I have frequently asserted that telomere lengths do not decline in a uniform manner through life but depend on the interaction of multiple endogenous and extrinsic factors.  Two important factors are stress and exercise.  A new study in Plos One lends light on the relationship among these factors and telomere lengths:  The Power of Exercise: Buffering the Effect of Chronic Stress on Telomere Length.  This blog entry reviews interesting previous research relating to the impacts of stress and exercise on telomere lengths and describes the new findings.  For background, there is the Telomere Shortening and Damage theory of aging described in my treatise and a number of previous posts relating to telomere lengths which you can find by searching in this blog.

Stress

There are many forms of stress, social, physical and emotional, and these may be temporary, recurring or chronic.  Stress can result from external sources like injury or loss of a relative or result from internal conditions like a disease or mood disorder.    In the blog entries Stress and longevity and Hormesis and age retardation I discussed how some manageable forms of body stress can lead to a hormetic response, mobilization of heat shock proteins that actually confer health and longevity benefits, while more intense or more prolonged forms of stress may lead to multiple pathological conditions and premature aging.  For example, the stress of parachute jumping could possibly be good for you as suggested in the publication Emotional stress induced by parachute jumping enhances blood nerve growth factor levels and the distribution of nerve growth factor receptors in lymphocytes.  On the other hand, stress of prolonged unemployment could be bad for you even after you find a job, as suggested in the report History of unemployment predicts future elevations in C-reactive protein among male participants.  I also discussed how several dietary supplements like curcumin exercise positive effects in part through activating the body’s heat-shock stress response. 

There is a significant body of research literature associated with the negative effects of chronic or excess stress including immune system dysregulation, premature immunosenescence, elevated blood pressure and cortisol response, and undetected Type 2 diabetes(ref)(ref)(ref)(ref)(ref)(ref)(ref)(ref) which I will not review here.  Such stress is often viewed as associated with accelerated aging.

 Telomeres, telomerase and stress

The 2004 publication Accelerated telomere shortening in response to life stress was one of the first of many to link telomere shortening to stress. “We investigated the hypothesis that stress impacts health by modulating the rate of cellular aging. Here we provide evidence that psychological stress–both perceived stress and chronicity of stress–is significantly associated with higher oxidative stress, lower telomerase activity, and shorter telomere length, which are known determinants of cell senescence and longevity, in peripheral blood mononuclear cells from healthy premenopausal women. Women with the highest levels of perceived stress have telomeres shorter on average by the equivalent of at least one decade of additional aging compared to low stress women.” 

A 2006 study suggested that telomere shortening could be a mechanism through which stress leads to accelerated aging: Telomere shortening and mood disorders: preliminary support for a chronic stress model of accelerated aging.  “Little is known about the biological mechanisms underlying the excess medical morbidity and mortality associated with mood disorders. Substantial evidence supports abnormalities in stress-related biological systems in depression. Accelerated telomere shortening may reflect stress-related oxidative damage to cells and accelerated aging, and severe psychosocial stress has been linked to telomere shortening. We propose that chronic stress associated with mood disorders may contribute to excess vulnerability for diseases of aging such as cardiovascular disease and possibly some cancers through accelerated organismal aging. METHODS: Telomere length was measured by Southern Analysis in 44 individuals with chronic mood disorders and 44 nonpsychiatrically ill age-matched control subjects. RESULTS: Telomere length was significantly shorter in those with mood disorders, representing as much as 10 years of accelerated aging. — These results provide preliminary evidence that mood disorders are associated with accelerated aging and may suggest a novel mechanism for mood disorder-associated morbidity and mortality.”

The 2006 report Insulin resistance, oxidative stress, hypertension, and leukocyte telomere length in men from the Framingham Heart Study suggests another key link between telomere lengths, insulin resistance and hypertension.  ”Collectively, these observations suggest that hypertension, increased insulin resistance and oxidative stress are associated with shorter leukocyte telomere length and that shorter leukocyte telomere length in hypertensives is largely due to insulin resistance.”

A May 2010 publication Childhood adversities are associated with shorter telomere length at adult age both in individuals with an anxiety disorder and controls advances the hypothesis that stress leads to accelerated aging via telomere shortening.   “Accelerated leukocyte telomere shortening has been previously associated to self-perceived stress and psychiatric disorders, including schizophrenia and mood disorders. We set out to investigate whether telomere length is affected in patients with anxiety disorders in which stress is a known risk factor. We also studied the effects of childhood and recent psychological distress on telomere length. We utilized samples from the nationally representative population-based Health 2000 Survey that was carried out between 2000-2001 in Finland to assess major public health problems and their determinants. — Our results suggest that childhood stress might lead to accelerated telomere shortening seen at the adult age.”

Another slightly earlier 2010 study Childhood maltreatment and telomere shortening: preliminary support for an effect of early stress on cellular aging had the same theme, that childhood stress can lead to shorter telomeres and perhaps accelerated aging in adult life. “Based on previous evidence linking psychosocial stress to shorter telomere length, this study was designed to evaluate the effect of childhood adversity on telomere length. — These results extend previous reports linking shortened leukocyte telomere length and caregiver stress to more remote stressful experiences in childhood and suggest that childhood maltreatment could influence cellular aging.”

Telomere lengths stress and exercise

The first articles to show up relating telomerase and telomere lengths to exercise seemed to express surprise that exercise did not affect observed levels of telomerase, like the 2001 paper Telomerase activity is not altered by regular strenuous exercise in skeletal muscle or by sarcoma in liver of rats.  “We conclude that mild and strenuous exercise training does not significantly affect the activity of telomerase in the systems studied. Exercise training during sarcoma significantly retards the development of tumors and could possibly serve as a positive adjunct to treatment.”

A 2003 study looked at the result of exercise fatigue:  Athletes with exercise-associated fatigue have abnormally short muscle DNA telomeres. “In this study, we have compared a marker of skeletal muscle regeneration of athletes with exercise-associated chronic fatigue, a condition labeled the “fatigued athlete myopathic syndrome” (FAMS), with healthy asymptomatic age- and mileage-matched control endurance athletes. — The minimum value of TRF lengths (4.0 +/- 1.8 kb) measured on the DNA from vastus lateralis biopsies from these athletes were significantly shorter than those from 13 age- and mileage-matched control athletes (5.4 +/- 0.6 kb, P < 0.05). Three of the FAMS patients had extremely short telomeres (1.0 +/- 0.3 kb). The minimum TRF lengths of the remaining 10 symptomatic athletes (4.9 +/- 0.5 kb, P < 0.05) were also significantly shorter that those of the control athletes.”

The 2008 paper The effects of regular strength training on telomere length in human skeletal muscle reported “A recent study has reported abnormally short telomeres in skeletal muscle of athletes with exercise-associated fatigue. This important report raises the question of whether long-term practice of sports might have deleterious effects on muscle telomeres. Therefore, we aimed to compare telomere length of a group of power lifters (PL; N = 7) who trained for 8 +/- 3 yr against that of a group of healthy, active subjects (C; N = 7) with no history of strength training. — CONCLUSION: These results show for the first time that long-term training is not associated with an abnormal shortening of skeletal muscle telomere length. Although the minimum telomere length in PL remains within normal physiological ranges, a heavier load put on the muscles means a shorter minimum TRF length in skeletal muscle.”

A recent 2010 paper looks at the long-term consequences of strenuous exercise on telomere lengths: Skeletal muscle telomere length in healthy, experienced, endurance runners.  “Measuring the DNA telomere length of skeletal muscle in experienced endurance runners may contribute to our understanding of the effects of chronic exposure to endurance exercise on skeletal muscle. This study compared the minimum terminal restriction fragment (TRF) length in the vastus lateralis muscle of 18 experienced endurance runners (mean age: 42 +/- 7 years) to those of 19 sedentary individuals (mean age: 39 +/- 10 years). The runners had covered almost 50,000 km in training and racing over 15 years. Minimum TRF lengths measured in the muscle of both groups were similar (P = 0.805) and within the normal range. Minimum TRF length in the runners, however, was inversely related to their years spent running (r = -0.63, P = 0.007) and hours spent training (r = -0.52, P = 0.035). Therefore, since exposure to endurance running may influence minimum TRF length, and by implication, the proliferative potential of the satellite cells, chronic endurance running may be seen as a stressor to skeletal muscle.”

Telomere length, stress and exercise

Finally, a new twist is suggested in the publication:  The Power of Exercise: Buffering the Effect of Chronic Stress on Telomere Length.  “Chronic psychological stress is associated with detrimental effects on physical health, and may operate in part through accelerated cell aging, as indexed by shorter telomeres at the ends of chromosomes. However, not all people under stress have distinctly short telomeres, and we examined whether exercise can serve a stress-buffering function. We predicted that chronic stress would be related to short telomere length (TL) in sedentary individuals, whereas in those who exercise, stress would not have measurable effects on telomere shortening. — Participants were categorized into two groups-sedentary and active (those getting Centers for Disease Control-recommended daily amount of activity). The likelihood of having short versus long telomeres was calculated as a function of stress and exercise group, covarying age, BMI and education. Logistic regression analyses revealed a significant moderating effect of exercise. As predicted, among non-exercisers a one unit increase in the Perceived Stress Scale was related to a 15-fold increase in the odds of having short telomeres (p<.05), whereas in exercisers, perceived stress appears to be unrelated to TL (B = −.59, SE = .78, p = .45).”  This was a relatively small (63 healthy post-menopausal women aged between 54 and 82) and short (3 days) study using a self-evaluation 10-item questionnaire to measure psychological stress.  Nonetheless the implication is most interesting: exercise can nullify erosion in telomere lengths due to psychological stress.

I remind my readers of the January 2010 blog post Vitamins, supplements and telomerase – upregulation or downregulation? That post points to a study in which telomere lengthening was observed over a long period of time for a sizeable portion of the population studied. As stated in my treatise, “It appears that taking a number of popular supplements in the anti-aging firewalls supplement regimen like Vitamin E, fish oils, Vitamin D3 and resveratrol can lead to telomeres being longer than they otherwise might be, possibly because they induce the production of telomerase, possibly for other reasons. And, several of these supplements actually turn off telomerase in cancer cells.”

To sum it up

o   Childhood stress resulting in shorter telomeres may result in accelerated cellular aging later in life.

o   There is a “sweet spot” range for exercise stress within which the impact of the exercise is positive and health-producing and there is no stress-related telomere erosion, even if the exercise is repeated over the long term.  The sweet spot range may depend on the state of the individual.

o   If exercise is pursued too vigorously, to the point where it produces chronic fatigue, or pursued consistently as an endurance activity, telomere erosion may ensue, at least in muscles.

o   Psychological stress can produce telomere erosion even within a few days, but “sweet spot” exercising can prevent such erosion.

Final comments: 

–         There is much complication involved with telomere shortening or lengthening involving diet, health and age as well as stress and exercise.  The interplay of these and multiple internal factors is not well understood. Most likely there are epigenetic regulators of telomere length and every day, perhaps every hour or minute, complicated programs throughout the body readjust telomere lengths, perhaps making them shorter, leaving them the same or even making them longer.

–      How telomerase activators like cycloastragenol play into the situation of lengthening telomeres is also not well understood.  See my April 2010 post Telomerase activators – what do they really do?

Ever hear of humanin?  At the Paul K Glenn Symposium on Aging yesterday at Harvard, Dr. Pinchas Cohen gave a talk on The New World of Mitochondrial Proteins featuring humanin and other closely related proteins.  The subject is important because it points to a new and important function of mitochondria – generating protective proteins.  This blog post draws on material from that talk heavily augmented with materials from a number of published sources. 

Humanin is a mitochondria-derived peptide.  That is, humanin is expressed by mitochondrial genes in the 16s ribosomal RNA coding region.  The evidence suggests that gene transcription takes place in the mitochondria but translation into protein form takes place in the cytoplasm.  Humanin was independently cloned by three different groups in 2001.  It is one of some seven health-related peptides encoded by mitochondrial genes.

Several things have been learned about humanin:

·        Humanin is created by an evolutionary-conserved gene sequence and homolog versions of this sequence can be found in humans, mice, horses, nematodes, zebrafish and no doubt in many other species.

·        Humanin suppresses apoptosis by interfering with Bax activation (ref).  “HN prevents the translocation of Bax from cytosol to mitochondria. Conversely, reducing HN expression by small interfering RNAs sensitizes cells to Bax and increases Bax translocation to membranes. HN peptides also block Bax association with isolated mitochondria, and suppress cytochrome c release in vitro. Notably, the mitochondrial genome contains an identical open reading frame, and the mitochondrial version of HN can also bind and suppress Bax. We speculate therefore that HN arose from mitochondria and transferred to the nuclear genome, providing a mechanism for protecting these organelles from Bax(ref).” “The anti-apoptotic potential of HN appears to be dependent upon the formation of homodimers, as interfering with this process completely blocks its ability to suppress cell death [10]. Once dimerized, HN directly interacts with a variety of pro-apoptotic proteins, including Bax-related proteins [2] and insulin-like growth factor binding protein-3 (IGFBP-3) [11](ref).”

·        Humanin also works through ERK1/2 to mobilize calcium and provide an anti-inflammatory effect(ref)(ref). 

·        Tyrosine kinases and STAT3 in are involved in humanin-mediated neuroprotection(ref), particularly in the brain.

·        Humanin suppresses hepaptic glucose production, its main site of action being the hypothalamus(ref).

·        Humanin also works through STAT3 to exercise metabolic effects(ref).

·        Expression of humanin declines with age.  “Based upon the link of HN with two age-related diseases (AD and diabetes), we examined if there were age associated changes in HN levels. Indeed, the amount of detectable HN in hypothalamus, skeletal muscle, and cortex was decreased with age in rodents, and circulating levels of HN were decreased with age in humans and mice. — We conclude that the decline in HN with age could play a role in the pathogenesis of age-related diseases including AD and T2DM(ref).”

·        Humanin appears in multiple tissue types “Endogenous HN is both an intracellular and secreted protein and has been detected in normal mouse testis and colon at specific stages of development [3]. In addition to brain, colon and testis, we have shown the presence of HN by western blot in rodent heart, ovary, pancreas and kidney (unpublished data). In addition, our group has demonstrated the presence of HN in cerebral spinal fluid (CSF), seminal fluid and plasma, with levels in the biologically active range (Cohen & Hwang, unpublished data)(ref).”

·        Finally, the mitochondrial genes that make humanin may also transmigrate out of mitochondria into cell nucleuses and be incorporated into chromosomal DNA.   So, some of the humanin observed in tissues may result from ordinary protein-generating mechanisms.

Humanin, neural protection and Alzheimer’s disease

·        Humanin was from the start recognized to be a powerful neuroprotective substance.  Humanin blocks the process through which beta-amyloid causes neuronal death and was thought to be possibly useful for prevention or treatment of Alzheimer’s disease.  As outlined in one of the initial 2001 papers Mechanisms of Neuroprotection by a Novel Rescue Factor Humanin from Swedish Mutant Amyloid Precursor Protein: “We report a novel gene, designated Humanin (HN) cDNA, that suppresses neuronal cell death by K595N/M596L-APP (NL-APP), a mutant causing familial Alzheimer’s disease (FAD), termed Swedish mutant. — Therefore, HN suppressed neuronal cell death by NL-APP not through inhibition of Aβ42 secretion, but with two targets for its inhibitory action: (i) the intracellular toxic mechanism directly triggered by NL-APP and (ii) neurotoxicity by Aβ. HN will contribute to the development of curative therapy of AD, especially as a novel reagent that could mechanistically supplement Aβ-production inhibitors.” 

·        Humanin rescues cortical neurons treated with beta-amaloid in a concentration-dependent manner(ref).

·        The 2001 publication Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer’s disease-relevant insults stated “A novel factor, termed Humanin (HN), antagonizes against neurotoxicity by various types of familial Alzheimer’s disease (AD) genes [V642I and K595N/M596L (NL) mutants of amyloid precursor protein (APP), M146L-presenilin (PS) 1, and N141I-PS2] and by Abeta1-43 with clear action specificity ineffective on neurotoxicity by polyglutamine repeat Q79 or superoxide dismutase 1 mutants. Here we report that HN can also inhibit neurotoxicity by other AD-relevant insults: other familial AD genes (A617G-APP, L648P-APP, A246E-PS1, L286V-PS1, C410Y-PS1, and H163R-PS1), APP stimulation by anti-APP antibody, and other Abeta peptides (Abeta1-42 and Abeta25-35).”  However, it was not clear how it worked to do that.

·        The 2008 publication A rescue factor for Alzheimer’s diseases: discovery, activity, structure, and mechanism states the practical case for humanin in AD.  “While understanding the mechanism of AD and the involvement of key players should lead to rational drug discovery against this disease, a traditional screening approach should also work for identifying drugs using AD models. We have used a cellular AD model, in which a cell death was induced by AD-causing neurotoxicities, and then screened the genes, which rescued the cells from the cell death. This resulted in isolation of a gene encoding a novel 24-amino acid long peptide, termed Humanin (HN), which protected neuronal cells at approximately microM level. Surprisingly, these gene products and the synthetic peptides not only protected neurons from cell death induced by Abeta-related neurotoxicities, but also Abeta-unrelated neurotoxicities. While a broad range of activities of HN against AD-related insults is discovered, the detailed mechanism of its action is still obscure.”  In other words, “we don’t know how it works, but it works.”

·        A 2009 publication suggests a mechanism of action: Humanin inhibits neuronal cell death by interacting with a cytokine receptor complex or complexes involving CNTF receptor alpha/WSX-1/gp130.  “Together, these results indicate that HN protects neurons by binding to a complex or complexes involving CNTFR/WSX-1/gp130.”

·        Humanin and protect against prion-induced and other forms of drug-induced neuron apoptosis(ref)(ref) and can also inhibit death in other types of cells.

Humanin and diabetes

Humanin appears to have several salutary effects with respect to diabetes

·        Humanin is an insulin secretalog.  Humanin is A Novel Central Regulator of Peripheral Insulin Action.  “Decline in insulin action is a metabolic feature of aging and is involved in the development of age-related diseases including Type 2 Diabetes Mellitus (T2DM) and Alzheimer’s disease (AD). A novel mitochondria-associated peptide, Humanin (HN), has a neuroprotective role against AD-related neurotoxicity. Considering the association between insulin resistance and AD, we investigated if HN influences insulin sensitivity. — Using state of the art clamp technology, we examined the role of central and peripheral HN on insulin action. — HN represents a novel link between T2DM and neurodegeneration and along with its analogues offers a potential therapeutic tool to improve insulin action and treat T2DM.”

·        HNG-F6a, an analog of humanin, has a powerful capability to suppress blood sugar in Zucker rats(ref). 

·        In non-obese diabetic mice, humanin both prevents and treats Type 1 Diabetes.  According to the 2009 paper The neurosurvival factor Humanin inhibits β-cell apoptosis via signal transducer and activator of transcription 3 activation and delays and ameliorates diabetes in nonobese diabetic mice “Humanin normalized glucose tolerance in NOD mice treated for 6 weeks, and their pancreata revealed decreased lymphocyte infiltration and severity. In addition, Humanin delayed/prevented the onset of diabetes in NOD mice treated for 20 weeks. In summary, Humanin treatment decreases cytokine-induced apoptosis in β-cells in vitro and improved glucose tolerance and onset of diabetes in NOD mice in vivo. This indicates that Humanin may be useful for islet protection and survival in a spectrum of diabetes-related therapeutics.”

Humanin and vascular processes

·         The 2010 publication Humanin is Expressed in Human Vascular Walls and Has a Cytoprotective Effect against Oxidized LDL-Induced Oxidative Stress suggests other roles for humanin in vascular and possibly cardiovascular health and disease processes.  “The current study demonstrates for the first time the expression of Humanin in the endothelial cell layer of human blood vessels. Exogenous addition of Humanin to endothelial cell cultures was shown to be effective against Ox-LDL-induced apoptosis. These findings suggest that Humanin may play a role and may have protective effect in early atherosclerosis in humans.” 

Other SHLPs – SHLP6

There are six additional small humanin-like mitochondria-derived peptides (SHLPs) that Dr. Cohen talked about.  Four of these are somewhat similar in their effects to Humanin but one, SHLP6 is very different and interesting in its effects.  SHLP1 through SHLP5 line humanin are anti-apoptosis pro cell-survival substances, although they have varied neuro-protective and other effects.   I will not discuss those here.  SHLP6, however, is strongly pro-apoptosis, inhibits cancer cell growth in-vitro, inhibits VEGF, inhibits tumor growth and angiogenesis in vivo.  Its levels are reduced in prostate cancer – pretty much the opposite profile of humanin and the first five SHLPs.  One important difference is that levels of humanin and the first 5 SHLPs in the brain are reduced to half or less in the process of aging, but the level of SHLP6 may actually increase.  In blood plasma, the level of humanin drops to less than half with age but the level of SHLP6 remains nearly the same.   SHLP6 is also a highly conserved 20 amino-acid peptide.  It could be that, evolutionary-speaking, there is an anti-cancer survival advantage to high levels of SHLP6 with age.  It is effective in inducing apoptosis in prostate, breast and other cancer cell lines.  Potentially, SHLP6 could provide a basis for new cancer prevention or treatment approaches. To sum it up, mitochondria-derived peptides, humanin and its cousins, appear to define a new and largely unexploited field in biology with particular implications related to two important diseases of the elderly – Alzheimer’s disease and diabetes.  Humanin and some of its cousins seem to have similar health and longevity effects as SIRT1 and certain heat-shock proteins involved in hormesis responses(ref)(ref).  As time goes on I plan to explore possible relationships between these pro-life proteins and report on them in this blog.Somebody reading this blog is bound to ask me “Where can I get humanin or humanin-promoting dietary substances or supplements?”  I don’t know, except to suggest that keeping mitochondria healthy is probably the best way to keep humanin levels up with aging.  So, please see the Mitochondrial Damage Firewall in my treatise.

The free radical theory of aging, also known as the Oxidative Damagetheory of aging is over 50 years old and is perhaps the most-studied and most venerable of all the theories of aging.   But at least one line of research suggests that whatever this theory is and however important that it might be, in fact it may not be a theory of aging.   It does not appear to apply to mice living in luxury mouse resorts.

The free radical theory of aging

The free radical theory of aging holds that aging is due to the accumulation of DNA, tissue and organ damage created by free radicals.  Free radicals (ROS or Reactive Oxygen Species) are produced as a result of natural metabolism, by exposure to UV and X-rays, by exposure to certain toxic chemicals including heavy metals, and by consuming certain foods.  ROS ions steal electrons from lipids in cell membranes, a process called lipid peroxidation.  A chain of damaging events can be let loose from a single ROS molecule as unstable fatty acid radicals propagating in tissues and within cells produce other unstable radicals.  The result can be cell death, damage to DNA or mitochondrial DNA, mangled chromosomes, protein cross-linking, cell apoptosis (suicide), genetic mutations, damaged mitochondria, mutated germ cells and other forms of cell havoc. The damage can show up in many ways including skin erythema, hair loss, atherosclerosis and other forms of vascular damage, internal bleeding, cataracts, cancers, hypertension, type 2 diabetes, weakened immune systems, sterility, mutations in offspring, cancers, Alzheimer’s disease, premature aging and death.   The idea of avoiding stress and radiation, eating foods rich in antioxidants (ref)(ref) or taking antioxidant supplements (ref) is to avert such damage.

Up to a few years ago, most students of aging thought that oxidative damage was the primary cause of aging.  The evidence implicating free radicals in degenerative biological processes is overwhelming(1998 ref)(1983 ref (1991 ref) )(1999 ref) (2001 ref)(citation list ref).  The newer theories of aging continue to see oxidative damage as very important in the aging process, but part of a larger picture.  But the research described here questions whether the theory is dead, at least as a theory of aging.

Mouse experiments

Arlan Richardson is a well-known researcher who has long been studying free radicals and their biologic effects.  See, for example, his 2006 Powerpoint Presentationon The Basics of Free Radical Chemistry and Their Biology.  Richardson gave a presentation at the recent AAAS meeting describing mouse research that throws into question whether free radicals really affects aging.  The presentation called “Current Status of the Free Radical Theory of Aging” is not available in print or online but I will repeat some of the information in the slides here and cite applicable earlier publications by Richardson.

Part of the research Richardson described involved working with transgenic or knockout mice whose natural anti-oxidant defenses were disabled and checking their lifespans against those of comparable normal mice.  Only 1 of 20 Tg/KO mouse models tested showed a shortened lifespan as predicted by the free radical theory of aging, that model being SOD-/- mice.  And there is a special explanation for the shortened lifespans in that one strain.  Survival graphs were shown for mice in which CuZnSOD, Catalase, MnSOD, Gpx-4 antioxidant defenses were knocked out.  Not only did the mice have the same maximum lifespan as normal mice, but also at any age roughly the same number of mice survived.  The knockouts did, however, show signs of oxidative damage, increased cancers and sickness.  Inserting extra antioxidant defense genes also seemed to have no significant effects on the lifespans of these mice. 

These mice were all kept in clean cages with exercise wheels and were well fed.  They lived in mouse-equivalents of 4-star resorts with exercise club facilities.  In these gated resorts the mice were free of predators and free of many disease conditions to be found in the wild.

Conclusions of the study were:

 1.      Oxidative damage/stress plays a role in aging ONLY in a stressful environment (For example, suboptimal environment or short-lived genotype).”

 2.       Oxidative damage/stress plays a minimal role in aging in long-lived mice maintained in an optimal environment.”

Actually the theme described in the Richardson presentation goes back some time.  One of the key findings was telegraphed in a 2003 paper he co-authored Life-long reduction in MnSOD activity results in increased DNA damage and higher incidence of cancer but does not accelerate aging and in the 2009 publication he co-authored Mice Deficient in Both Mn Superoxide Dismutase and Glutathione Peroxidase-1 Have Increased Oxidative Damage and a Greater Incidence of Pathology but No Reduction in Longevity.  “Thus, these data do not support a significant role for increased oxidative stress as a result of compromised mitochondrial antioxidant defenses in modulating life span in mice and do not support the oxidative stress theory of aging.” Nonetheless, the knockout mice got sicker. “Consistent with the important role for oxidative stress in tumorigenesis during aging, the incidence of neoplasms was significantly increased in the older Sod2+/−Gpx1−/− mice (28–30 months).”

The idea that oxidative damage may not be a cause of aging but rather is a consequence of aging was raised in an earlier blog entry Oxidative damage – cause or effect?

Do the results apply to humans? 

I speculate that the answer is “possibly” if not “probably.” Most of us in the US live pretty well-protected lives.  We do not have to worry about large predators that can eat us.  We get enough food even if we do not choose healthy food and most of us do not have to worry about shelter.  So what happens is a) the ravages of oxidative damage build up with age including all kinds of diseases and cancers, and b) yet our average lifespans continue to increase.  We get a lot of sicker older people.  If this line of reasoning is correct, strengthening our antioxidant defenses through diet, supplements and lifestyle interventions might not lead us to live longer, but may well result in us living healthier in old age.

Have you heard about Ghrelin?  No, I am not talking about a new grill cleaner or a men’s girl-attracting perfume or a new shortstop for the Yankees.  Ghrelin is a hormone deeply involved in a very important current health condition, obesity, and it also plays critical roles in other key processes including neural functioning and learning. This blog is an introduction to Ghrelin and some of the important current research related to it.

About Ghrelin

The 2006 paper Gastrointestinal hormones (not including pancreatic hormones): Drug Insight: the functions of ghrelin and its potential as a multitherapeutic hormone summarizes some key aspects of the ghrelin story.  “The endogenous ligand for the growth-hormone (GH) secretagogue receptor was purified from stomach and named ghrelin. It has potent GH-releasing activity and stimulates appetite by acting on the hypothalamic arcuate nucleus, a region known to control food intake. Ghrelin thus plays important roles in maintaining GH release and energy homeostasis in vertebrates. Ghrelin, moreover, stimulates gastric motility and acid secretion, shows positive cardiovascular effects, and has direct actions on bone formation. The diverse functions of ghrelin raise the possibility of its clinical application for GH deficiency, eating disorders, gastrointestinal disease, cardiovascular disease, osteoporosis and aging.”

“Ghrelin is a hormone produced mainly by P/D1 cells lining the fundus of the human stomach and epsilon cells of the pancreas that stimulates hunger.^[1] Ghrelin levels increase before meals and decrease after meals. It is considered the counterpart of the hormone leptin, produced by adipose tissue, which induces satiation when present at higher levels(ref).”  “The hypothalamus in the brain is another significant source of ghrelin; smaller amounts are produced in the placenta, kidney, and pituitary gland(ref).”

“Ghrelin’s activity in modulating feeding behavior and energy balance are best explained by the presence of ghrelin receptors in areas of the hypothalamus long known to be involved in appetite regulation. Receptors are also found concentrated in other areas of the brain, including the hippocampus and regions known to be involved in reward systems (e.g. tegmental area); indeed, ghrelin appears to activate some of the same circuits that are involved in drug reward, which may also be related to this hormone’s effects on appetite(ref).”

“At least two major biologic activities have been ascribed to ghrelin:  Stimulation of growth hormone secretion: Ghrelin, as the ligand for the growth hormone secretagogue receptor, potently stimulates secretion of growth hormone. The ghrelin signal is integrated with that of growth hormone releasing hormone and somatostatin to control the timing and magnitude of growth hormone secretion. – Regulation of energy balance: In both rodents and humans, ghrelin functions to increase hunger though its action on hypothalamic feeding centers. – (ref)”

Ghrelin is a key actor in the hypothalamic melanocortin system.  “As an important part of that model, the gut hormone ghrelin is believed to inform the brain about energy availability and has been shown to increase adiposity, raise blood pressure and promote hyperglycemia(ref). The overwhelming majority of ghrelin’s effects on metabolism are mediated via CNS circuits, with the hypothalamic melanocortin system arguably being its most important direct target(ref)³. In turn, the melanocortin system is an essential and potent regulator of body adiposity, glucose metabolism and blood pressure(ref). Furthermore, mutations of melanocortin receptors are strongly correlated with human obesity(ref) and alterations in cholesterol transport are a common occurrence in obesity and the metabolic syndrome(ref). We hypothesized that a gut-brain axis integrates all of the primary physiological components known to be affected in the metabolic syndrome, that, in addition to regulating glucose homeostasis, blood pressure, food intake and body weight, it also likely controls cholesterol metabolism. We found that a gut-brain axis including ghrelin, glucagon-like peptide 1 (GLP-1) and the central melanocortin system directly regulates the hepatic synthesis and re-uptake of cholesterol(ref).”

Ghrelin and growth hormone secretion

The 2000 paper Preliminary evidence that Ghrelin, the natural GH secretagogue (GHS)-receptor ligand, strongly stimulates GH secretion in humans was one of the first to point to actions of ghrelin going beyond simple appetite-regulating and metabolic effects. “In conclusion, this preliminary study shows that Ghrelin exerts a strong stimulatory effect on GH (growth hormone) secretion in humans releasing more GH than GHRH.” Of course, growth hormone exercises multiple effects of its own. “Growth hormone (GH) is a protein-based poly-peptide hormone. It stimulates growth, cell reproduction and regeneration in humans and other animals. It is a 191-amino acid, single-chain polypeptide hormone that is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland(ref).”

Ghrelin, hunger and obesity

The 2001 publication Minireview: Ghrelin and the Regulation of Energy Balance—A Hypothalamic Perspective  “The first published evidence for the involvement of ghrelin in the regulation of appetite was provided by Ghigo and co-workers (ref)). They described that 3 out of 4 healthy volunteers spontaneously reported hunger following ghrelin administration as a “side effect” in a clinical study analyzing GH release (ref). This hunger-inducing effect of ghrelin has now been confirmed in two more studies, where, again, 3 out of 7 (33) and 9 out of 11 individuals report hunger as the only sensation after ghrelin injection –. A large number of animal studies added strength to the argument that ghrelin is involved in the regulation of energy balance. For example, exogenous ghrelin induces adiposity in rodents by stimulating an acute increase in food intake, as well as a reduction in fat utilization –. Adipogenic as well as orexigenic effects of ghrelin are independent from its ability to stimulate GH secretion — and are most likely mediated by a specific central network of neurons that is also modulated by leptin –.” 

The 2007 article Meal suppression of circulating ghrelin is normalized in obese individuals following gastric bypass surgery examines ghrelin levels in obese individuals, indicating effects of gastric bypass surgery.  “Design:  Cross-sectional study with repeated blood samples in 40 subjects after 14 h of prolonged overnight fasting followed by a standardized mixed meal (770 kcal).  Subjects: Twenty men and 20 women were included: 10 middle-aged morbidly obese (body mass index (BMI) 43.9 3.3 kg/m2), 10 middle-aged subjects who had undergone RYGBP (Roux-en-Y gastric bypass surgery) at the Uppsala University Hospital (BMI 34.7 5.8 kg/m2), 10 middle-aged non-obese (BMI 23.5 2.2 kg/m2) and 10 young non-obese (BMI 22.7 1.8 kg/m2).  Measurements: Ghrelin, glucose and insulin levels were analyzed pre- and postprandially. Results: In the morbidly obese, ghrelin concentrations were lower in the morning than in the RYGBP group and did not change following the meal. In the RYGBP group, fasting ghrelin levels fell after meal intake and showed similar suppression as both age-matched and young non-obese controls. The RYGBP surgery resulted in an increased meal-induced insulin secretion, which was related to the degree of postprandial ghrelin suppression.  Conclusion: The present study demonstrates low circulating concentrations of ghrelin and blunted responses to fast and feeding in morbidly obese subjects. Marked weight reduction after RYGBP at our hospital is followed by a normalization of ghrelin secretion, illustrated by increased fasting levels compared to the preoperative obese state and regain of meal-induced ghrelin suppression.”

The 2010 publication Plasma ghrelin levels and polymorphisms  of ghrelin gene in Chinese obese children and adolescents indicates “AIM: To evaluate the role of fasting plasma ghrelin levels [ln(ghrelin)] and polymorphisms of ghrelin gene in Chinese obese children. METHODS: Genotyping for ghrelin polymorphism was performed in 230 obese and 100 normal weight children. Among them, plasma ghrelin levels were measured in 91 obese and 23 health subjects. RESULTS: (1) Bivariate correlation analysis showed the ln(ghrelin) was inversely correlated with abnormality of glucose metabolism (r = -0.240, P = 0.023). Stepwise multiple regression analysis showed that abnormality of glucose metabolism was an independent determinant of plasma ghrelin levels (P = 0.023). — CONCLUSION: Ghrelin is associated with obesity in childhood, especially associated with the glucose homeostasis. Lower ghrelin levels might be a result of obesity, but not a cause of obesity.  –” 

The 2009 publication Ghrelin and growth hormone secretagogues, physiological and pharmacological aspect looks at the possibility of pharmacological interference with expression of ghrelin as a strategy to control appetite and obesity.  “At least theoretically ghrelin receptor antagonists could be anti-obesity drugs, as blockers of the orexigenic signal from the gastrointestinal tract to the brain. Inverse agonists of the ghrelin receptor, by blocking the constitutive receptor activity, might lower the set-point for hunger between meals.”   

The 2006 publication Ghrelin and the short- and long-term regulation of appetite and body weight came to a similar conclusion. “Chronic ghrelin administration increases body weight via diverse, concerted actions on food intake, energy expenditure, and fuel utilization. Congenital ablation of the ghrelin or ghrelin-receptor gene causes resistance to diet-induced obesity, and pharmacologic ghrelin blockade reduces food intake and body weight. Ghrelin levels are high in Prader-Willi syndrome and low after gastric bypass surgery, possibly contributing to body-weight alterations in these settings. Extant evidence favors roles for ghrelin in both short-term meal initiation and long-term energy homeostasis, making it an attractive target for drugs to treat obesity and/or wasting disorders.” 

Ghrelin, inflammatory diseases and NF-kappaB 

In a number of previous blog entries and in my treatise I have written about inhibition of expression of NF-kappaB as a strategy for controlling inflammatory diseases and perhaps even for life extension(ref)(ref)(ref)(ref).  It appears that ghrelin is an inhibitor of NF-kappaB and helps control at least some inflammatory diseases.  The 2007 publication Ghrelin attenuates sepsis-induced acute lung injury and mortality in rats reports “Our study has shown that plasma levels of ghrelin, a stomach-derived peptide, are significantly reduced in sepsis, and that ghrelin administration improves organ blood flow via a nuclear factor (NF)-kappaB-dependent pathway. — Ghrelin administration restored pulmonary levels of ghrelin, reduced lung injury, increased pulmonary blood flow, down-regulated proinflammatory cytokines, inhibited NF-kappaB activation, and improved survival in sepsis. — CONCLUSIONS: Ghrelin can be developed as a novel treatment for severe sepsis-induced ALI. The protective effect of ghrelin is mediated through inhibition of NF-kappaB.” 

Ghrelin and radiation injury

A 2009 paper Human ghrelin ameliorates organ injury and improves survival after radiation injury combined with severe sepsis points to another potential role for ghrelin.  “Administration of human ghrelin attenuated tissue injury markedly, reduced proinflammatory cytokine levels, decreased tissue myeloperoxidase activity, and improved survival after RCI. Furthermore, elevated plasma levels of norepinephrine (NE) after RCI were reduced significantly by ghrelin. However, vagotomy prevented ghrelin’s beneficial effects after RCI. In conclusion, human ghrelin is beneficial in a rat model of RCI. The protective effect of human ghrelin appears to be attributed to re-balancing the dysregulated sympathetic/parasympathetic nervous systems.”

And, a  2010 paper Ghrelin as a novel therapy for radiation combined injury indicates “In this review, we describe briefly the pathological consequences of ionizing radiation and provide an overview of the animal models of radiation combined injury. We highlight the combined radiation and sepsis model we recently established and suggest the use of ghrelin, a novel gastrointestinal hormone, as a potential therapy for radiation combined injury.”

Ghrelin and aging

Expression of ghrelin changes with aging and ghrelin expression affects the aging process.  It contributes to energy balance.  According to the 2010 publication Thermoregulation, energy balance, regulatory peptides: recent developments, “Some peripheral peptides (e.g. leptin, insulin, ghrelin) acting at either peripheral or cerebral sites also contribute to the regulation of energy balance. The prevailing thermoregulatory status, the substances or neural signals representing actual feeding vs. established nutritional states, and the aging process may modify the expression and/or activity of peripheral and central peptides and peptide receptors.” 

Another 2010 publication concludes “In healthy elderly people relatively large amounts of fat increase the satiety signal from GLP-1 and lower the acylated to desacylated ratio of ghrelin, consequently decreasing hunger. This condition may lead to a reduction in calorie intake.” 

Yet-another 2010 e-publication Impaired postprandial response of active ghrelin and prolonged suppression of hunger sensation in the elderly has related findings.  “RESULTS: Our results showed that older participants felt postprandially less hungry and more full. Although basal levels were not significantly different, active and total ghrelin levels declined postprandially only in the younger study participants. Highly significant differences between the two age groups were shown for the changes of the area under the curve for active ghrelin (p = .024). CONCLUSIONS: Our study demonstrates for the first time that differences in hunger and satiety sensations in relation to age are paralleled by a substantially different response of acylated and total ghrelin, that is, the absence of a postprandial decline in ghrelin levels.”

Wrapup 

Research into ghrelin and its related pathways and activities appears to be mostly in the last 10 years and definitely accelerating.  Part of the impetus is the current concern with obesity.  It is likely that therapies will be proposed for a number of disease conditions based on manipulation of ghrelin.  At present, however, there are only hints of anti-aging interventions based on manipulation of ghrelin.  I would not be surprised to see publications on this topic appearing in the coming year or so. 

I wrote about blueberries back in September 2009 in the blog entry Blueberries and health – the research case, citing 11 research citations there on their positive health effects.  I have long been in the habit of eating a cupful of wild blueberries every morning together with raisin bran and walnuts.  However, my mind has been concerned with more exotic longevity things reported in this blog. At the AAAS meeting I was exposed to some interesting new research relating to those little purple critters.    In this post I review mostly-new research relating blueberries to cognition, neural inflammation,  Alzheimer’s disease, cancer, and cardiovascular health.

Blueberries and microglial activation

I have discussed emerging knowledge about the role of activated microglia in blog entries relating to inflammatory nervous system disorders including Spinal cord injury pain, and New views of Alzheimer’s disease and new approaches to treating it.  “Microglia are a type of glial cell that are the resident macrophages of the brain and spinal cord, and thus act as the first and main form of active immune defense in the central nervous system (CNS)(ref).” Microglia can become activated due to physical injury, toxic assault, the presence of pathogens, or disease conditions such as Alzheimer’s disease.  When activated, microglia release pro-inflammatory cytokine signaling molecules like TNF-alpha, NO, IL-1 and IL-6, and this process can excite neurons to further excite the microglia.  The loop of mutual excitement can feed on itself contributing to a worsening pathological inflammatory phenotype.  It appears that blueberries can help block the process.

The 2007 publication Inhibitory effects of blueberry extract on the production of inflammatory mediators in lipopolysaccharide-activated BV2 microglia was one of the first relating blueberries to microglia.  “Sustained microglial activation in the central nervous system (CNS) has been extensively investigated in age-related neurodegenerative diseases and has been postulated to lead to neuronal cell loss in these conditions. Recent studies have shown that antiinflammatory drugs may suppress microglial activation and thus protect against microglial overactivation and subsequent cell loss. Research also suggests that fruits such as berries may contain both antioxidant and antiinflammatory polyphenols that may be important in this regard. Our previous research showed that blueberry extract was effective in preventing oxidant-induced calcium response deficits in M1 (muscarinic receptor)-transfected COS-7 cells. Extrapolating from these findings, the current study investigated the effect of blueberry extract on preventing inflammation-induced activation of microglia. Results indicated that treatments with blueberry extract inhibited the production of the inflammatory mediator nitric oxide (NO) as well as the cytokines interleukin-1beta and tumor necrosis factor-alpha, in cell conditioned media from lipopolysaccharide (LPS)-activated BV2 microglia. Also, mRNA and protein levels of inducible nitric oxide synthase and cyclooxygenase-2 in LPS-activated BV2 cells were significantly reduced by treatments with blueberry extract. The results suggest that blueberry polyphenols attenuate inflammatory responses of brain microglia and could be potentially useful in modulation of inflammatory conditions in the CNS.”  LPS is an endotoxin often used in laboratory and mouse experiments to elicit powerful immune responses and excite microglia. 

The 2010 publication Blueberry supplementation attenuates microglial activation in hippocampal intraocular grafts to aged hosts is one of several that expands on this theme. “Transplantation of central nervous tissue has been proposed as a therapeutic intervention for age-related neurodegenerative diseases and stroke. However, survival of embryonic neuronal cells is hampered by detrimental factors in the aged host brain such as circulating inflammatory cytokines and oxidative stress. We have previously found that supplementation with 2% blueberry in the diet increases graft growth and neuronal survival in intraocular hippocampal grafts to aged hosts. In the present study we explored possible biochemical mechanisms for this increased survival, and we here report decreased microglial activation and astrogliosis in intraocular hippocampal grafts to middle-aged hosts fed a 2% blueberry diet. Markers for astrocytes and for activated microglial cells were both decreased long-term after grafting to blueberry-treated hosts compared with age-matched rats on a control diet. Similar findings were obtained in the host brain, with a reduction in OX-6 immunoreactive microglial cells in the hippocampus of those recipients treated with blueberry. In addition, immunoreactivity for the pro-inflammatory cytokine IL-6 was found to be significantly attenuated in intraocular grafts by the 2% blueberry diet. These studies demonstrate direct effects of blueberry upon microglial activation both during isolated conditions and in the aged host brain and suggest that this nutraceutical can attenuate age-induced inflammation.”

In my previous post on blueberries I mentioned a related citation: Blueberry polyphenols attenuate kainic acid-induced decrements in cognition and alter inflammatory gene expression in rat hippocampus, “These results indicate that blueberry polyphenols attenuate learning impairments following neurotoxic insult and exert anti-inflammatory actions, perhaps via alteration of gene expression.” It was observed that blueberries attenuated the expression of NF-kappaB induced by the neurotoxic kainic acid and augmented the expression of IGF-1. Blueberries can be protective of mental capability when exposed to brain toxins.”  While this 2005 document does not mention the role of microglia, their involvement can now clearly by hypothesized.

The mechanisms of action of blueberries appear to be shared by other anthocyanin-expressing flavonoids such as found in other berries.  The 2009 publication Flavonoids and brain health: multiple effects underpinned by common mechanisms outlines some of those actions that go along with inhibition of microglial activation. “The neuroprotective actions of dietary flavonoids involve a number of effects within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation, and the potential to promote memory, learning and cognitive function. This multiplicity of effects appears to be underpinned by two processes. Firstly, they interact with important neuronal signalling cascades leading to an inhibition of apoptosis triggered by neurotoxic species and to a promotion of neuronal survival and differentiation. These interactions include selective actions on a number of protein kinase and lipid kinase signalling cascades, most notably the PI3K/Akt and MAP kinase pathways which regulate pro-survival transcription factors and gene expression. Secondly, they induce peripheral and cerebral vascular blood flow in a manner which may lead to the induction of angiogenesis, and new nerve cell growth in the hippocampus. Therefore, the consumption of flavonoid-rich foods, such as berries and cocoa, throughout life holds a potential to limit the neurodegeneration associated with a variety of neurological disorders and to prevent or reverse normal or abnormal deteriorations in cognitive performance.”

The 2010 publication Blueberry supplementation improves memory in older adults reports on a small human study confirming what we have pretty much known all along.  “The prevalence of dementia is increasing with expansion of the older adult population. In the absence of effective therapy, preventive approaches are essential to address this public health problem. Blueberries contain polyphenolic compounds, most prominently anthocyanins, which have antioxidant and anti-inflammatory effects. In addition, anthocyanins have been associated with increased neuronal signaling in brain centers, mediating memory function as well as improved glucose disposal, benefits that would be expected to mitigate neurodegeneration. This study investigated the effects of daily consumption of wild blueberry juice in a sample of nine older adults with early memory changes. At 12 weeks, improved paired associate learning (p = 0.009) and word list recall (p = 0.04) were observed. In addition, there were trends suggesting reduced depressive symptoms (p = 0.08) and lower glucose levels (p = 0.10). We also compared the memory performances of the blueberry subjects with a demographically matched sample who consumed a berry placebo beverage in a companion trial of identical design and observed comparable results for paired associate learning. The findings of this preliminary study suggest that moderate-term blueberry supplementation can confer neurocognitive benefit and establish a basis for more comprehensive human trials to study preventive potential and neuronal mechanisms.”  Clearly, larger-scale studies are needed to confirm the results. 

Pterostilbene

Blueberries contain pterostilbene. “Pterostilbene is a stilbenoid chemically related to resveratrol. It is thought to be the key compound found predominantly in blueberries (as well as grapes) that exhibit anti-cancer, anti-hypercholesterolemia, anti-hypertriglyceridemia properties, as well as fight off and reverse cognitive decline. It is believed that the compound also has anti-diabetic properties, but so far very little has been studied on this issue. Additionally, it is also touted as a potent anti-fungal(ref).”

Pterostilbene, inflammation, glycation and diabetes

Pterostilbene is a powerful anti-inflammatory, responsible for some of the effects of blueberries in controlling a range of inflammatory disease conditions.  The 2008 publication Pterostilbene suppressed lipopolysaccharide-induced up-expression of iNOS and COX-2 in murine macrophages reports “Pterostilbene, an active constituent of blueberries, is known to possess anti-inflammatory activity and also to induce apoptosis in various types of cancer cells. Here, we investigated the inhibitory effects of pterostilbene on the induction of NO synthase (NOS) and cyclooxygenase-2 (COX-2) in murine RAW 264.7 cells activated with lipopolysaccharide (LPS).”  Continuing, “Treatment with pterostilbene resulted in the reduction of LPS-induced nuclear translocation of the nuclear factor-kappaB (NFkappaB) subunit and the dependent transcriptional activity of NFkappaB by blocking phosphorylation of inhibitor kappaB (IkappaB)alpha and p65 and subsequent degradation of IkappaB alpha. Transient transfection experiments using NFkappaB reporter constructs indicated that pterostilbene inhibits the transcriptional activity of NFkappaB in LPS-stimulated mouse macrophages. We found that pterostilbene also inhibited LPS-induced activation of PI3K/Akt, extracellular signal-regulated kinase 1/2 and p38 MAPK. Taken together, these results show that pterostilbene down regulates inflammatory iNOS and COX-2 gene expression in macrophages by inhibiting the activation of NFkappaB by interfering with the activation of PI3K/Akt/IKK and MAPK. These results have an important implication for using pterostilbene toward the development of an effective anti-inflammatory agent(ref).”  Inhibition of the transcriptional activity of NFkappaB is not only a useful anti-inflammatory strategy but has been proposed as a powerful anti-aging strategy as discussed several times in this blog and in my treatise(ref).  See the discussion under the subheading Increase in aberrant NF-kappaB signaling.  Thirty nine supplements in my anti-aging combined supplement firewall are inhibitors of NFkappaB.  To these can be added blueberries and a number of other phyto-substances in my normal food diet.

Pterostilbene and cancer

A number of recent studies have focused on the anti-cancer properties of pterostilbene. I cite a selected few here. 

–         The 2010 report Pterostilbene inhibits pancreatic cancer in vitro,

–         The 2010 report Dietary intake of pterostilbene, a constituent of blueberries, inhibits the β-catenin/p65 downstream signaling pathway and colon carcinogenesis in rats,

–         The 2010 report Pterostilbene inhibits breast cancer in vitro through mitochondrial depolarization and induction of caspase-dependent apoptosis,

–         The 2010 report Pterostilbene inhibits lung cancer through induction of apoptosis,

–         The 2009 publication Pterostilbene inhibited tumor invasion via suppressing multiple signal transduction pathways in human hepatocellular carcinoma cells,

–         The 2009 publication Anti-inflammatory action of pterostilbene is mediated through the p38 mitogen-activated protein kinase pathway in colon cancer cells, and

–         The 2007 report Pterostilbene, an active constituent of blueberries, suppresses aberrant crypt foci formation in the azoxymethane-induced colon carcinogenesis model in rats.

Pterostilbene, inflammation, glycation and diabetes

One of the theories of aging is Tissue Glycation, a process deeply implicated in diabetes.  Tissue glycation involves cross linking of tissue proteins with sugars resulting in the formation of Advanced Glycation Endproducts (AGEs).  The result of AGEs can be self-propagating systemic or “silent” tissue inflammation. AGEs are recognized by cell RAGE receptors which result in the production of cytokine chemicals that can induce unwanted and potentially deadly inflammation in blood vessels, nerve, liver and other tissues. Atherosclerosis can be a consequence. AGEs are responsible for much bodily mischief related to aging leading to deterioration of function and structure of organs. They play important roles in diabetes, atherosclerosis, vascular disease, kidney failure, and neuropathy including Alzheimer’s disease. The presence of AGEs also appears to negatively impact on immune system functioning.  Diabetes in particular appears to have its roots due to glycation and people with high blood sugar levels are particularly susceptible to glycation.

The 2006 publication Wild blueberry (Vaccinium angustifolium) consumption affects the composition and structure of glycosaminoglycans in Sprague-Dawley rat aorta is one of several that speaks to the effects of blueberries on tissue glycation.  “Our results demonstrate for the first time that a diet rich in blueberries results in structural alterations in rat aortic tissue GAGs. These changes may affect cellular signal transduction pathways and could have major consequences for the biological function of GAG molecules within the vascular environment.”  Other relevant documents include the 2009 publication Seasonal phytochemical variation of anti-glycation principles in lowbush blueberry (Vaccinium angustifolium) and Anti-diabetic properties of the Canadian lowbush blueberry Vaccinium angustifolium Ait.

The previous blog post on blueberries contains a number of additional research citations worth reviewing. One clear conclusion from reading those citations and the citations quoted here is that researchers are taking the anti-inflammatory, brain-health, cancer-fighting, diabetes-fighting and anti-aging properties of blueberries most seriously.  Eating blueberries goes far beyond being a health food fad.

Putting it all together

My daily cup of blueberries is an important dietary component of my total anti-aging firewalls regimen, taking its place along with olive oil, walnuts, green tea, garlic and many other healthy foods.  In general, I go along with the philosophy expressed in the 2009  publication Grape juice, berries, and walnuts affect brain aging and behavior.  “Numerous studies have indicated that individuals consuming a diet containing high amounts of fruits and vegetables exhibit fewer age-related diseases such as Alzheimer’s disease. Research from our laboratory has suggested that dietary supplementation with fruit or vegetable extracts high in antioxidants (e.g. blueberries, strawberries, walnuts, and Concord grape juice) can decrease the enhanced vulnerability to oxidative stress that occurs in aging and these reductions are expressed as improvements in behavior. Additional mechanisms involved in the beneficial effects of fruits and vegetables include enhancement of neuronal communication via increases in neuronal signaling and decreases in stress signals induced by oxidative/inflammatory stressors (e.g. nuclear factor kappaB). Moreover, collaborative findings indicate that blueberry or Concord grape juice supplementation in humans with mild cognitive impairment increased verbal memory performance, thus translating our animal findings to humans. Taken together, these results suggest that a greater intake of high-antioxidant foods such as berries, Concord grapes, and walnuts may increase “health span” and enhance cognitive and motor function in aging.”

I end this note with a speculation relating to my personal health.  In the blog post Spinal cord injury pain, I reported on an extremely painful injury to my spine incurred about a year ago while swimming in extremely cold water.  I reported how terrible pains were showing up in unrelated parts of my body – e.g. my knee, my back, my hips, and my rotator cuff.  A runaway inflammatory condition in my spine was sending out large quantities of cytokines creating debilitating havoc in all those places.  My personal diagnosis was microglial over-activation due to injury in my spine, though none of the medical personnel treating me had ever heard of that.   They thought my diverse pains were psychosomatic because the painful parts were not direct;y connected to my spine.  Finally, I reported how the pains went away when I started taking gabapentin, and how I came across an obscure publication indicating that gabapentin reverses spinal microglial activation in a rat model(ref).  Since then, all pains or problems associated with the spinal injury have vanished though I have kept myself on a low dose of gabapentin ever since, just-in-case.  What I have to say now is that, having read the research about blueberries and microglia cited above, perhaps blueberries get some or all of the credit for having quenching my activated microglia – instead of or in addition to the gabapentin.  About when the accident happened, I had raised my daily blueberry consumption to a small cupful and this may have helped a lot.  I will soon phase the gabapentin out completely and see what happens.

Please see the medical disclaimer for this blog.

I have written about calorie restriction (CR) a number of times in this blog. CR is the most-proven approach to life extension, involving an ancient biological pathway that works across a variety of species. This blog entry focuses on CR mimetics, substances that can presumably produce the same results as CR, and focuses particularly on mannoheptulose, a sugar in avocados that seems to do the trick.

Ample background on CR can be found in my blog entries Calorie Restriction, longevity, and waiting for proof of what works,  Calorie restriction research roundup – Part I, and Calorie restriction research roundup – Part II.  These entries discuss the molecular pathways involved in CR, and the Part II entry touches on the possible use of resveratrol as a CR mimetic.  These three blog entries ref, ref, and ref touch on the SIRT1 longevity gene and how it activates the CR pathway and on resveratrol and other drugs being developed as likely CR mimetics.  However the blog entry What does Resveratrol do? cites research that throws into question whether resveratrol really activates SIRT1 and is in fact a CR mimetic. 

Actually, a number of substances have been proposed as CR mimetics including several in the anti-aging firewalls combined supplement regimen: resveratrol, carnosine, alpha-lipoic acid, acetyl-l-carnitine, and mixed antioxidants(ref).  The proposed list also includes metformin, gymnema, 2-deoxyglucose, aminoguanidine, hydroxycitrate, thiazolidinediones, lodoacetate, modulators of NPY, exandin, PYY3-36, leptin, oxaloacetate, cinnamon and avocado extract.  I focus here on avocado extract because there seems to be some solid research relating to it.   

A 2009 publication from researchers at several collaborating institutions tells the story, Mannoheptulose: glycolytic inhibitor and novel caloric restriction mimetic. “Caloric restriction (CR) is the most robust and reproducible strategy for retarding aging. Benefits of CR have been demonstrated in multiple species, but application to human or companion animal aging represents a challenge. In 1998 the concept of CR “mimetic” (CRM) was introduced as a method to obtain “anti-aging” and health-promoting benefits of CR without reducing food intake. We hypothesized that an effective CRM would best mimic the effects of CR if it impeded initial stages of energy metabolism. We focused initially on glycolytic inhibition using 2-deoxyglucose (2DG). Upon entry into cells, this glucose analog is phosphorylated and becomes a strong competitive inhibitor of phosphohexose isomerase. 2DG effectively induces a CR-like state in rats based on metabolic effects such as reduced plasma glucose, insulin, body temperature, pulse, heart rate and inhibiting tumor growth. Results show 2DG has a narrow window between efficacy and toxicity so recently we shifted our focus to mannoheptulose (MH), a seven-carbon sugar that reduces glycolysis via hexokinase inhibition. MH appears non-toxic with negligible effects on food intake and BW, and increased insulin tolerance by 25% in mice. MH extends median and maximal lifespan (~15%) in D. melanogaster and median lifespan (~30%) in C3H/HeJ mice. These findings, coupled with simple extraction from avocados, suggest that MH may be a practical, highly effective CRM.”    

“Mannoheptulose is a hexokinase inhibitor. It is a heptose, a monosaccharide with seven carbon atoms. By blocking the enzyme hexokinase, it prevents glucose phosphorylation. As a result less dextrose units are broken down into smaller molecules in an organism. It is found as D-mannoheptulose in avocado(ref).^[1] ” In simple terms, it works to block the metabolism if glucose.
 

From ScienceNews April 20, 2009  “So Roth and his team (from P&G Pet Care, Wayne State University, Southern Illinois University and the Pennington Biomedical Research Institute) have been mining avocados for an alternative — MH (for mannoheptulose). It’s a fairly simple sugar with a 7-carbon backbone. — When fed to mice in fairly concentrated doses (roughly 300 milligrams per kilogram of an animal’s body weight), it improved insulin sensitivity and the clearance of glucose from the blood. Meaning it helped overcome diabetes-like impairments to blood-sugar control. MH supplementation also improved the ability of insulin, a hormone, to get cells throughout the body to do its bidding (and that’s a good thing). –MH revved up the burning of fats in muscle. That’s the opposite of fat deposition and something that these scientists note “would be an expected effect of a calorie restriction mimetic.”  — Treated mice also lived longer — some 30 percent longer than untreated animals. And they were happier, I’m guessing, because they didn’t have to give up most of their chow to achieve this life extension. Indeed, their food intake and weight matched that of untreated mice.”

Virtually everybody has taken acetaminophen (also known as paracetamol) from time to time; probably a lot of it over the years.  It is of course the key ingredient in the over-the-counter pain-killer Tylenol®,  is widely sold as an inexpensive over-the-counter analgesic and is included in a very large number of proprietary OTC pain formulations.  Could something so familiar and so mundane as acetaminophen have anti-aging properties?  That is the story I explore here. 

This is the first blog entry inspired by what I came across in the last few days at the American Aging Association’s 39^th annual meeting in Portland Oregon.  Wandering through the poster presentations I came across one entitled  Acetaminophen improves Protein Translational Signaling in Aged Skeletal Muscle prepared by a team including Miazong Wu and Hua Liu from Marshall University.    Scanning the poster quickly I came across an assertion that grabbed my attention, and that is that “Acetaminophen improves mTOR-related signaling in aged skeletal muscles.”  Further, in a rat study “Compared to 26 and 27 month old F344BN rats the expression of the mTOR complex proteins raptor and GbetaL and the phosphorylation of the negative regulator tuberin/TSC2 (Thr1462) were reduced in the soleus muscles of very aged animals (33 months old).” I thought “improves” the signaling meant reduces it, but I was wrong.

Background on mTOR

I will come back to this research shortly but first want to say why I so-quickly became excited when I scanned the poster.  Inhibition of mTOR signaling is one of extremely few interventions known that can extend lives across a variety of species including mammals.  I have mentioned mTOR frequently in this blog.  See the earlier blog post Longevity genes, mTOR and lifespan.  I repeat “Mammalian target of rapamycin (mTOR) is a protein encoded in humans by the FRAP1 gene.  As the name suggests, mTOR is targeted by the immunosuppressive drug rapamycin, a drug used clinically to treat graft rejection and restenosis and being tested as a treatment for autoimmune diseases.“ Inhibiting the expression of mTOR produces all kinds of health effects in experimental animals.  The blog entry Viva mTOR! Caveat mTOR! tells how feeding  rapamycin to mice late in life inhibits mTOR expression and extends their lives, so rapamycin is being considered as a longevity drug for humans.  I also  describes potential hazards of taking it.  The blog entry More mTOR links to aging theories looks at the involvement of the mTOR pathway in the hypoxic response biochemical pathway involved in aging and in the stem cell supply chain theory of aging.  Finally, I added a candidate theory of aging to my treatise Increasing mTOR signalling. 

A bottom-line conclusion of all this background is that if we could find a safe way to inhibit the expression of mTOR we might well live longer – 14% for males and 8% for females both average and maximum if we are like mice(ref).  So the possibility of being able to extend our lives using something so simple and relatively safe as acetaminophen came across as very exciting to me.  Unfortunately, looking a bit more carefully this possibility seems unlikely.   “Improving” mTOR signaling as described in the poster meant increasing it, not reducing it. So, I am afraid that taking acetaminophen is likely to work at cross-purposes and on the whole be a negative intervention.

Getting back to the current presentation at the AAAS meeting, the poster presentation appeared to document the case for acetaminophen “improving” the expression of TOR in mouse tissues and having rejuvenating effects on mouse muscle tissues fairly well, but that latest work still remains unpublished even in abstract form.  However, some of the same the authors published a paper in 2009 describing earlier stages of their work Aging-Associated Dysfunction of Akt/Protein Kinase B: S-Nitrosylation and Acetaminophen Intervention.  “Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention.  –  Principal Findings — Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, along with decreased levels of insulin receptor beta (IR-β), phosphoinositide 3-kinase (PI3K), phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1) (Ser241). In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS). Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month) animals with acetaminophen (30 mg/kg body weight/day) for 6-months “Our results show that aging skeletal muscle exhibits impaired Akt kinase activity and that acetaminophen-induced improvements in Akt signaling are associated with increases in myocyte size and the expression of myosin and actin, along with decreases in muscle apoptosis.”   

A number of protein analyses studies described in the 2009 paper demonstrated  “Dysfunction of Akt in the very aged muscle can be corrected by acetaminophen,” “Akt dysfunction is associated with increases in iNOS and Akt S-nitrosylation,” “Akt hyper-phosphorylation is associated with a loss of PTEN protein,” “Akt dysfunction is associated with decreases of muscle fiber cross-sectional area,” and “Akt dysregulation is associated with increases in myocyte apoptosis.” 

Of relevance to our focus on mTOR, the 2009 paper reported “Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308) was higher in soleus muscles of very aged rats (33-months). Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR) phosphorylation, — The abundance of phosphorylated mTOR (pmTOR) (Ser2448) and mTOR total protein in the very aged soleus were lower than that in the adult animals (−86.3% and −86.8%, respectively; P<0.05.”  In other words there was an age-related decline in mTOR expression in these muscle cells that accompanied accelerated expression of certain Akt proteins and loss of functionality.   Moreover “chronic acetaminophen treatment restored the amount of phosphorylated and total mTOR to a level equivalent to that seen in 6- and 27-month old animals (P>0.05).” 

The ability of acetaminophen to correct age-related problems in aged muscle was reported on in the above-mentioned 2009 paper, but the role of mTOR in the process is only clarified in the current 2010 yet-unpublished poster presentation.  I quote only selectively from what was on the poster or the printed abstract for it.  “Here we hypothysize that age-related impairments in Akt/mTOR function are associated with reduced translational signaling and that these age-associated alterations, if present, can be attenuated by acetaminophen treatment.” — “Compared to 6 and 27 month old F344BN rats, the expression of the mTOR-complex proteins raptor and GbetaL and the phosphorylation of negative regulator tuberin/TSC2 (Thr1462) were reduced in the soleus muscle of very aged animals (33months old).  These changes in Akt/mTOR pathway signaling proteins were in turn associated with decreased phosphorylation.” “ —  Age-associated alterations in the Akt/mTOR pathway signaling and in the phosphorylation of the stress-responsive eIF2alpha protein were attenuated by chronic acetaminophen treatment –Conclusion: Aging is associated with impairments in the regulation  of proteins thought to be important in controlling mRNA translation and acetaminophen may be useful for the treatment of age-related muscle atrophy by reducing oxidative stress.”  (Whoops, in this condensed presentation I not quite sure where the last conclusion related to oxidative stress comes from.) 

The 2009 publication Impaired overload-induced hypertrophy in Obese Zucker rat slow-twitch skeletal muscle provides independent confirmation that inadequate mTOR expression may be responsible for stress or age-related atrophy in the soleus muscle.  “Taken together, these data suggest that IR or other related co-morbidities may impair the ability of the soleus to activate mTOR signaling and undergo load-induced muscle hypertrophy.”  If you are further interested in the molecular signaling pathways that affect muscle atrophy and hypertrophy, you can start with Akt signalling through GSK-3β, mTOR and Foxo1 is involved in human skeletal muscle hypertrophy and atrophy, and then work your way through the citations. 

So the situation appears to be: 

–         The study described in the poster and its antecedent publication  suggest that age-related muscle atrophy is likely to be due to (or at least associated with) age-related decline in Akt/mTOR signaling and that this atrophy can be at least partially offset by increasing Akt/mTOR signaling using acetaminophen.  In aged muscle cells a strategy for longevity of capability is increasing mTOR signaling.

–         Other studies covered in previous blog entries suggest that median and maximal lifespan of organisms including mice and possibly people can be increased by inhibiting mTOR signaling.  

The paradox is one I have possibly encountered before.  As I recall, there are many who argue that HGH administration can increase muscle strength, vitality and several indicators of youth but there is also evidence that it does not extend and possibly shortens lifespans(ref)(ref). 

Both the poster and the earlier report record feeding the mice with acetaminophen (30 mg/kg body weight/day) for 6-months.  For a 150 pound human being this is equivalent to a 2,045mg daily dose.  Taking large chronic doses of acetaminophen is potentially risky.  “While generally safe for use at recommended doses (1,000 mg per single dose and up to 4,000 mg per day for adults, up to 2,000 mg per day if drinking alcohol^[2]), acute overdoses of paracetamol can cause potentially fatal liver damage and, in rare individuals, a normal dose can do the same; the risk is heightened by alcohol consumption. Paracetamol toxicity is the foremost cause of acute liver failure in the Western world, and accounts for most drug overdoses in the United States, the United Kingdom, Australia and New Zealand(ref).^[3][4][5][6] ” 

I have put the giant bottle of acetaminophen tablets from Costco back under the bathroom sink.

I have written a number of times in this blog about iPSCs (induced pluripotent stem cells) including the exciting possibility of closing the loop in the stem cell supply chain and thereby enabling very long lives.  See the posts IPSCs, telomerase, and closing the loop in the stem cell supply chain, Progress in closing the stem cell supply chain loop and The stem cell supply chain – closing the loop for very long lives.  I have also pointed out in a recent presentation(ref) that, for this concept to become real, a number of technical challenges must be overcome including: a) obtaining iPSCs that are free of DNA contamination, and that have long telomeres and full hESC pluripotency, b) developing reliable means for assuring differentiation into adult stem cells of various types, and c) developing reliable and safe means for introducing  those cells into their respective body niches.  I further stated that although much research is being devoted to these approaches, 10-20 years are likely to be required before the stem cell supply chain can truly be closed in humans.   I believe the main challenges that will have to be faced are of a bioengineering nature. 

There is another application for iPSCs which is likely to become very important in the immediate future: supplying large quantities of specialized body cells for research and drug testing purposes.  It appears that the engineering challenges of producing industrial-quantities of high grade cardiomyocytes suitable for drug-testing purposes have already been solved.  The article iPSC-Derived Human Cardiomyocytes appearing in the May 15 issue of Genetic Engineering & Biotechnology News describes the development, the work of a company Cellular Dynamics International.  Also, the development is covered in an article in PharmTech.  Cardiomyocytes are the cells that make up cardiac muscle.

The engineering challenges of obtaining large numbers of pure cardiomyocytes are not simple.  According to the Gen article “The pharmaceutical industry requires large numbers of purified cell types for screening candidate molecules for efficacy and unintentional toxicity, and the industrialized use of terminal cell types derived from iPSCs has been severely hampered, if not prohibited, by the difficulties of culturing stem cells. — iPSCs, while highly proliferative, are sensitive to manipulation; improper handling can severely restrict their pluripotency and drastically reduce the numbers of subsequently differentiated healthy cells. — Furthermore, while producing terminally differentiated cell types from stem cells using embryoid body (EB) and directed differentiation techniques are well known, the efficiency with which these methods produce terminally differentiated cells is highly variable; a common theme to both techniques is difficulty in producing highly pure (>90%) populations of terminally differentiated cells.”

“Therefore, the key to utilizing stem cell technology on an industrial scale is to develop processes that are both scalable and standardizable for both iPSC maintenance and differentiation. — Cellular Dynamics International’s (CDI) iCell™ Cardiomyocytes are human iPSC-derived cardiomyocytes that possess expected cardiac characteristics, form electrically connected syncytial layers, and exhibit expected electrophysiological and biochemical responses upon exposure to exogenous agents.  CDI’s new technology overcomes barriers in both iPSC maintenance, terminal cell type differentiation, and purification by generating standardized and scalable protocols. The primary production constraint of iPSC husbandry was eliminated by developing a culture system that uses standard single-cell splitting techniques and small molecules to minimize operator-specific effects(ref).”

Cardiomyocytes are highly specialized muscle cells(ref) which contract in a coordinated manner producing heart beats.  This video on the CDI web site shows a monolayer of iPSC-produced cardiomyocytes that is spontaneously beating.  The iPSC-produced cardiomyocytes’ biochemical and electrophysiological properties and gene expression profiles have been tested and appear to match those of human-derived cardiomyocytes(ref).  The cardiomyocytes can be customized.  “–because iPSCs can be derived from individuals with identifiable phenotypes and genotypes, targeted human subpopulation models can be employed early in the discovery and toxicity screening processes(ref).”

CDI has developed technology for directing differentiation of iPSCs, for their proliferation, and for purifying them.  Their basic breakthrough has been in engineering an accurate and efficient production process.  “CDI’s new technology overcomes barriers in both iPSC maintenance, terminal cell type differentiation, and purification by generating standardized and scalable protocols. The primary production constraint of iPSC husbandry was eliminated by developing a culture system that uses standard single-cell splitting techniques and small molecules to minimize operator-specific effects.– iPSC culture scalability was incorporated into the process by building the cell culture system in a parallel fashion to enable the production of billions of iPSCs through the use of CellSTACK® culture chambers (Corning).  — Differentiation of iPSCs into iCell Cardiomyocytes is built on CDI’s platform that utilizes recombinant genetic engineering and antibiotic selection. Prior to iPSC clonal expansion, genes encoding antibiotic resistance and an optional marker under control of a cell-type specific promoter (pan-cardiac for iCell Cardiomyocytes) are introduced into the iPSCs through homologous recombination. — After curation and quality control (QC), the iPSC clone carrying the selectable marker is expanded using iPSC maintenance procedures, harvested, and placed into the directed differentiation protocol of choice. Subsequent to differentiation initiation, the cultures are exposed to the selection agent to leave a pure, targeted cell population. — In the case of iCell Cardiomyocytes, the directed differentiation method produces cardiomyocyte purities greater than 50%, while antibiotic selection subsequently increases this purity to approximately 100%, a level that is necessary to ensure that the observed experimental outcome is due to an effect on cardiomyocytes rather than noncardiac “contaminating” cells. — This process, as currently practiced at CDI, is capable of meeting the foreseeable demand for purified iPSC-derived human cardiomyocytes and is scalable by more than two orders of magnitude, without difficulty, if necessary(ref).”

It is likely that variants of this technique can be used to produce a variety of other cell types “”We intend to launch liver, nerve, and blood vessel cell products over the next 18 months,” said Chris Parker, CDI’s Chief Commercial Officer(ref).”  CDI has recently raised $40.6 million in private equity funding with a total of $70 million since 2004(ref).

The iPSC-derived cells should be more reliable for drug toxicity testing than existing cell lines.  “Drug toxicity that emerges either in late-stage clinical trials or following market launch has been a long-term problem for the pharmaceutical industry. The fundamental challenge is that existing preclinical models do not adequately predict the toxicity of new chemical entities. Current cell models are either primary cell cultures derived from non-human animals or immortal cell lines derived from tumors. Because of their non-human nature or neoplastic life history, they are imperfect predictors of drug toxicity in humans(ref).” 

Developing a viable and robust cardiomyocyte product line based on iPSCs basically required overcoming a number of serious bioengineering challenges.  I think the same will be the case for closing the loop in the stem cell supply chain.

In my presentation Towards a Systems Theory of Aging I argue that the two theories Programmed epigenomic changes and Decline in functioning of the stem cell supply chain are complimentary and equivalent and have the potential for providing a framework for an overall systems view of aging that knits together a large collection of traditional special theories of aging.  In this post, I review some research that is relevant to this assertion, especially with respect to the relationships between the Programmed epigenomic changes theories and the aging theories 6. Chronic Inflammation, 7. Immune System Compromise, 8. Neurological Degeneration, 10. Susceptibility to Cancers, and 11. Susceptibility to Cardiovascular Disease.   

The 2010 publication Epigenetics in atherosclerosis and inflammation is a review study.  “Atherosclerosis is a multifactorial disease with a severe burden on western society. Recent insights into the pathogenesis of atherosclerosis underscore the importance of chronic inflammation in both the initiation and progression of vascular remodelling. — Besides genetic factors also epigenetic mechanisms play an essential and fundamental role in the transcriptional control of gene expression.  –. The concept of epigenetic regulation is gradually being recognized as an important factor in the pathogenesis of atherosclerosis. Recent research provides an essential link between inflammation and reprogramming of the epigenome.” The Programmed epigenomic changes theory of aging asserts that age-related reprogramming of the epigenome increases susceptibility to inflammation and inflammation-related diseases. 

The 2008 publication Epigenetic regulation of gene expression in the inflammatory response and relevance to common diseases highlights the same points, extending their scope to autoimmune diseases and cancers. “It is clear that the epigenetic state is a central regulator of cellular development and activation. Emerging evidence suggests a key role for epigenetics in human pathologies, including in inflammatory and neoplastic disorders. The epigenome is influenced by environmental factors throughout life. Nutritional factors can have profound effects on the expression of specific genes by epigenetic modification, and these may be passed on to subsequent generations with potentially detrimental effects. Many cancers are associated with altered epigenetic profiles, leading to altered expression of the genes involved in cell growth or differentiation. Autoimmune and neoplastic diseases increase in frequency with increasing age, with epigenetic dysregulation proposed as a potential explanation. In support of this hypothesis, studies in monozygotic twins revealed increasing epigenetic differences with age. Differences in methylation status of CpG sites, monoallelic silencing, and other epigenetic regulatory mechanisms have been observed in key inflammatory response genes. The importance of the epigenome in the pathogenesis of common human diseases is likely to be as significant as that of traditional genetic mutations.”A number of studies have been concerned with identifying epigenomic changes associated with particular cancers.  Although they are often highly technical, they show that complicated epigenomic/epigenetic changes are involved in cancer processes. 

 The 2007 publication Epigenetic profiling of multidrug-resistant human MCF-7 breast adenocarcinoma cells reveals novel hyper- and hypomethylated targets is an example.  “Presently, two hypotheses, genetic and epigenetic, have been proposed to explain mechanisms of acquired cancer drug resistance. In the present study, we examined the alterations in epigenetic mechanisms in the drug-resistant MCF-7 human breast cancer cells induced by doxorubicin (DOX) and cisplatin (cisDDP), two chemotherapeutic drugs with different modes of action. Despite this difference, both of the drug-resistant cell lines displayed similar pronounced changes in the global epigenetic landscape showing loss of global DNA methylation, loss of histone H4 lysine 20 trimethylation, increased phosporylation of histone H3 serine 10, and diminished expression of Suv4-20h2 histone methyltransferase compared with parental MCF-7 cells. In addition to global epigenetic changes, the MCF-7/DOX and MCF-7/cisDDP drug-resistant cells are characterized by extensive alterations in region-specific DNA methylation, as indicated by the appearance of the number of differentially methylated DNA genes. A detailed analysis of hypo- and hypermethylated DNA sequences revealed that the acquisition of drug-resistant phenotype of MCF-7 cells to DOX and cisDDP, in addition to specific alterations induced by a particular drug only, was characterized by three major common mechanisms: dysfunction of genes involved in estrogen metabolism (sulfatase 2 and estrogen receptor alpha), apoptosis (p73, alpha-tubulin, BCL2-antagonist of cell death, tissue transglutaminase 2 and forkhead box protein K1), and cell-cell contact (leptin, stromal cell-derived factor receptor 1, activin A receptor E-cadherin) and showed that two opposing hypo- and hypermethylation processes may enhance and complement each other in the disruption of these pathways. These results provided evidence that epigenetic changes are an important feature of cancer cells with acquired drug-resistant phenotype and may be a crucial contributing factor to its development. Finally, deregulation of similar pathways may explain the existence and provide mechanism of cross-resistance of cancer cells to different types of chemotherapeutic agents.”

The 2008 publication Epigenetic mapping and functional analysis in a breast cancer metastasis model using whole-genome promoter tiling microarrays states “Breast cancer metastasis is a complex, multi-step biological process. Genetic mutations along with epigenetic alterations in the form of DNA methylation patterns and histone modifications contribute to metastasis-related gene expression changes and genomic instability. — . RESULTS: We integrated data from the tiling microarrays with targets identified by Ingenuity Pathways Analysis software and observed epigenetic variations in genes implicated in epithelial-mesenchymal transition and with tumor cell migration. We identified widespread genomic hypermethylation and hypomethylation events in these cells and we confirmed functional associations between methylation status and expression of the CDH1, CST6, EGFR, SNAI2 and ZEB2 genes by quantitative real-time PCR. Our data also suggest that the complex genomic reorganization present in cancer cells may be superimposed over promoter-specific methylation events that are responsible for gene-specific expression changes. CONCLUSION: This is the first whole-genome approach to identify genome-wide and gene-specific epigenetic alterations, and the functional consequences of these changes, in the context of breast cancer metastasis to lymph nodes. This approach allows the development of epigenetic signatures of metastasis to be used concurrently with genomic signatures to improve mapping of the evolving molecular landscape of metastasis and to permit translational approaches to target epigenetically regulated molecular pathways related to metastatic progression.”

The 2009 publication  Pituitary tumours: all silent on the epigenetics front states “Investigation of the epigenome of sporadic pituitary tumours is providing a more detailed understanding of aberrations that characterise this tumour type. Early studies, in this and other tumour types adopted candidate-gene approaches to characterise CpG island methylation as a mechanism responsible for or associated with gene silencing. However, more recently, investigators have adopted approaches that do not require a priori knowledge of the gene and transcript, as example differential display techniques, and also genome-wide, array-based approaches, to ‘uncover’ or ‘unmask’ silenced genes. Furthermore, through use of chromatin immunoprecipitation as a selective enrichment technique; we are now beginning to identify modifications that target the underlying histones themselves and that have roles in gene-silencing events. Collectively, these studies provided convincing evidence that change to the tumour epigenome are not simply epiphenomena but have functional consequences in the context of pituitary tumour evolution. Our ability to perform these types of studies has been and is increasingly reliant upon technological advances in the genomics and epigenomics arena. In this context, other more recent advances and developing technologies, and, in particular, next generation or flow cell re-sequencing techniques offer exciting opportunities for our future studies of this tumour type.”Relating to almost all areas of medicine, starting in the early-2000s more and more research publications have been appearing pointing out the importance of epigenomic regulation in disease etiology and progression. 

 For example, the 2008 review publication Epigenetic Regulation of Vascular Endothelial Gene Expression “Epigenetics has emerged as an increasingly powerful paradigm to understand complex non-Mendelian diseases. For example, epigenetics provides a newer perspective for understanding how gene expression is perturbed in prevalent diseases of the human vascular system characterized by a dysfunctional endothelium.⁴” 

The 2009 publication Epigenetics and periodontal disease: future perspectives states “Periodontitis is a multifactorial infection characterized by inflammation and destruction of tooth supporting tissues, as a result of the response of a susceptible host to bacterial challenge. Studies have demonstrated that epigenetic events are able to influence the production of cytokines, contributing to the development of inflammatory diseases. Epigenetic events act through the remodeling of chromatin and can selectively activate or inactivate genes, determining their expression. The epigenetic process, by inducing a change in cytokine profile, may subsequently influence the pathogenesis and determine the outcome of many infectious diseases. These findings may have relevance for inflammatory diseases in which the expression of cytokines is unregulated. The purpose of this review is to show evidence that supports the hypothesis that epigenetic alterations, such as hyper and hypomethylation, of cytokine genes, could help to understand the mechanisms related to periodontal disease activity. Therefore, epigenetics may have future impact on diagnosis and/or therapeutics of periodontal disease.“

The 2009 publication Epigenetic mechanisms that underpin metabolic and cardiovascular diseases relates to other critical disease fronts and focuses on lifelong changes in the epigenome and their affect on disease susceptibilities:

·     “Developmental plasticity enables an organism to respond to environmental cues and adjust its phenotypic development to match its environment.

·     Developmental plasticity is effected, at least in part, by epigenetic changes that are established in early life and modulate gene expression during development and maturity.

·     In mammals, the window during which the epigenome is susceptible to nutritional cues extends from conception to at least weaning.

·     Mismatch between the early and mature environments may result in inappropriate patterns of epigenetic changes and gene expression that increase subsequent susceptibility to metabolic and cardiovascular diseases.

·     The available evidence suggests that interventions to prevent metabolic and cardiovascular diseases should focus on the prenatal and early postnatal periods.”   

The 2008 publication Epigenetic principles and mechanisms underlying nervous system functions in health and disease states “Epigenetics and epigenomic medicine encompass a new science of brain and behavior that are already providing unique insights into the mechanisms underlying brain development, evolution, neuronal and network plasticity and homeostasis, senescence, the etiology of diverse neurological diseases and neural regenerative processes. Epigenetic mechanisms include DNA methylation, histone modifications, nucleosome repositioning, higher order chromatin remodeling, non-coding RNAs, and RNA and DNA editing. RNA is centrally involved in directing these processes, implying that the transcriptional state of the cell is the primary determinant of epigenetic memory. This transcriptional state can be modified not only by internal and external cues affecting gene expression and post-transcriptional processing, but also by RNA and DNA editing through activity-dependent intracellular transport and modulation of RNAs and RNA regulatory supercomplexes, and through trans-neuronal and systemic trafficking of functional RNA subclasses. These integrated processes promote dynamic reorganization of nuclear architecture and the genomic landscape to modulate functional gene and neural networks with complex temporal and spatial trajectories. Epigenetics represents the long sought after molecular interface mediating gene-environmental interactions during critical periods throughout the lifecycle. The discipline of environmental epigenomics has begun to identify combinatorial profiles of environmental stressors modulating the latency, initiation and progression of specific neurological disorders, and more selective disease biomarkers and graded molecular responses to emerging therapeutic interventions. Pharmacoepigenomic therapies will promote accelerated recovery of impaired and seemingly irrevocably lost cognitive, behavioral, sensorimotor functions through epigenetic reprogramming of endogenous regional neural stem cell fate decisions, targeted tissue remodeling and restoration of neural network integrity, plasticity and connectivity.”

The above is just a small sample of the river of applicable literature but enough to make the point: The Programmed epigenomic changes theory of aging is critically implicated in a large number of disease and disease progression processes. It will clearly take a lot more research to establish that this theory is capable of explaining all the phenomena described by all the other theories of aging but I strongly suspect that this will in time happen.

Epigenomic interventions to deal with various diseases are now in the clinical trials phase.  Specifically, a number of histone deacetylase inhibitors are now in clinical trials, basically substances that keep apoptosis and other critical genes active.  Describing these will be the subject of a subsequent post.  I also hope to characterize some interesting research relating lifelong nutrition patterns to epigenomic changes.

The p21 gene has long been known for its role in cell cycle arrest and apoptosis.  In case of DNA damage it signals to the p53 gene to initiate apoptosis of the cell, averting the possibility of tumorgenesis.  Very-recent research indicates something else – expression of P21 is part of what keeps us from growing new limbs or other body parts like salamanders or newts.  This blog post reviews the new research in the context of what has long been known about P21.

Some background on P21

P21 is a cell cycle regulator, specifically a CDK (cyclin-dependent kinase) inhibitor(ref).  It has long been known to impede stem cell differentiation and proper embryonic development.  For example, the 1996 publication Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration states “The CDK inhibitor p21 (WAF-1/CIP-1/SDI-1) has been implicated in DNA damage-induced p53-mediated G1 arrest, as well as in physiological processes, such as cell differentiation and senescence, that do not involve p53 function. — These results provide the first in vivo evidence that appropriate p21 levels are critical in normal development and further implicate p21 in the control of multiple cell-cycle phases.”

P21 has been studied for its role in fibrosis and other lung diseases.  A 2004 publication is entitled Induction of CDK inhibitor p21 gene as a new therapeutic strategy against pulmonary fibrosis.  The 2008 publication P21 regulates TGF-beta1-induced pulmonary responses via a TNF-alpha-signaling pathway relates “Transforming growth factor (TGF)-beta(1) is an essential regulatory cytokine that has been implicated in the pathogenesis of diverse facets of the injury and repair responses in the lung. The types of responses that it elicits can be appreciated in studies from our laboratory that demonstrated that the transgenic (Tg) overexpression of TGF-beta(1) in the murine lung causes epithelial apoptosis followed by fibrosis, inflammation, and parenchymal destruction. Because a cyclin-dependent kinase inhibitor, p21, is a key regulator of apoptosis, we hypothesized that p21 plays an important role in the pathogenesis of TGF-beta(1)-induced tissue responses. — Collectively, our studies demonstrate that p21 regulates TGF-beta(1)-induced apoptosis, inflammation, fibrosis, and alveolar remodeling by interacting with TNF-alpha-signaling pathways.”

Expression of P21 is a barrier to stem cell differentiation.   The 2000 publication Hematopoietic Stem Cell Quiescence Maintained by p21^cip1/waf1 states “Therefore, p21 is the molecular switch governing the entry of stem cells into the cell cycle, and in its absence, increased cell cycling leads to stem cell exhaustion. Under conditions of stress, restricted cell cycling is crucial to prevent premature stem cell depletion and hematopoietic death.”  In the absence of P21, hematopoietic stem cells would not remain quiescent in their niches but would instead prematurely differentiate when stress occurs exhausting the pools of those cells and interrupting the normal functioning of the stem cell supply chain leading to premature death.  The 2009 paper Accelerating stem cell proliferation by down-regulation of cell cycle regulator p21 offers a consistent message.  “Inhibition of the cell cycle regulator p21 results in significant acceleration of mesenchymal stem cell proliferation without promoting spontaneous cellular differentiation.”

P21 is also implicated in active disease processes especially in certain cancers.  For example, the 2009 paper Cell-cycle restriction limits DNA damage and maintains self-renewal of leukemia stem cells argues that p21 gives cancer cells the chance to repair their DNA and keep living.  “Here we demonstrate that expression of the cell-cycle inhibitor p21 is indispensable for maintaining self-renewal of leukemia stem cells. Expression of leukemia-associated oncogenes in mouse haematopoietic stem cells (HSCs) induces DNA damage and activates a p21-dependent cellular response, which leads to reversible cell-cycle arrest and DNA repair.  Activated p21 is critical in preventing excess DNA-damage accumulation and functional exhaustion of leukemic stem cells. These data unravel the oncogenic potential of p21 and suggest that inhibition of DNA repair mechanisms might function as potent strategy for the eradication of the slowly proliferating leukemia stem cells.”

There is a lot more that can be said about P21 but the above is sufficient for this post.

P21 and organ renewal

Interesting news relating P21 to limb and appendage regeneration is reported in a March 2010 report Lack of p21 expression links cell cycle control and appendage regeneration in mice.  “Animals capable of regenerating multiple tissue types, organs, and appendages after injury are common yet sporadic and include some sponge, hydra, planarian, and salamander (i.e., newt and axolotl) species, but notably such regenerative capacity is rare in mammals. The adult MRL mouse strain is a rare exception to the rule that mammals do not regenerate appendage tissue. Certain commonalities, such as blastema formation and basement membrane breakdown at the wound site, suggest that MRL mice may share other features with classical regenerators.  As reported here, MRL fibroblast-like cells have a distinct cell-cycle (G2/M accumulation) phenotype and a heightened basal and wound site DNA damage/repair response that is also common to classical regenerators and mammalian embryonic stem cells.”  MRL mice do not express P21 and are like salamanders in an important respect.  When wounded “The super-healing mice form a “blastema”, a clump of immature cells that behave like stem cells, at the injury site. The blastema cells differentiate into the proper cells, leaving virtually no scar tissue or other trace of the injury. Such regenerative power is almost unknown in mammals. But it is common in amphibians such as the newt and axolotl, which can regrow entire limbs(ref).”  MRL mice are known to be able to at least partially regenerate digits and “have a far superior ability to regenerate cardiac tissue than do regular mice, and humans(ref).”  

The researchers discovered that the wound-injury response of P21-knockout mice was like that of MRL mice.  In response to a hole punched in a mouse’s ear, the MRL and P21- mice formed healing blastemas which closed up the holes like they were never there while ordinary P21+ mice formed scars and the holes remained open. 

What about the absence of the protective role of P21 in P21- and MRL mice?  Are they more cancer prone? “DNA damage is a hallmark of cancer, and regeneration in the healer mice and p21 knockout mice features an increase in DNA damage in the dividing cells at the blastema. — The link between regeneration and cancer notwithstanding, the researchers who originally created the p21 knockout mice haven’t found any evidence that the lack of a p21 gene leads to increased rates of cancer. — Along with the cell proliferation and DNA damage in the heightened regeneration, there’s an increased rate of apoptosis, which kills off cells too severely damaged to be repaired, —  “The combined effects of an increase in highly regenerative cells and apoptosis may allow the cells of these organisms to divide rapidly without going out of control and becoming cancerous,” Heber-Katz (an author of the study) said. “In fact, it is similar to what is seen in mammalian embryos, where p21 also happens to be inactive after DNA damage. The down regulation of p21 promotes the induced pluripotent state in mammalian cells, highlighting a correlation between stem cells, tissue regeneration, and the cell cycle(ref).”

The line of research was conducted in the Wistar Institute in the laboratory of Dr. Ellen Heber-Katz.  Dr. Heber Katz has been involved in highly related research since the 1990s.  In fact they accidentally discovered the regenerative capability of MRL mice back in the mid 90s when they punched holes in the ears of some of these animals to identify them, a standard laboratory procedure.  In normal mice the holes stay permanently open.  In the MRL mice the holes healed closed without a trace.  This set Dr. Katz and her group off on a chain of discoveries based on working with normal and super-healer mice.  In 1998 the group reported on multiple regions in 5 chromosomes connected with wound healing.  The latest discovery is that reported here(ref).The hope is that it may ultimately be possible to regenerate damaged or missing organs, possibly even fingers or limbs in human beings through temporary inactivation of P21. “If humans can be induced to heal like these healer mice, it would be possible to repair skin wounds without scarring, and to induce regrowth of cartilage. Internal healing could be improved, including that of damage from heart attacks, as a 2001 study in PNAS said(ref).”  Spinal cord tissue injury is another potential area of application.

On a personal note, I have been concerned about loss of nerve connections in two fingers and spinal cord tissue damage.  See my posts Nerve regeneration and Spinal cord injury pain – a personal story and a new paradigm.  And I have been looking forward to the still far-off days when these parts of me can be regenerated.  I believe the current research about P21 moves us a step further up the long ladder leading to practical regenerative medicine.