This morning, a reader raised a question in a blog comment: do resveratrol and resveratrol homologs really activate SIRT1 or is that mostly hype promoted by Sirtris Pharmaceuticals and the scientists who made many millions of dollars from the acquisition of Sirtris by GlaxoSmithKline(ref)?  This is an issue I will be looking into. 

Some of the popular media touted new April 2010 research as heralding the discovery of a new gene(e.g., AOL news(ref)), but what the new piece of research does is only add another perspective to a long-developing story on how the DAF-16 gene contributes to longevity in nematode worms, and possibly – only possibly – in higher organisms like us. 

I have mentioned the DAF-16 gene in previous blog entries.  See FOXO genes and protecting stem cells — What does resveratrol do?, Calorie restriction research roundup – Part II, and my comment MicroRNAs in cancers and aging, and back-to-the-nematode.  It is an evolutionary-conserved gene belonging to what is called the forkhead family involved in the insulin/IGF-1 pathway and the response to calorie restriction and possibly to taking resveratrol.  Most of the research on DAF-16 the gene has been on members of the Caenorhabditis  family of nematodes, but since the related pathways seem to be evolutionary-conserved, have been thought possibly to apply to us as well. 

Background

For over 22 years it has been known that mutations in certain genes can drastically extend the lives of nematodes.  For example, the 1988 publication A mutation in the age-1 gene in Caenorhabditis elegans lengthens life and reduces hermaphrodite fertility reports “age-1(hx546) is a recessive mutant allele in Caenorhabditis elegans that results in an increase in mean life span averaging 40% and in maximal life span averaging 60% at 20 degrees; at 25 degrees age-1(hx546) averages a 65% increase in mean life span (25.3 days vs. 15.0 days) and a 110% increase in maximum life span (46.2 days vs. 22.0 days for wild-type hermaphrodites).”  It was known back then that the IGF1 insulin-like signaling pathway was involved and that two other key genes involved on both longevity and reproduction of caenorhabditis elegans were DAF-2 and DAF-16(ref).  So, forget the idea that DAF-16 is “a newly discovered gene.”

Identification of the potential life-expanding role of DAF-16 in C. elegans is evidenced in the 1993 paper A C. elegans mutant that lives twice as long as wild type: “We have found that mutations in the gene daf-2 can cause fertile, active, adult Caenorhabditis elegans hermaphrodites to live more than twice as long as wild type. This lifespan extension, the largest yet reported in any organism, requires the activity of a second gene, daf-16. — daf-2 and daf-16 provide entry points into understanding how lifespan can be extended.” Also, DAF-16 is discussed in the 1997 paper The Fork head transcription factor DAF-16 transduces insulin-like metabolic and longevity signals in C. elegans.   The 2006 review article Worming pathways to and from DAF-16/FOXO summarizes the knowledge as of that time: “Caenorhabditis elegans, the insulin/IGF-1 signaling pathway controls many biological processes such as life span, fat storage, dauer diapause, reproduction and stress response . This pathway is comprised of many genes including the insulin/IGF-1 receptor (DAF-2) that signals through a conserved PI 3-kinase/AKT pathway and ultimately down-regulates DAF-16, a forkhead transcription factor (FOXO). DAF-16 also receives input from several other pathways that regulate life span such as the germline and the JNK pathway –. JNK regulates lifespan in by modulating nuclear translocation of forkhead transcription factor/DAF-16. Therefore, DAF-16 integrates signals from multiple pathways and regulates its downstream target genes to control diverse processes. Here, we discuss the signals to and from DAF-16, with a focus on life span regulation.” So, DAF-16 has long been known to be involved in lifespan regulation in caenorhabditis elegans. 

The new research 

The this-week publication of interest is Phenotypic Covariance of Longevity, Immunity and Stress Resistance in the Caenorhabditis Nematodes.  Basically, the research looked at four different members of the Caenorhabditis family of nematodes that have widely different levels of expression of DAF-16 and widely different disease resistance and longevities.  Higher levels of expression of DAF-16 were found to be correlated with increased disease resistance and longevity. “It is known that lifespan varies significantly among the Caenorhabditis species but, although DAF-16 signaling is highly conserved, it is unclear whether this phenotypic linkage occurs in other species. Here we investigate this phenotypic covariance by comparing longevity, stress resistance and immunity in four Caenorhabditis species.  — We show using phenotypic analysis of DAF-16 influenced phenotypes that among four closely related Caenorhabditis nematodes, the gonochoristic species (Caenorhabditis remanei and Caenorhabditis brenneri) have diverged significantly with a longer lifespan, improved stress resistance and higher immunity than the hermaphroditic species (C. elegans and Caenorhabditis briggsae). Interestingly, we also observe significant differences in expression levels between the daf-16 homologues in these species using Real-Time PCR, which positively correlate with the observed phenotypes. Finally, we provide additional evidence in support of a role for DAF-16 in regulating phenotypic coupling by using a combination of wildtype isolates, constitutively active daf-16 mutants and bioinformatic analysis.”

Going on, “The gonochoristic species display a significantly longer lifespan (p<0.0001) and more robust immune and stress response (p<0.0001, thermal stress; p<0.01, heavy metal stress; p<0.0001, pathogenic stress) than the hermaphroditic species. Our data suggests that divergence in DAF-16 mediated phenotypes may underlie many of the differences observed between these four species of Caenorhabditis nematodes. These findings are further supported by the correlative higher daf-16 expression levels among the gonochoristic species and significantly higher lifespan, immunity and stress tolerance in the constitutively active daf-16 hermaphroditic mutants(ref).”

So, an interspecies comparison among closely related nematodes establishes a correlation between expression level of DAF-16 and disease resistance/lifespan.  It was already known that “In the nematode Caenorhabditis elegans, these phenotypes are molecularly linked such that activation of the forkhead transcription factor DAF-16 both extends lifespan and simultaneously increases immunity and stress resistance(ref).”  The new research is interesting but, given what was already known, hardly earth shaking or even surprising. 

Implications for humans of the new research are not completely clear.  It appears that “sir-2.1 and daf-16 have both overlapping and distinct functions in regulation of C. elegans life span(ref).” But I have had difficulty finding any research directly related to DAF-16 in mice or rats, let alone in humans.  Will resveratrol activate DAF-16 in humans?  Probably not.  A 2008 publication reports “Treatment of C. elegans with the small molecule resveratrol, however, extends life span in a manner fully dependent upon sir-2.1, but independent of daf–16.”  So, it looks like it is going to be a while before implications in humans are known.  First, we will have to hear about implications in mice.

This blog entry is intended to clarify what aging, life extension and anti-aging are about.  It suggests a new way of looking at the theories of aging in my treatise ANTI-AGING FIREWALLS THE SCIENCE AND TECHNOLOGY OF LONGEVITY.

It is easiest to start out with distinguishing between life extension and postponing aging (or anti-aging), which are not quite the same things.  Aging is the most difficult of these matters to define and I will get to it a bit later.

First of all, life-extension approaches are not sure things; they negatively affect the probability of death of a member of a defined population.  Even if you could halt and reverse all causes of aging, the next day you could get run over and killed by a bus.  A lot of things will extend the average lifespan of a population, including quality sewage systems, unpolluted air, and living where there is a law-abiding citizenry.  Included in this list can be pursuance of personal choices such as exercising daily, eating a Mediterranean diet and taking certain supplements.  However, not all things that extend average lifespans also postpone aging.  For example requiring people to wear seatbelts in cars and observe speed limits can extend average lifespans but not affect aging which I see to be a biological process.

In this blog, I have mainly been concerned with life extension approaches that involve biological processes and that slow, stop or reverse aging.  So, the next question is “What is aging?”  Aging starts with birth (actually before birth) and lasts through life.  The part of it I have mainly been concerned with is post-maturational aging, aging that happens after an organism reaches reproductive age.   This later-stage aging is characterized by a number of degenerative processes that accelerate with advancing age, and these are the degenerative processes described by the 14 theories of aging in my treatise and an additional 7 “candidate” theories of aging:

1.     Oxidative Damage

2.     Cell DNA Damage

3.     Mitochondrial Damage

4.     Tissue Glycation

5.     Lipofuscin Accumulation

6.     Chronic Inflammation

7.     Immune System Compromise

8.     Neurological Degeneration

9.     Declines in Hormone Levels

10.   Susceptibility to Cancers

11.   Susceptibility to Cardiovascular Disease

12.   Telomere Shortening and Damage

13.  Programmed Epigenomic Changes

14.  Stem Cell Supply Chain Breakdown

15.   Incorrect protein folding

16.   Accumulation of progerin

17.   Gene mutations leading to hellicase abnormalities

18.   Increasing mTOR signaling

19.   Declining hypoxic response 

20.  Epigenomic changes in DNA methylation and histone acetylation 

21.   Micronutrient triage with aging 

As pointed out in the treatise and many blog discussions, many of these “theories of aging” are highly interdependent, existing in feedback loops where one promotes another, or are results of one or more common underlying processes.  In fact, it is possible to add additional theories of aging in the same spirit such as

22.    Metabolic degradation affecting the expression of sirtuins(ref)(ref), and

23.     Decline in effectiveness of autophagy(ref), and, probably many more yet.

A few key points are:

·        These are not so much theories of aging as they are corollaries of aging or processes that go on during aging. They are all processes that, once one of them is started, it can then can affect multiple body systems and unchain rapid aging in each of them.

·        There are many more corollaries of aging everyone is familiar with such as bone loss, grey hair, loss of hair, wrinkled skin, far-sightedness, loss of teeth, eyesight, hearing, and balance, etc.  While these are not usually seen as theories of aging because they each seem to involve only one system, this is not strictly true in all cases.  Hair stem cells, for example, can differentiate into epidermal and other kinds of cells(ref) and the same is true of stem cells living in tooth pulp(ref).  Bone marrow houses important mesenchymal and haemopoietic stem cells needed for renewal of cells in the blood and other parts of the body.  So, loss of hair or bone or even natural teeth could affect body renewal elsewhere.

.·        It might be possible to extend expected lifespan by interventions that address only one or several of these processes, just as it is possible to do so by diligently wearing seatbelts in a car.  Wearing eyeglasses and hearing aids may extend the lives of those who wear them by preventing accidents.  So, life-extension does not need to address fundamental aging.  

·        Since body systems and the theories of aging are so inter-related, a biologically-oriented antiaging intervention addressed to one theory, such as taking antioxidants(ref), is likely to affect aging with respect to several other theories as well, such as tissue glycation, inflammation and susceptibility to cancers.  The actions in my anti-aging lifestyle regimen affect aging with respect to several of the theories. The same is true of almost all of the supplements in the supplement regimen.  For this reason I call them anti-aging interventions though individually they may not slow or halt aging according to all the corollary theories.

·        The life extension interventions of most interest to me are the ones that slow or halt aging from the viewpoint of most of the 23 theories of aging listed.   In that category appear to be systematic exercise, weight management, and most of the other interventions suggested in my lifestyle regimen. 

·        On the supplement side, to the extent that resveratrol can activate SIRT1 producing the well-studied longevity results of calorie restriction(ref)(ref)(ref), it too affects most of the theories of aging.  Some others of the supplements might do that as well.
 

 What is aging?

This is a question I have ducked up to this point: What is aging?  Actually, we don’t know for sure.  We know all the corollaries of aging as listed, but we are not sure if any of these or any other as-yet unknown process is basic to all of the others.  The later could be the case, or it simply could be that we will never know aging except as through its corollaries.  More or less the same holds for anti-aging interventions.  If we could find an anti-aging intervention that halts or reverses aging according to all of the listed corollaries of aging, then the mechanisms of that intervention would go a long way towards telling us what aging is.  As I have mentioned above activation of SIRT1 via taking resveratrol probably comes as close to being a full anti-aging intervention as we have now.

Some of the 23 theories of aging listed are without doubt more fundamental than others as underlying drivers of aging.  That is, some of the theories are sufficient to explain what goes on according to some of the other theories of aging.   My favorites at the moment are Epigenomic changes in DNA methylation and histone acetylation and Stem Cell Supply Chain Breakdown with Telomere Shortening and Damage being in third place.  Since I have discussed each of these extensively elsewhere I comment only briefly on them here.  You can follow the hyperlinks to find out more about them. 

Epigenomic changes in DNA methylation and histone acetylation is actually a special case of Programmed Epigenomic Changes.  It is a global theory relating to changes that accumulate over a lifetime and that are capable of regulating all genes.  Anti-aging interventions could involve many specialized approaches to protein or histone demethylation or deacetylation.  It is interesting that SIRT1 mentioned above works through being a powerful deacetylase.  To see past blog posts related in some respect to this theory of aging start out with Feb 2009 blog entry Epigenetics, Epigenomics and Aging.  To see more just enter the phrase “epigenomic changes” into the search box to the left of this main text.  

Stem Cell Supply Chain Breakdown is a favorite theory of mine, and not just because because I have formulated it myself.  It says that we are charged up with a supply of pluripotent stem cells at conception (cells capable of differentiating into any body cell type), and these differentiate into our normal body cells (somatic cells) as well as various troves of adult stem cells which throughout life differentiate to make new replacement somatic cells.  The process runs down when the adult stem cells get old and are not replaced, so old senescent and near-senescent somatic cells are not replaced and aging results.  Anti-aging interventions according to this theory could include introduction of embryonic stem cells (ESCs) or “closing the loop” by reverting a few cells taking from a person to induced pluripotent stem cell (iPSC) status(ref)(ref).  It too is a global theory that could explain everything.  To see multiple past blog posts on this theory of aging just enter the word “supply chain” into the search box to the left of this main post.  

Telomere Shortening and Damage is the theory all over the news since the last Nobel prizes were given out, but it has fallen to third place in my listing because 1.  It is not clear to me that telomere shortening is the critical factor limiting human lifespans.  In a healthy young human if telomeres get too short in a cell and apoptotic processes are working well, the cell dies and is replaced by a new cell that is differentiated from an adult stem cell.  The same is not true for adult stem cells because there are no pluripotent cells around to differentiate to replace them, so we are back to the previous theory of aging, and 2.  Despite all the commercial hoop-la about supplements that induce “telomerase activation,” there is no published experimental evidence that people, monkeys, rats, mice or even worms live longer when they are subject to telomerase activation.  And we know for sure that there are a lot of other interventions that make worms, mice and rats live a lot longer than normal. To see multiple past blog posts related to telomeres and telomerase activation just enter the word “telomerase” into the search box to the left of this main text.  

I note that the second and third of these theories could in fact be highly complementary.  Adult stem cells could age and beome depleted because of shortening of their telomeres. If controlled telomerase expression could be activated in adult stem cells, in theory at least it might take a lot longer before they become depleted.

I expect that in another year it will be possible to refine or update the above observations in a significant way.  I could never have written the above when I first drafted my treatise in May 2008.  At that point I had identified only the first dozen of the above 23 theories/corollaries of aging.

It was disturbing to some readers when I characterized niacinamide as a pro-aging substance in the March 24 blog post  SIRT1, mTOR, NF-kappaB and resveratrol, as it was disturbing to me when I first came to that realization years ago.  In response to several blog comments I promised some readers to further research niacinamide and niacin for both health benefits and possible pro-aging hazards, and this blog post reports on that research. I will report on a number of research studies and finally I will give my opinions on how it all comes together and I will even disclose my past secret affair with niacin.

Niacin and Niacinimide

“Nicotinamide, also known as niacinamide and nicotinic acid 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, and though the two are identical in their vitamin functions, nicotinamide does not have the same pharmacologic and toxic effects of niacin, which occur incidental to niacin’s conversion. Thus nicotinamide does not reduce cholesterol or cause flushing,^[1] although nicotinamide may be toxic to the liver at doses exceeding 3 g/day for adults.^[2] In cells, niacin is incorporated into nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), although the pathways for nicotinamide and nicotinic acid are very similar. NAD+ and NADP+ are coenzymes in a wide variety of enzymatic oxidation-reduction reactions.^[3] (ref)” 

Continuing, “Nicotinamide adenine dinucleotide (NAD) and its relative nicotinamide adenine dinucleotide phosphate (NADP) are two of the most important coenzymes in the cell. NADP is simply NAD with a third phosphate group attached – (ref).  — NAD plays several key roles in metabolism.  It “participates in many redox reactions in cells, including in glycolysis and most of those in the citric acid cycle of cellular respiration(ref).”  And, as I have discussed previously, SIRT1 requires NAD for its actions(ref)(ref). 

General information on dietary sources of niacin/niacinamide and problems associated with deficiency of vitamin B3 can be found here and here.   “Niacin can be found in nuts, dairy products, lean meats, poultry, fish, and eggs. Some niacin is also supplied by legumes and enriched breads and cereals. The best dietary sources of vitamin B3 are found in beets, brewer’s yeast, beef liver, beef kidney, pork, turkey, chicken, veal, fish, salmon, swordfish, tuna, sunflower seeds, and peanuts. The body can synthesize niacin from the essential amino acid tryptophan, but the synthesis is extremely slow; 60 mg of tryptophan are required to make one milligram of niacin. For this reason, eating lots of tryptophan is not an adequate substitute for consuming niacin. As serotonin synthesis is reliant on tryptophan availability, inadequate dietary intake of vitamin B3 may also therefore lead to depression. The liver is the main storage area for this vitamin and absorption of vitamin B3 takes place in the intestines(ref).”  

Further,  “Niacin works closely with vitamin B1, vitamin B2, vitamin B6, pantothenic acid, and biotin to break the carbohydrates, fats, and proteins in food .  — Vitamin B3 is essential in the metabolism of carbohydrates (to produce energy), fats, and proteins. It also aids in the production of hydrochloric acid, needed for proper digestion. Additionally, vitamin B3 facilitates the body’s ability to eliminate toxins.” —  Vitamin B3 is required by the body for digestive processes, activating enzymes which nourish the brain, regulating blood pressure and regulating cholesterol levels.”  “Niacin is a water-soluble vitamin that participates in more than 50 metabolic functions, all of which are important in the release of energy from carbohydrates. Because of its pivotal role in so many metabolic functions, niacin is vital in supplying energy to, and maintaining the integrity of, all body cells. Niacin also assists in antioxidant and detoxification functions, and the production of sex and adrenal hormones. Vitamin B3 (niacin, niacinamide, nicotinic acid) lowers cholesterol by preventing its buildup in the liver and arteries. Niacin moves fat from tissues for fat metabolism, burning it for energy. It promotes healthy skin, the health of the myelin sheath (the protective covering of the spinal nerves), and good digestion, where it is also vital for the production of hydrochloric (stomach) acid. It is an aid in protecting the pancreas, and is necessary for the health of all tissue cells(ref).”

The government-recommended daily allowance (RDA) for niacin/niacinamide is 16 mg a day for men, 14 mg for women.  The 95^th percentile intake for both food and supplements is estimated to be 40-70 mg for both food and supplements(ref).  Pharmacological doses of either form of B3 can range from 500mg to 2gm daily.  There is a possibility of liver endangerment at the level of 3gms or more daily.

The case for niacinamide promoting health and longevity

B3 has been used now for over 40 years, often in large doses as a drug(ref).  A major application has been control blood cholesterol levels.  According to a 1986 publication: “The Coronary Drug Project was conducted between 1966 and 1975 to assess the long-term efficacy and safety of five lipid-influencing drugs in 8,341 men aged 30 to 64 years with electrocardiogram-documented previous myocardial infarction. The two estrogen regimens and dextrothyroxine were discontinued early because of adverse effects. No evidence of efficacy was found for the clofibrate treatment. Niacin treatment showed modest benefit in decreasing definite nonfatal recurrent myocardial infarction but did not decrease total mortality. With a mean follow-up of 15 years, nearly 9 years after termination of the trial, mortality from all causes in each of the drug groups, except for niacin, was similar to that in the placebo group. Mortality in the niacin group was 11% lower than in the placebo group (52.0 versus 58.2%; p = 0.0004). This late benefit of niacin, occurring after discontinuation of the drug, may be a result of a translation into a mortality benefit over subsequent years of the early favorable effect of niacin in decreasing nonfatal reinfarction or a result of the cholesterol-lowering effect of niacin, or both.”  Later it has become popular to combine niacin with a statin to control blood lipids.  A 2009 study concludes: “ Combined use of extended-release niacin with atorvastatin was superior to atorvastatin monotherapy alone in lipid profile regulation. Combination therapy with niacin ER and atorvastatin was well tolerated and safe in patients with coronary artery disease.” 

Several researchers think that pathways activated by niacin/niacinamide could offer hope for development of new drug treatments for several diseases, diabetes being prime among them,  but further understanding of the complex metabolic pathways involved is a prerequisite.   

The 2008 review article NAD+ and vitamin B3: from metabolism to therapies espouses this viewpoint.  The 2008 publication Triple play: promoting neurovascular longevity with nicotinamide, WNT, and erythropoietin in diabetes mellitus, states “Here we discuss the novel application of nicotinamide, Wnt signaling, and erythropoietin that modulate cellular oxidative stress and offer significant promise for the prevention of diabetic complications in the nervous and vascular systems. Essential to this process is the precise focus upon diverse as well as common cellular pathways governed by nicotinamide.” 

The 2010 publication Diabetes Mellitus: Channeling Care through Cellular Discovery comes to essentially the same conclusion “For these reasons, innovative strategies are necessary for the implementation of new treatments for DM that are generated through the further understanding of cellular pathways that govern the pathological consequences of DM. In particular, both the precursor for the coenzyme beta-nicotinamide adenine dinucleotide (NAD(+)), nicotinamide, and the growth factor erythropoietin offer novel platforms for drug discovery that involve cellular metabolic homeostasis and inflammatory cell control.” 

Health and potential life-extending properties of niacin/niacinamide 

“More recently, the health benefits of niacin have been shown to be far more extensive than previously appreciated — Numerous reports indicate niacin might help prevent atherosclerosis, diabetes and hypercholesterolemia. Niacin is effective in assisting burn-wound recovery, and in the prevention of cataracts and skin cancer. Unfortunately, the molecular basis for most of these health benefits remains unclear(ref).”

The 2006 e-publication Nicotinamide extends replicative lifespan of human cells reports “We found that an ongoing application of nicotinamide to normal human fibroblasts not only attenuated expression of the aging phenotype but also increased their replicative lifespan, causing a greater than 1.6-fold increase in the number of population doublings. Although nicotinamide by itself does not act as an antioxidant, the cells cultured in the presence of nicotinamide exhibited reduced levels of reactive oxygen species (ROS) and oxidative damage products associated with cellular senescence, and a decelerated telomere shortening rate without a detectable increase in telomerase activity. Furthermore, in the treated cells growing beyond the original Hayflick limit, the levels of p53, p21WAF1, and phospho-Rb proteins were similar to those in actively proliferating cells. The nicotinamide treatment caused a decrease in ATP levels, which was stably maintained until the delayed senescence point. Nicotinamide-treated cells also maintained high mitochondrial membrane potential but a lower respiration rate and superoxide anion level. Taken together, in contrast to its demonstrated pro-aging effect in yeast, nicotinamide extends the lifespan of human fibroblasts, possibly through reduction in mitochondrial activity and ROS production. 

Observation 2:  The research literature related to therapeutic use of niacin/niacinamide is at first confusing expressing different viewpoints that have emerged during different periods of time and representing different ways of looking at the effects of substance. Some articles cite reasons why large doses of niacin may promote longevity, and other articles cite reasons why such use may shorten life.

Observation 3:  For about 40 years large doses of niacin have been used for several therapeutic purposes.  Many physicians currently prescribe large doses of niacin for lipid control and other purposes. Mild benefits seem to exist in some areas like raising HDL cholesterol.  The long-term consequences of repeatedly taking large doses are unknown.

Observation 4:  Large scale niacin dosage profoundly affects multiple genes through multiple pathways producing both wanted and unwanted results.  While many researchers are excited by the possibilities of niacin-related therapies for a variety of conditions, the one thing they agree on is a need for further understanding of the pathways involved.

Observation 5:  The sirtuin-related pathways involving the niacin metabolites NAD and NADP are among those related to niacin most intensely studied in recent years.  Large doses of niacin/niacinamide inhibit the expression of SIRT1 and therefore prevent the health and longevity benefits associated with expression of SIRT1.   

Niacin and my personal regimen I have been taking niacin included in a B-complex “B-50” pill taken twice daily, for a total of 100 mg daily.  And added to this is niacin/niacinamide included in my breakfast cereals and other food, perhaps another 40mg.  So, with food I have probably been ingesting  140 mg a day of B3.  This is considerably over the minimum USRDA of 16mg and is possibly excessive for my optimal health.  I am changing my personal regimen and my suggested anti-aging Supplement Regimen to include only one B-50 a day and am looking into different B-complex supplements.  And  clearly, I do not plan to take larger doses of either niacin or niacinamide.

Personal note on my affair with niacin

In the spirit of disclosure, I need to own up to the fact that I once had a torrid affair with niacin.   And I really mean torrid because the stuff gives you hot flushes.  In the late 1970s there were some psychiatric and medical practitioners prescribing megadoses of niacin for all kinds of conditions, including chronic hypoglycemia and what later has come to be called bipolar disease.  It was a period where megavitamin therapy and orthomolecular medicine seemed to be the latest and best things for health.  Proponents of this approach like Dr. Allan Cott were respected people to be paid attention to.   I came under the influence of a New Hampshire medical practitioner who tested me and several of my sons for hypoglycemia, “evaluated” our mental states, and soon the whole bunch of us were taking megadoses of niacin, 1,000-1,500 mg a day.  This at first may have actually benefitted one of my sons who had been having problems with his schoolwork.  Reading some of the orthomolecular medicine literature of the day, I thought niacin was wonderful stuff.  After about three years, however, two things happened that resulted in the affair with niacin ending.  The first is that the son mentioned started taking larger and larger doses after he entered college, hoping it would help him cope with harder and harder schoolwork.  He ended up being seriously ill with a very inflamed liver and had to be hospitalized.  The diagnosis was overdose on Niacin.  The second thing is that we learned that the New Hampshire practitioner was not really a medical doctor as he had represented himself to be.  He was a fake.  We stopped taking megadoses of niacin.  In essence looking back, I think the spirit and intent of orthomolecular medicine was and continues to be a good thing, but that the scientific knowledge required to allow effective orthomolecular therapy to become a systematic practice is only now emerging.

Please see the medical disclaimer for this blog.