Important research published in late 2010 and 2011 relates to a protein JDP2 that plays a key role in cell-cycle processes.   JDP2 is involved with epigenetic modifications to histones relevant to age-related changes in stem cell differentiation and cell senescence.  Like the previously-discussed Smurf2 gene, JDP2  is involved in the regulation of the differentiation and proliferation of cells.  Its presence or absence affects whether cells differentiate or become senescent.  The new research has implications related to organismal aging and for the Programmed Epigenomic Changes  theory of aging.  I review some of the new publications here and relate new findings to matters I have discussed previously.  The subject is highly technical and the actions of JDP2 are complex   So, I have framed some of the explanations  and interpretations I put forward here in simplified terms.

I start with the abstract of the 2011 electronic publication Jun dimerization protein 2 controls senescence and differentiation via regulating histone modification, and then go on to interpret and amplify certain key points.  “Transcription factor, Jun dimerization protein 2 (JDP2), binds directly to histones and DNAs and then inhibits the p300-mediated acetylation both of core histones and of reconstituted nucleosomes that contain JDP2 recognition DNA sequences. JDP2 plays a key role as a repressor of adipocyte differentiation by regulation of the expression of the gene C/EBPδ via inhibition of histone acetylation. Moreover, JDP2-deficient mouse embryonic fibroblasts (JDP2(-/-) MEFs) are resistant to replicative senescence. JDP2 inhibits the recruitment of polycomb repressive complexes (PRC1 and PRC2) to the promoter of the gene encoding p16(Ink4a), resulting from the inhibition of methylation of lysine 27 of histone H3 (H3K27). Therefore, it seems that chromatin-remodeling factors, including the PRC complex controlled by JDP2, may be important players in the senescence program. The novel mechanisms that underline the action of JDP2 in inducing cellular senescence and suppressing adipocyte differentiation are reviewed.” 

JDP2 is an epigenetic modifier, a transcription factor that inhibits gene expression

In binding to histones and preventing acetylation of core histones, JDP2 keeps the corresponding chromatin tightly wrapped up inhibiting accessibility of promoter regions of certain genes and therefore inhibiting expression of those genes.  “We found that acetylation by p300 is inhibited in a dose-dependent manner by JDP2, when added exogenously. We also found that JDP2 was not acetylated by p300 under our experimental conditions. The inhibitory effect of JDP2 was detected on histone acetylation induced by p300, CREB-binding protein (CBP), p300/CBP-associated protein (PCAF), and general control nonrepressive 5 (GCN5). The overexpression of JDP2 apparently represses the RA-induced acetylation of lysines 8 and 16 of histone H4 and some amino terminal lysine residues of histone H3(ref).”

JDP2 is a repressor of cell differentiation

As stated in the 2010 publication Histone chaperone Jun dimerization protein 2 (JDP2): role in cellular senescence and aging, “Thus JDP2 plays a key role as a repressor of cell differentiation by regulating the expression of genes with an activator protein 1 (AP-1) site via inhibition of histone acetylation and/or assembly and disassembly of nucleosomes.”  Going back to the 2007 publication JDP2 suppresses adipocyte differentiation by regulating histone acetylation, “–JDP2 inhibited both the acetylation of histone H3 in the promoter of the gene for C/EBPdelta and transcription from this promoter. Our data indicate that JDP2 plays a key role as a repressor of adipocyte differentiation by regulating the expression of the gene for C/EBPdelta via inhibition of histone acetylation.”   Additional insight is provided in the November 2010 publication Suppression of cell-cycle progression by Jun dimerization protein-2 (JDP2) involves downregulation of cyclin-A2.  “Fibroblasts derived from embryos of Jdp2KO mice proliferated faster and formed more colonies than fibroblasts from wild-type mice. JDP2 was recruited to the promoter of the gene for cyclin-A2 (ccna2) at the AP-1 site. Cells lacking Jdp2 had elevated levels of cyclin-A2 mRNA. Furthermore, reintroduction of JDP2 resulted in the repression of transcription of ccna2 and of cell-cycle progression. Thus, transcription of the gene for cyclin-A2 appears to be a direct target of JDP2 in the suppression of cell proliferation.” JDP2 is a driver of cell senescence, and does by activating the gene p16(Ink4a)The 2010 publication Epigenetic regulation of p16Ink4a and Arf by JDP2 in cellular senescence recapitulates the complex mechanisms through which JDP2 is a regulator of cellular senescence.   “In response to accumulating cellular stress, cells protect themselves from abnormal growth by entering the senescent stage. Senescence is controlled mainly by gene products from the p16^Ink4a/Arf locus. In mouse cells, the expression of p16^Ink4a and Arf increases continuously during proliferation in cell culture. Transcription from the locus is under complex control. p16^Ink4a and Arf respond independently to positive and negative signals, and the entire locus is epigenetically suppressed by histone methylation that depends on the Polycomb repressive complex-1 and -2 (PRC1 and PRC2). In fact, the PRCs associate with the p16^Ink4a/Arf locus in young proliferating cells and dissociate in aged senescent cells. Thus, it seems that chromatin-remodeling factors that regulate association and dissociation of PRCs might be important players in the senescence program. Here, we summarize the molecular mechanisms that mediate cellular aging and introduce the Jun dimerization protein 2 (JDP2) as a factor that regulates replicative senescence by mediating dissociation of PRCs from the p16^Ink4a/Arf locus.” 

According to the 2009 publication JDP2 (Jun Dimerization Protein 2)-deficient mouse embryonic fibroblasts are resistant to replicative senescence “Senescence protects normal cells from abnormal growth signals and oncogenic transformation by interrupting the cell cycle. Senescence is induced not only by cellular aging but also by the forced activation of the MAPK³ pathway and by genotoxic stressors, such as peroxide and certain DNA-damaging compounds. There is evidence to suggest that, in mice, two inhibitors of progression of the cell cycle, p16^Ink4a and p19^Arf (Arf and p14^ARF in humans), are the main regulators of senescence. These proteins are encoded by overlapping reading frames at the CDKN2A (MTS1) locus (1–3).  The expression of both p16^Ink4a and p19^Arf is enhanced in rodent cells with aging (1, 2). By contrast, in human cells, senescence is generally associated with the increased expression of p16^Ink4a but not of Arf (1–3).” 

The same publication reports “JDP2 (Jun dimerization protein 2, an AP-1 transcription factor) is involved in the regulation of the differentiation and proliferation of cells. We report here that JDP2-deficient mouse embryonic fibroblasts (Jdp2(-/-) MEF) are resistant to replicative senescence. In the absence of JDP2, the level of expression of p16(Ink4a), which is known to rise as normal fibroblasts age, fell significantly when cells were cultured for more than 2 months. Conversely, the overexpression of JDP2 induced the expression of genes for p16(Ink4a) and p19(Arf). Moreover, at the promoter of the gene for p16(Ink4a) in Jdp2(-/-) MEF, the extent of methylation of lysine 27 of histone H3 (H3K27), which is important for gene silencing, increased. Polycomb-repressive complexes (PRC-1 and PRC-2), which are responsible for histone methylation, bound efficiently to the promoter to repress the expression of the gene for p16(Ink4a). As a result, JDP2-deficient MEF became resistant to replicative senescence. Our results indicate that JDP2 is involved in the signaling pathway for senescence via epigenetic regulation of the expression of the gene for p16(Ink4a).” 

The October 2010 publication Histone chaperone Jun dimerization protein 2 (JDP2): role in cellular senescence and aging relates “Senescent cells show a series of alterations, including flatten and enlarged morphology, increase in nonspecific acidic β-galactosidase activity, chromatin condensation, and changes in gene expression patterns. The onset and maintenance of senescence are regulated by two tumor suppressors, p53 and retinoblastoma proteins. The expression of p53 and retinoblastoma proteins is regulated by two distinct proteins, p16(Ink4a) and Arf, respectively, which are encoded by cdkn2a. JDP2 inhibits recruitment of the polycomb repressive complexes 1 and 2 (PRC-1 and PRC-2) to the promoter of the gene that encodes p16(Ink4a) and inhibits the methylation of lysine 27 of histone H3 (H3K27). The PRCs associate with the p16(Ink4a)/Arf locus in young proliferating cells and dissociate from it in senescent cells. Therefore, it seems that chromatin-remodeling factors that regulate association and dissociation of PRCs, and are controlled by JDP2, might play an important role in the senescence program.” 

JDP2 contributes to aging via activation of p16(Ink4a)

I have discussed the role of p16(Ink4a) as an epigenetic driver of aging before in this blog and in my treatise as part of the discussion of the Programmed Epigenomic Changes theory of aging.  Specifically, levels of p16(Ink4a) increase with aging and p16(Ink4a) inhibits the differentiation of adult stem cells.  “Buildup of levels of Ink4a/P16 associated with aging slows down the rate of differentiation of adult stem cells.  “Recent evidence shows that loss of Bmi-1, a polycomb transcriptional repressor of the Ink4a-Arf locus, results in progressive loss of HSCs in adult mice with subsequent failure of hematopoiesis.” – “ These results show that either both p16Ink4a and p19Arf can inhibit HSC self-renewal in a serial transplant setting, or that only p16(Ink4a) is necessary(ref).“

The new research suggests that the smoking gun driving p16(Ink4a) expression and therefore cell senescence is JDP2. 

The 2009 publication Expression of p16(INK4a) in peripheral blood T-cells is a biomarker of human aging relates “Expression of the p16(INK4a) tumor suppressor sharply increases with age in most mammalian tissues, and contributes to an age-induced functional decline of certain self-renewing compartments. These observations have suggested that p16(INK4a) expression could be a biomarker of mammalian aging. To translate this notion to human use, we determined p16(INK4a) expression in cellular fractions of human whole blood, and found highest expression in peripheral blood T-lymphocytes (PBTL). We then measured INK4/ARF transcript expression in PBTL from two independent cohorts of healthy humans (170 donors total), and analyzed their relationship with donor characteristics. Expression of p16(INK4a), but not other INK4/ARF transcripts, appeared to exponentially increase with donor chronologic age. Importantly, p16(INK4a) expression did not independently correlate with gender or body-mass index, but was significantly associated with tobacco use and physical inactivity. In addition, p16(INK4a) expression was associated with plasma interleukin-6 concentration, a marker of human frailty. These data suggest that p16(INK4a) expression in PBTL is an easily measured, peripheral blood biomarker of molecular age.”

The usefulness  of p16(INK4a) as a biomarker of aging is also related in the 2008 publication p16INK4A is a robust in vivo biomarker of cellular aging in human skin.  “To determine whether p16INK4A expression in human skin correlates with donor age, p16INK4A expression was analyzed by immunohistochemistry as well as the expression of the p16INK4A repressor BMI1. Samples from the age groups 0-20, 21-70, and 71-95 years were selected from a bank of healthy human skin. We show that the number of p16INK4A positive cells is significantly higher in elderly individuals compared to the younger age groups. The number of p16INK4A positive cells was found to be increased in both epidermis and dermis, compartments with strictly different proliferative activities. BMI1 gene expression was significantly down-regulated with increasing donor age, whereas no striking age differences were observed for Ki67.  — In conclusion, we provide for the first time evidence that p16INK4A expression directly correlates with chronological aging of human skin in vivo. p16INK4A therefore is a biomarker for human aging in vivo. The data reported here suggest a model for changes in regulatory gene expression that drive aging in human skin.” 

JDP2 affects responses of cells to stress and growth signals by repressing ATF3 

The 2009 publication The ubiquitously expressed bZIP inhibitor, JDP2, suppresses the transcription of its homologue immediate early gene counterpart, ATF3 reports “JDP2 is a ubiquitously expressed bZIP repressor protein. JDP2 binds TPA response element and cyclic AMP response element located within various promoters. JDP2 displays a high degree of homology to the immediate early gene ATF3. ATF3 plays a crucial role in the cellular adaptive response to multiple stress insults as well as growth stimuli. We have identified ATF3 as a potential target gene for JDP2 repression. JDP2 regulates the ATF3 promoter potentially through binding to both the consensus ATF/CRE site and a non-consensus ATF3 auto-repression DNA-binding element. Expression of ATF3 protein in wild-type mouse embryo fibroblast (MEF) cells is below the detectable levels, whereas, JDP2 disrupted MEF cells display noticeable level of ATF3 protein. Following either serum or ER stress stimulation, ATF3 expression is potentiated in JDP2-KO fibroblast cells as compared with wild-type cells. Mice with either JDP2 over-expression or JDP2 disruption display undetectable level of ATF3 protein. However, ATF3 induction in response to either growth or stress signals is dependent on JDP2 expression level. ATF3 induction is attenuated in JDP2 over-expressing mice whereas is potentiated in JDP2-KO mice as compared with the corresponding wild-type mice. Collectively, the data presented strongly suggest that JDP2 plays a role in the determination of the ATF3 adaptive cellular threshold response to different stress insults and growth stimuli.”  

JDP2 may play mixed roles in cancer processes.   

“It is also interesting that JDP2 is one of the candidate oncoproteins that collaborate in the oncogenesis associated with the loss of p27 as the result of insertional mutations [29]. Recent study of tumor cells demonstrated that JDP2 was a tumor suppressor [30](ref).”  The 2002 publication Identification of oncogenes collaborating with p27Kip1 loss by insertional mutagenesis and high-throughput insertion site analysis reported “This analysis identified a remarkable number of putative protooncogenes in these lymphomas, which included loci that were novel as well as those that were overrepresented in p27-/- tumors.  We found that Myc activations occurred more frequently in p27-/- lymphomas than in p27+/+ tumors. We also characterized insertions within two novel loci: (i) the Jun dimerization protein 2 gene (Jundp2), and (ii) an X-linked locus termed Xpcl1. Each of the loci that we found to be frequently involved in p27-/- lymphomas represents a candidate oncogene collaborating with p27 loss.”  The 2004 publication The c-Jun dimerization protein 2 inhibits cell transformation and acts as a tumor suppressor gene reported “we show for the first time the potential role of JDP2 in inhibition of cell transformation and tumor suppression. The mechanism of tumor suppressor action of JDP2 can be partially explained by the generation of inhibitory AP-1 complexes via the increase of JunB, JunD, and Fra2 expression and decrease of c-Jun expression.” 

The 2010 publication The AP-1 repressor protein, JDP2, potentiates hepatocellular carcinoma in mice provides a later take: “RESULTS: JDP2-transgenic mice display normal liver function. JDP2-transgenic mice displayed potentiation of liver cancer, higher mortality and increased number and size of tumors. The expression of JDP2 at the promotion stage was found to be the most critical for enhancing liver cancer severity.– CONCLUSIONS: This study suggests that JDP2 expression may play a critical role in liver cancer development by potentiating the compensatory proliferative response and increased inflammation in the DEN liver cancer model.” 

Comments

There is much more to the literature of JDP2 than I have been able to cover.  However, it is clear that JDP2 is an important actor in the epigenetic cell-cycle regulatory system that relaters to stem cell differentiation capability, cell senescence and aging.   I believe this literature tends to support the Programmed Epigenomic Changes  theory of aging.  According to this theory, aging involves progressive and somewhat systematic changes in the chromatin.  JDP2 is a key player in the program as are the polycomb repressive complexes, P16(INK4a), Arf, P53  and a number of other regulatory proteins.  Yet, the extent and exact functioning of the program remains far from clear.  Most of the literature I have reviewed was focused on what JDP2 does and I saw little if any discussion of what promotes or inhibits JDP2 itself in-vivo.  Do any of the sirtuins impact on JDP2 expression?  Is JDP2 expression conditioned by exercise, by diet, by phytochemicals?  I saw no answers to these questions or a host of others I can think of.  So, I expect there will be much more to report as time progresses.

The US is falling behind other advanced countries in longevity gains.  This is the essential content of stories that appeared this week in almost every major newspaper.  The stories are based on a carefully-crafted study released by the National Academy of Sciences.   I first comment on where the US stands compared to other countries in terms of a few key health statistics.  Then I go on to summarize key findings of the new report.  Finally, largely in an editorial mode I comment on some of the deep-seated cultural factors that are involved, factors that may contribute to the US falling even further behind other leading countries when it comes to health and longevity.

Health statistics –where does the US stand?

Many US citizens like to think of our country as “having the most advanced medical system in the world.”  In terms of health statistics, however, we are very far from a leader.  We are in the middle of the pack and in some cases worse off than developing countries.  Here are a few example statistics:

–         Infant mortality:  According to the CIA World Factbook (2009 statistics) the US ranks 46^th, just below Cuba and Guam.  We have 6.26 deaths per thousand births, nearly three times as many as the first ranking nation Singapore with 2.31 deaths per thousand births.  Other countries doing better than us include Slovenia, the Czech Republic and Iceland.

–         Maternal mortality: 41 countries do better than the US in terms of deaths of mothers upon giving birth according to World Health Organization statistics.  Among the countries with fewer deaths per 100,000 births are Belarus, Bulgaria, Croatia, the Czech Republic, Estonia, Hungary, Kuwait  and Iceland.

–         Percent of rural population having access to improved drinking water:  57 countries rank better than the US including the Ukraine, Tuvalu, Turkey, Tonga, Slovinia, Slovakia, Egypt, Malasia, Macedonia, Thailand, Serbia, Qatar, Greece, Guyana and Montenegro, and the Democratic People’s Republic of Korea according to WHO statistics.

–         People living with HIV or AIDS.  According to the CIA World Factbook we have the world’s 8^th largest population of these people, 1.2 million.  Russia, China and India rank way below us.

Changes in life expectancy

A few days ago the National academy of Sciences issued a report “Explaining Divergent Levels of Longevity in High-Income Countries.”  The findings should be disturbing to US citizens.  We are trailing other advanced countries in both longevity and rate of increase of longevity.

According to the introduction to the report “Over the last 25 years, life expectancy at age 50 in the U.S. has been rising, but at a slower pace than in many other high-income countries, such as Japan and Australia. This difference is particularly notable given that the U.S. spends more on health care than any other nation. Concerned about this divergence, the National Institute on Aging asked the National Research Council to examine evidence on its possible causes. — According to Explaining Divergent Levels of Longevity in High-Income Countries, the nation’s history of heavy smoking is a major reason why lifespans in the U.S. fall short of those in many other high-income nations. Evidence suggests that current obesity levels play a substantial part as well. The book reports that lack of universal access to health care in the U.S. also has increased mortality and reduced life expectancy, though this is a less significant factor for those over age 65 because of Medicare access. For the main causes of death at older ages — cancer and cardiovascular disease — available indicators do not suggest that the U.S. health care system is failing to prevent deaths that would be averted elsewhere. In fact, cancer detection and survival appear to be better in the U.S. than in most other high-income nations, and survival rates following a heart attack also are favorable.”

The report was covered widely in the popular press with headlines like Why is America’s life expectancy lagging?  “Despite spending more on health care than any other country in the world, the U.S. is lagging in life expectancy. American men and women do not live as long as people in France, Japan, and other wealthy nations, and the gap is widening. Now, the National Research Council has attempted to find out why that is. Here, a quick guide to their findings:  — How much does our life expectancy lag? We rank 36th in the world, behind even South Korea and Cuba, with an average life expectancy of 78.3 years. Japan, whose citizens can expect to live to the ripe old age of 83, ranks first. But what is especially worrying is how we’re failing to keep up with other developed nations. Between 1980 and 2006, the life expectancy for men born in the U.S. rose by only 5.5 years — a lower rate than in 21 other developed countries. Women didn’t fare much better, gaining six years in life expectancy since the 1950s, while women from nine other high-income countries gained eight years. — What did the National Research Council say was behind the gap? Smoking, primarily. Even though only about 21 percent of U.S. adults smoke these days, reports Katherine Hobson in The Wall Street Journal, that number was closer to 37 percent back in 1980. “Smoking-related illnesses such as lung cancer can take decades to have an impact on mortality rates.” Men will likely see their average longevity increase in the next few years, as their “smoking habits are well past the peak,” says NPR. Women, however, “took longer to cut back, and the toll of smoking will be around longer.”  — Are there other factors? Obesity also plays some part, but just how much is a “controversial” question, says The Boston Globe. The NRC estimates that obesity could “account for up to a third of the shortfall compared with other rich nations.” Lack of exercise is also a factor. Americans are “among the most sedentary people” in the developed world, says Nathan Seppa at Science News, “vying with Poland for the dubious status of topping that category.”

Looking deeper

The picture is that we are falling behind other advanced countries in both health and longevity.  These statistics are not static.  A few years back the US was at or near the top of the list in most categories of health as well as longevity.  I do not think we in the US are getting worse off; in fact we have been improving.  However, much of the rest of the world is moving a lot faster.   We are falling further and further behind the leaders.   Even some countries we have thought of as “third world” are catching up with and surpassing us when it comes to health and longevity.    I note that the same trends apply to a number of other specific public health measures as well as levels and effectiveness of education, investment in public infrastructure and general quality of life. 

The report described above and most of the press articles blame our relatively poor longevity performance on smoking, a sedentary lifestyle, a non-universal medical system and poor nutrition.  I agree these are the important proximate causes.  However, I believe understanding what is really going on requires looking deeper.   Our society is subject to underlying operating conditions having to do with the cultural situation in the US, conditions rooted deeply in our mindsets and systems that will not go away easily.  I comment editorially on some of these conditions here.

Though the US cultural picture is complex and multi-faceted, it contains strong remnants of a frontier culture, valuing individual initiatives and private solutions over public ones.  While many cultural traditions such as in Europe and Japan value community initiatives to address shared problems, we have tended to value individualistic initiatives.  Distrust of government-based public solutions runs deep in many of us.  And initiatives of giant corporations, even ones bigger than many governments, are seen by many to be preferable to government initiatives.  This value system can be seen very clearly as prevalent in a large sector of our political system.  It also shows up in many dimensions that affect the public health and longevity pictures:

–         Compared to European countries we have fewer and smaller public spaces and a transportation system which is automobile-based with much poorer public transportation options.  This translates into many fewer opportunities or requirements for walking, less exercise, and poorer health in many dimensions(ref)(ref)(ref)(ref).    There is less opportunity for exercise-related stress that lead to cardiovascular and other exercise-related health benefits and hormesis-derived  longevity-enhancing effects. 

 –         Powerful lobbying groups representing private economic interests keep our health care system fragmented, non-universal, and far less cost-effective than single-payer health care systems.  So, many people who cannot afford expensive insurance or private health care do not get their basic health needs taken care of and die early.  We are virtually the only advanced country that does not have universal health care.

     –         There is a general reluctance to invest in infrastructure, be that in education, public spaces, clean water systems or sanitary landfills.  There is a preference for privatized solutions wherever possible – or no solutions at all.  

–         The media system, particularly TV, being commercial puts great emphasis on expensive proprietary drugs for treating diseases and relatively little emphasis on general health, good diet, lifestyle elements and dietary supplements. 

 –         We do not know how effective dietary supplements are as compared to expensive drugs because nobody is willing to underwrite the expensive clinical trials required to find out. –         There is a premium on as little regulation as possible and substantial delays in public health regulations when those regulations affect important industries and economic interests having important political lobbying power:            

o   High-taxation of tobacco products and effective regulation of tobacco use were delayed many decades after the public health issue and life-shortening effect of tobacco use were first identified.  The article Tobacco Smoking and Longevity was published in 1938.  And smoking is still largely condoned and second-hand smoke in the homes of tobacco smokers is still an issue.

               o   The coal industry and electric utilities that burn dirty high-sulphur  coal have effectively lobbied for decades against clean air regulations.  The result is a relatively high incidence of a variety of sometimes-fatal lung diseases, including lung cancer. 

            o   Only in recent years has there been limited control of toxic pollution, and many rivers and lakes and tracks of land remain with high concentrations of toxic substances.  Thousands of toxic waste sites remain not cleaned up threatening water supplies.  Airborn mercury from burning coal and mercury in fish remain largely-unaddressed issues.  The life-shortening effects of toxins are well documented. 

            o   Even the practice of medicine has clearly unhealthy elements such as a drastic increase in CATscan diagnostic X-rays despite the known life-shortening effects of radiation.  See my article Protection Against Radiation – The Second Line of Defense.

             o   Regulation of clearly-unhealthy foods, certain processed foods and junk foods,  is now only getting started and may take decades before it is completely implemented.  Again, poor diets translate into higher prevalence of diseases like diabetes and cardiovascular failure and the consequence is shorter average lifespans.  See the previous  blog entry Public health longevity developments – focus on foods.

Our political system is now deeply divided between those who seek greater government participation for the common good and those who seek less.  At stake is whether we look to government for improving public spaces and our public transportation system, for improving our educational system, for deeper research in the life sciences, for implementing  further health-promoting regulations and for a host of related matters.  The alternative is to reduce government involvements in public health issues, continue to allow industries to regulate themselves and to look to private industries for our solutions.  In my opinion, following this second course is likely to cause us to fall further and further behind the rest of the world with regard to the health and wellbeing of our citizens – eventually into third-world status.

Public health measures like cleaner water, public sanitation systems, cleaner air and barriers to cigarette smoking have been major contributing to longevity in the last 200 years. These measures have contributed significantly to the average lifespan in the US more than doubling during that period. High-tech end-of-life medical developments have had only minor impacts on overall longevity compared to these public initiatives. How do such public health longevity-enhancing interventions get put into place? There is usually first a long period of scientific awareness and then growing public awareness that something can and should be done. After that, what typically follows are initial efforts that are only partially effective, an example being small warnings that were first placed on cigarette boxes. Finally, as a result of innovative leadership, effective action steps are taken, often steps that are controversial like outright smoking bans. In the final phases, the public health issue is often characterized as an economic one where the cost of proposed public health measures are very small compared to the costs of inaction. Here, I review several news items that appeared only during the last week describing the states of public health initiatives related to mass-distributed foods.

Dangerous foods, diet, obesity, disease and shortened lifespans

Research over decades has established the life-shortening impacts of obesity(ref) (ref)(ref) and the roles of saturated fats, trans-fats and excess sugar in soft drinks and processed foods in promoting obesity, cardiovascular diseases, diabetes and cancers, and leading to shorter lives(ref)(ref)(ref)(ref)(ref)(ref). Countless blogs and websites are devoted to these issues and to dietary advice(ref). My focus in this blog entry is not to review that research, but rather to discuss what is being done about these issues from a public health viewpoint. Major public health focus has been on foods likely to lead to an alarming increase in childhood obesity including sweetened sodas, fast foods containing saturated and trans-fats, and on unhealthy school lunches.

Sin taxes

One of the ways of dealing with public health problems associated with marketed substances is to impose “sin taxes” on products that generate public health problems. The idea is that raising the cost of an unhealthy item will result in lesser consumption of that item. The two primary current examples are high taxes on tobacco products and on alcoholic beverages.

The this-week article Tax sugar-sweetened beverages in a British Columbia newspapers is one on a many that have called for an extra tax on sugary soda pop.

But sin taxes tend not to work unless they are set painfully high. An April 2010 Science Daily article Small Soda Taxes Insufficient to Curb onsumption Among Children, Study Finds reports “Small sales taxes on soft drinks in the range currently in force in some states are insufficient to reduce consumption of soda or curb obesity among children, according to a new RAND Corporation study. — Such small taxes may reduce consumption in some subgroups such as children at greater risk for obesity, but reducing consumption for all children would require larger taxes, according to the study published by the journal Health Affairs. — “If the goal is to noticeably reduce soda consumption among children, then it would have to be a very substantial tax” said Roland Sturm, the study’s lead author and a senior economist at RAND, a nonprofit research organization. “A small sales tax on soda does not appear to lead to a noticeable drop in consumption, led alone reduction in obesity.” — Taxes on soft drinks and other sugar-sweetened beverages have been proposed as part of many anti-obesity efforts, with the goal being to discourage consumption of the high-calorie drinks in order to curb excess weight gain. — Researchers estimated the potential effect of soft drink taxes on children’s consumption and weight by examining differences in existing sales taxes on soft drinks between states. Details about state soda taxes were compared to information about weight and soda consumption among 7,300 children enrolled in the Early Childhood Longitudinal Study, which has been gathering information about a national group of children for many years. — Children studied reported drinking an average of six sodas per week, but there was wide variation among the group. Fifteen percent reported drinking no sodas in the prior week, while 10 percent consumed two or more sodas per day.  The amount of soda purchased at school was small. — The analysis could find no significant link between the consumption of soda or weight gain among children and differential taxes on sodas versus other foods. Existing differential taxes (taxes that are imposed on sodas and not other food items sold in grocery stores) are small, averaging 3.5 percent and none are larger than 7 percent.”

Nutrition labels do not necessarily affect eating patterns and can be misleading

Another approach favored by the food industry and the US government up to this point has been nutritional labeling. The logic seems very reasonable: “Tell consumers what they are getting and let them make their own choices.” This approach may be good for a few people who have the knowledge, eyesight and patience to read labels. But the approach tends to be relatively ineffective when it comes to the general public. Just like cigarette warning labels did not initially scare off many smokers, last week’s news reports indicate that calorie listing on restaurant menus have virtually no impact.

A publication appearing in the February 2011 issue of American Journal of Preventative Medicine is Mandatory menu labeling in one fast-food chain in King County, Washington: “As part of a comprehensive effort to stem the rise in obesity, King County, Washington, enforced a mandatory menu-labeling regulation requiring all restaurant chains with 15 or more locations to disclose calorie information at the point of purchase beginning in January 2009. The purpose of this study is to quantify the impact of the King County regulation on transactions and purchasing behavior at one Mexican fast-food chain with locations within and adjacent to King County. To examine the effect of the King County regulation, a difference-in-difference approach was used to compare total transactions and average calories per transaction between seven King County restaurants and seven control locations focusing on two time periods: one period immediately following the law until the posting of drive-through menu boards (January 2009 to July 2009) and a second period following the drive-through postings (August 2009 through January 2010). Analyses were conducted in 2010. No impact of the regulation on purchasing behavior was found. Trends in transactions and calories per transaction did not vary between control and intervention locations after the law was enacted. In this setting, mandatory menu labeling did not promote healthier food-purchasing behavior.”

A number of newspaper articles picked up on this research in the last few days.  Here is an excerpt from one in the Daily Mail, Nutrition labels on fast food ‘won’t stop you eating unhealthily: “Making fast food chains print nutritional facts on the packaging of burgers, fries and other fat laden products does not make an ounce of difference to diners’ choices, according to new research. — A 13-month study of restaurants after mandatory labelling legislation was brought in found customer tastes remained just the same. — Professor Eric Finkelstein, of Duke-National University of Singapore, said: ‘Given the results of prior studies, we had expected the results to be small, but we were surprised we could not detect even the slightest hint of changes in purchasing behaviour as a result of the legislation. — Right choice? Mandatory labelling of fast food products did not stop consumers eating them, a survey found ‘The results suggest mandatory menu labelling, unless combined with other interventions, may be unlikely to significantly influence the obesity epidemic.’ — As part of a comprehensive effort to stem the rise in obesity King County in Washington, which includes Seattle and surrounding areas, brought in the regulation on all restaurant chains with 15 or more outlets from January, 2009. Restaurants had to disclose calorie information at the point of purchase. Some companies in the UK, including McDonald’s, now print facts including the fat, salt, calorie and carbohydrate content of its foods to help people make healthy choices. — But Prof Finkelstein and his colleagues monitored a chain of Mexican restaurants called Taco Time for just over a year and found there was no difference in the eating habits of diners at those situated in King County and those who used ones outside the area where the rule was introduced. — No difference: ‘Traffic light’ health label on a pizza. The total number of sales and average calories per transaction were unaffected by the menu labelling, reports the American Journal for Preventive Medicine. — As part of health care reform, the US
government is planning a nationwide launch of mandatory nutrition information
at the point of purchase for fast-food chains with twenty or more outlets.”

Besides confusing fine-print nutrition labeling on supermarket foods, products often feature pseudo-health claims in much larger print on the fronts of packages. A “low fat” desert product may be taken as healthy when in fact it contains a large amount of sugar. An “all natural” product may contain an unhealthily large amount of fructose sugar. And a number of toxins are perfectly natural. Another news story this week was Front-of-Package Labels Hide Truth: 8-of-10 kids’ foods flunk nutrition standards. “Parents want healthy food for their kids, and they want accurate information to guide them. — But parents are being fundamentally misled, says Prevention Institute’s new study, released today through Strategic Alliance. Claiming Health: Front-of-Package Labeling of Children’s Food examined products with front-of-package labeling–those products that food companies choose to identify as healthier. Claiming Health found that 84% of products studied failed to meet basic nutritional standards. — Contrary to the claims on the labels, study findings reveal: – More than half (57%) of the study products qualified as high sugar, and 95% of products contained added sugar. – More than half (53%) were low in fiber. – More than half (53%) of products did not contain any fruits or vegetables; of the fruits and vegetables found, half came from just 2 ingredients – tomatoes and corn. – 24% of prepared foods were high in saturated fats. – More than 1/3 (36%) of prepared foods & meals were high in sodium.”

Banning junk foods in schools and playgrounds

A news story that appeared last week was WHO calls for junk food ban in schools, playgrounds, as reported by AFP: “GENEVA — Junk food should not be sold in schools and playgrounds, the World Health Organization said Friday in a series of recommendations aimed at promoting a healthy diet and cutting child obesity. — However it fell short of calling for a ban on advertising directed at children for foods high in saturated fats, sugars or salt, opting instead to ask member states to “consider the most effective approach to reduce” such marketing. — The non-binding recommendations will be put to a high-level meeting on the prevention and control of non-communicable diseases during September’s General Assembly in New York, WHO officials said. — “Settings where children gather should be free from all forms of marketing of foods high in saturated fats, trans-fatty acids, free sugars or salt,” said the UN health agency. — “Such settings include, but are not limited to, nurseries, schools, school grounds and pre-school centres, playgrounds, family and child clinics and paediatric services and during any sporting and cultural activities that are held on these premises,” it added . — Some 43 million pre-school children are obese or overweight, according to WHO data. — “Children throughout the world are exposed to marketing of foods high in fat, sugar or salt, which increases the potential of younger generations developing noncommunicable diseases during their lives,” it said. — Six out of ten deaths every year are due to cardiovascular diseases, cancers, diabetes and chronic lung diseases, the WHO warned, pointing out that a common factor of the four main diseases is poor diet.”

Another headline this week was USDA Proposes Cutting Fatty Foods From School Lunches. “According to government data, almost a third of all kids under 19 are obese. In an effort to curb the trend, the USDA is proposing new guidelines on the nutritional content of school lunches. — The plan would place a limit on calories, saturated fat content, and sodium on school meals. It would also ban trans-fats entirely. — Servings for starchy foods, like French fries or tater tots would be limited to one per week. — “I probably wouldn’t have school lunches anymore,” said Kya, a student at Turtle Bay School. “I would kind of eat a little bit.” — Other students say they already enjoy the vegetables. — “Really, I wouldn’t care because they’re still good,” said Cloe, another Turtle Bay student. — The school already offers fresh fruits and a salad bar, something Turtle Bay principal Linda Lawhon should make the transition easier. — “For our students, they already have the choices of greens and fruits that are available to them and they usually choose to take from the salad bar almost daily,” said Lawhon. “I don’t think it’ll be much of a change for them.” — Before the guidelines are finalized, the USDA is requesting input from the public on how to make school lunches both healthier and appealing to kids. Those interested in providing input can do so at Regulations.gov.”

A related headline was School nutrition-guideline changes sought to fight obesity. “Calling it not only a national health issue but also a military one, Agriculture Secretary Tom Vilsack on Thursday proposed to overhaul the nutrition guidelines for public school meals for public school meals for the first time since 1995, when Americans were mostly alarmed by the fat content of food. — The proposed rules are far more wide-ranging and would gradually reduce sodium, limit starchy vegetables, ban most trans fats, require fat-free or lowfat milk, increase whole grains, add more fruits and vegetables, and, for the first time, limit the number of calories children consume daily. The guidelines are consistent, Vilsack said, with first lady Michelle Obama‘s Let’s Move initiative, which promotes healthier eating for children. — “The numbers are rather troubling. We have today nearly a third of our youngsters at risk of being obese or, in fact, are obese in our schools,” he said in a conference call Thursday. He added: “If we do not get our hands around the obesity epidemic in the United States by the year 2018, we will face nearly $344 billion of additional health-care costs. That’s money we won’t be able to spend on innovation and creating jobs and improving our education system.”

Another related story was USDA unveils new school lunch rules “– U.S. Department of Agriculture Secretary Tom Vilsack unveiled Thursday what he called a series of “fundamental changes” his agency plans to make to the country’s school nutrition program. The changes are designed to stem the effects of childhood obesity. — Recognizing the financial burden the nutrition guidelines can place on a school system, in terms of increased equipment, personnel and ingredient costs, Vilsack said his agency has set aside more than $380 million of additional nutrition program funding each year for schools that comply with the new rules. — That funding increase mainly comes in the form of a 6 cent per meal increase in the amount of money the government pays schools through its free- and reduced-price lunch program, which helps students from low-income families across the country eat the meals served in their cafeteria. The government currently pays schools in the 48 contiguous states between $2.32 and $2.89 for each lunch they serve through the program and $1.18 and $1.76 for each breakfast.” It is not clear to me whether this program will be funded by congress.

The guidelines are just guidelines and are not necessarily followed. The Boston Globe in a January 23 2011 Sunday editorial School lunches get healthier commented “While the USDA deserves a pat on the back for updating its guidelines for the first time in 15 years, it should consider further updates, including restrictions on the amount of sugar and processed foods a school can serve. Increasing the number of apples kids consume is beneficial, but not if they are covered in caramel sauce. In short, the USDA should create incentives, and provide encouragement, for schools to provide healthy meals with fresh ingredients, without tilting too far toward food-police tastelessness. — That may be harder than it would appear. According to the USDA’s own estimates, up to 35 percent of schools are out of compliance with even current federal regulations. While schools that follow the new guidelines will get an extra 6 cents per meal from the agriculture department, some school-lunch advocates doubt this rate will cover the extra cost of healthier lunch options. Schools that already meet national standards will surely adapt to the new guidelines, and their students will be healthier for it. But those struggling to keep up with even the outdated regulations may need more help — and a stronger nudge — to stop dishing out so many fries.”

I suspect that these approaches related to school lunches are likely to have a slow but growing effect. Already a number of school districts have moved to better diets and some have even thrown out their soft drink machines. On the other hand, needed change is slow and partial and it may take 20-30 more years for a full effect to be felt. The food industry has traditionally been very effective in blunting USDA regulations they don’t like.

Banning junk food advertising

Another approach involves banning advertising of junk foods, advertising that appears in vast quantities on TV programs viewed by children. One headline that appeared in the press last week was World leaders to discuss junk food ad ban at UN. “The U.N. health agency says world leaders will discuss efforts to clamp down on junk food marketing to children when they meet in New York on Sept 19-20. — The World Health Organization says heads of state will use the U.N. General Assembly meeting to talk about limiting the number and type of ads that children are exposed to. — WHO says 43 million preschool children around the world are overweight or obese. Experts talk of a “fat tsunami” that is already causing millions of premature deaths each year. –Bjorn-Inge Larsen of the Norwegian Directorate of Health told reporters Friday that he expects voluntary measures limiting junk food advertising to eventually evolve into laws banning the practice in the same way that has occurred with tobacco.”

Another headline was Health officials eye junk food ad ban. “It may be time to ban ads for foods high in salt, sugar and trans fats that target children, international health officials say. — Voluntary measures to limit junk food ads eventually could evolve into legislated bans just as tobacco bans did, Bjorn-Inge Larsen of the Norwegian Directorate of Health told reporters Friday in Geneva. — Domestic laws might not work since so many ads reach children through international TV channels, Larsen said. — Non-communicable diseases such as cancer, diabetes and heart and lung disease will top the agenda when heads of state meet at the United Nations General Assembly in New York on Sept. 19 to 20. — About 43 million children aged five and under around the world are overweight or obese, according to the UN health agency. — WHO officials are consulting with food makers such as Coca-Cola, General Mills, Kellogg, Kraft, McDonald’s, Mars, Nestle, Pepsico, Unilever and the World Federation of Advertisers on drawing up a code of conduct that restricts marketing of unhealthy products to children under the age of 12.”

Despite the horrors of the situation involved for 43 million children, I suspect this approach of banning advertising will not get very far. That is because the advertising at stake is worth billions of dollars to gigantic media companies and because the product sales are worth many tens (or hundreds) of billion dollars to the food giants involved. Do I think these companies are more influential in political circles than the World Health Organization and all the nutritional do-gooders in the world combined? Yes, I do.

Wal-Mart move to healthier foods

It takes a giant to influence other giants like food packagers, and Wal-Mart is definitely a giant, the world’s largest retailer with about half of its sales in foods. “Retailing behemoth Wal-Mart Stores, Inc., operates 4404 stores in the U.S. (including Sam’s Club) and 8838 worldwide, at last count(ref).” The news was all over the world’s press three mornings ago. From a Bloomberg news report Wal-Mart to Stock Healthier, More Affordable Foods to Help Fight Obesity: “Wal-Mart Stores Inc., the world’s largest retailer, said it is joining first lady Michelle Obama’s anti-obesity campaign by stocking healthier foods. — The company said it will reformulate thousands of packaged food items by 2015, reducing the salt content by 25 percent and sugar content by 10 percent, and will remove all remaining industrially produced trans fats and partially hydrogenated oils. — “No family should have to choose between food that is healthier for them and food they can afford,” Bill Simon, chief executive officer of U.S. stores for Bentonville, Arkansas-based Wal-Mart, said in a statement. — With more than 140 million customer visits each week, Wal- Mart “is uniquely positioned to make a difference” by making healthy foods more affordable, said Simon, who joined Mrs. Obama for an event today in Washington. — The first lady said Wal-Mart’s initiative is a victory for parents and children that will give families more information and more opportunities to eat more healthy foods. She said that because of company’s size, the move “has the potential to transform the marketplace.” — Wal-Mart said it plans to reduce prices to save customers about $1 billion a year on fresh fruits and vegetables. The company said it would develop “strong criteria” for simple front-of-package seals that would help consumers identify healthier foods, including whole-grain cereals, whole-wheat pastas or unsweetened canned fruit. — Andrea Thomas, Wal-Mart’s senior vice president of sustainability, said lower costs will come in part from planned efforts to make the entire supply chain more efficient, including steps to stock more produce from local farmers to reduce shipping costs.”

I am not claiming the step will make all or even most of Wall Mart foods healthier. For example, it appears the company is doing nothing about sugar-infused soft drinks. But I believe it is an important step that is likely to have immense impact. Wal-Mart suppliers will have to modify their manufacturing processes and product mixes to meet Wal-Mart’s criteria. Once they do this to produce healthier products they will want to sell those products through other retailers as well. Everybody will benefit from more efficient supply chains for healthier food. And other retailers are likely to want to sell foods even healthier than Wal-Mart’s. A health-positive feedback loop is likely to be set off.

Wrapping it up

Almost all the above-quoted news stories appeared in the course of only one week, last week. So my take is on the whole optimistic:

– Obesity is now recognized as a major public health issue and there is good general awareness of what has to be done to address the situation. It seems displacing unhealthy foods is the next big public health issue after slowing tobacco smoking.

– A vital and growing health food industry offers affluent consumers many choices (over $3 billion in 2011 for the health food and supplement store sector alone(ref)). The public-health crosshairs are now on low-cost mass-distributed foods and on the fast food and school lunch sectors.

– The public health issue of foods is being tied to economic issues like future health care costs and national defense, and to the emotional issue of wellbeing of our children.

– Public health measures for providing healthier foods for children and adults are in the advanced planning and initial action phases involving all levels of government, giant corporations and tiny businesses, school systems of every size and households such as my own.

– Even fast-food companies are opting into the push for better nutrition. Three weeks ago McDonalds added oatmeal to their breakfast menu. “But now the stakes are much higher. The government is beating on their door, telling them that it is no longer politically correct to serve greasy, high in fat foods to the general public(ref).”

– For now, dietary suggestions are contained in the anti-aging anti-aging lifestyle regimen in my treatise. Also, for comments on a number of healthy as well as unhealthy foods, see my blog entry Diabetes Part 2: Lifestyle, dietary and supplement interventions

– The food industry is likely to play a mixed role, progressive as well as regressive. Providers of more healthy foods will exercise influence counterbalancing the influence of providers of unhealthy and junk foods. And, big food companies do not want to find themselves facing gigantic lawsuits in the future like cigarette companies are facing now.

– While we are seeing the start concerted action there is a long way to go. Further measures are likely to identified and implemented throughout the century as our science and knowledge and public awareness related to nutrition improves. The point is, the healthier-food train has already left the station.

Those of you who have put together jigsaw puzzles know that every once in a while a piece is found that links together several seemingly unrelated chunks of the puzzle.  The sirtuin SIRT3 is doing that for several chunks of the aging/longevity puzzle, showing a key way in which the Oxidative Damageand the Mitochondrial Damagetheories of aging fit together and how these fit with the known life-extending properties of calorie restriction, with the role of exercise, response to stress and PGC – 1alpha, cell metabolism, several age-related diseases including obesity, diabetes and Alzheimer’s Disease, the FOXO gene family, and actions of P53,  and with the actions of the dietary polyphenol resveratrol.  Much of the relevant research is quite recent. 

Preamble  

(From the introduction to Mitochondrial SIRT3 and heart disease) “The desire to live longer and probably forever has long fascinated mankind. Concoctions to prevent ageing and maintain youth have been described in medical books of ancient civilizations, including Charaka Samhita, the most ancient textbook of Ayurveda (an Indian system of traditional medicine), which is believed to have been written centuries before the birth of Christ. It seems our forefathers found a way to live longer and healthy by undergoing calorie restriction, a diet regimen that is considered to be impractical for modern society where food is surplus and time is scarce. Even though vaccination, antibiotics, better child care, and early disease-detection techniques in combination with modern drugs have helped us to increase our average lifespan, the quest to increase maximal lifespan still remains elusive. The major advances in ageing research that we have witnessed in the past two decades are the rediscovery of benefits of calorie restriction, and the delineation of the molecular mechanism involved in its protective effects. Many studies have proposed that the beneficial effect of calorie restriction is mediated through a set of genes collectively called sirtuins (SIRT1–7).1” 

Mitochondrial sirtuins – focus on SIRT3 

In humans, there are at least seven sirtuins (SIRT1–7), proteins  with diverse actions including the regulation of metabolism and chromatin structure, DNA repair and preservation of genomic integrity.  I have discussed actions of SIRT1 and SIRT6 in several previous blog entries (ref)(ref)(ref)(ref).   Lifespan extension has been linked to actions of sirtuins in various publications(ref).   

SIRT3 is a mitochondrial protein that serves to deacetylate acetyllysine-modified proteins in mitochondria 

From the 2010 publication Mitochondrial sirtuins  “Three sirtuins, SIRT3, 4 and 5, are located within the mitochondrial matrix. SIRT3 and SIRT5 are NAD(+)-dependent deacetylases that remove acetyl groups from acetyllysine-modified proteins and yield 2′-O-acetyl-ADP-ribose and nicotinamide. SIRT4 can transfer the ADP-ribose group from NAD(+) onto acceptor proteins. Recent findings reveal that a large fraction of mitochondrial proteins are acetylated and that mitochondrial protein acetylation is modulated by nutritional status. This and the identification of targets for SIRT3, 4 and 5 support the model that mitochondrial sirtuins are metabolic sensors that modulate the activity of metabolic enzymes via protein deacetylation or mono-ADP-ribosylation.” 

The 2007 publication Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation reports “Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.”

Expression of SIRT3 is controlled by diet and exercise 

The 2009 publication Diet and exercise signals regulate SIRT3 and activate AMPK and PGC-1alpha in skeletal muscle reports “SIRT3 is a member of the sirtuin family of NAD(+)-dependent deacetylases, which is localized to the mitochondria and is enriched in kidney, brown adipose tissue, heart, and other metabolically active tissues. We report here that SIRT3 responds dynamically to both exercise and nutritional signals in skeletal muscle to coordinate downstream molecular responses. We show that exercise training increases SIRT3 expression as well as associated CREB phosphorylation and PGC-1alpha up-regulation. Furthermore, we show that SIRT3 is more highly expressed in slow oxidative type I soleus muscle compared to fast type II extensor digitorum longus or gastrocnemius muscles. Additionally, we find that SIRT3 protein levels in skeletal muscle are sensitive to diet, for SIRT3 expression increases by fasting and caloric restriction, yet it is decreased by high-fat diet. Interestingly, the caloric restriction regimen also leads to phospho-activation of AMPK in muscle. Conversely in SIRT3 knockout mice, we find that the phosphorylation of both AMPK and CREB and the expression of PGC-1alpha are down regulated, suggesting that these key cellular factors may be important components of SIRT3-mediated biological signals in vivo.”

Cellular stress causes SIRT3 to translocate from the nucleus to the mitochondria and to be highly expressed in brown adipose tissue

The 2007 publication SirT3 is a nuclear NAD+-dependent histone deacetylase that translocates to the mitochondria upon cellular stress reports “SirT3 levels have been shown to correlate with extended life span, to localize to the mitochondria, and to be highly expressed in brown adipose tissue. — In humans, SirT3 exists in two forms, a full-length protein of approximately 44 kDa and a processed polypeptide lacking 142 amino acids at its N terminus. We found that SirT3 not only localizes to the mitochondria, but also to the nucleus under normal cell growth conditions. Both the full-length and processed forms of SirT3 target H4-K16 for deacetylation in vitro and can deacetylate H4-K16 in vivo when recruited to a gene. Using a highly specific antibody against the N terminus of SirT3, we found that SirT3 is transported from the nucleus to the mitochondria upon cellular stress. This includes DNA damage induced by Etoposide and UV-irradiation, as well as overexpression of SirT3 itself.”

There are two isoforms of SIRT3 (in mice at least) with somewhat different properties

As time progresses more and more of the detailed structure and workings of SIRT3 are being discovered.  The 2010 publication Characterization of the murine SIRT3 mitochondrial localization sequence and comparison of mitochondrial enrichment and deacetylase activity of long and short SIRT3 isoforms relates “SIRT3 is identified as the major mitochondrial deacetylase. Two distinct isoforms of the murine SIRT3 have been identified with the short isoform having no recognizable mitochondrial localization sequence (MLS) and the long isoform having a putative MLS. A recent study questions the mitochondrial deacetylase activity of this short isoform. In contrast, the long isoform has been shown to be predominantly mitochondrial with robust deacetylase activity.  In this study, we investigate whether the amino-terminus of the long SIRT3 isoform is a legitimate MLS and evaluate in-situ mitochondrial deacetylase activity of both isoforms. We confirm the presence of long and short isoforms in murine liver and kidney. —  Despite lower mitochondrial expression of the short isoform, the capacity to deacetylate mitochondrial proteins and to restore mitochondrial respiration is equally robust following transient transfection of either isoform into SIRT3 knockout embryonic fibroblasts. How these alternative transcripts are regulated and whether they modulate distinct targets is unknown. Furthermore, in contrast to exclusive mitochondrial enrichment of endogenous SIRT3, overexpression of both isoforms shows nuclear localization. This overexpression effect, may partially account for previously observed divergent phenotypes attributed to SIRT3.” 

PGC1-alpha is an upstream activator of SIRT3.  Further, SIRT3 suppresses mitochondrial ROS and promotes mitochondrial biogenesis

As discussed in the blog entry PGC-1alpha and exercise, the protein PGC1-alpha (Peroxisome proliferator-activated receptor gamma coactivator 1-alpha) appears to be the mediator of the health benefits produced by exercise, and plays an important role in the metabolism of both white and brown fat.  PGC1-alpha also plays a role in the the regulation of mitochondrial biogenesis, and is a major factor that regulates muscle fiber type determination.  This protein also appears to be implicated in the regulation of cellular cholesterol homoeostasis, control of blood pressure, and the development of obesity. 

The 2010 publication Sirtuin 3, a new target of PGC-1alpha, plays an important role in the suppression of ROS and mitochondrial biogenesisreports “(PGC-1alpha) plays important roles in adaptive thermogenesis, gluconeogenesis, mitochondrial biogenesis and respiration. PGC-1alpha induces several key reactive oxygen species (ROS)-detoxifying enzymes, but the molecular mechanism underlying this is not well understood.  — RESULTS: Here we show that PGC-1alpha strongly stimulated mouse Sirt3 gene expression in muscle cells and hepatocytes. —  Furthermore, Sirt3 was essential for PGC-1alpha-dependent induction of ROS-detoxifying enzymes and several components of the respiratory chain, including glutathione peroxidase-1, superoxide dismutase 2, ATP synthase 5c, and cytochrome c. Overexpression of SIRT3 or PGC-1alpha in C(2)C(12) myotubes decreased basal ROS level. In contrast, knockdown of mSIRT3 increased basal ROS level and blocked the inhibitory effect of PGC-1alpha on cellular ROS production. Finally, SIRT3 stimulated mitochondrial biogenesis, and SIRT3 knockdown decreased the stimulatory effect of PGC-1alpha on mitochondrial biogenesis in C(2)C(12) myotubes. — CONCLUSION: Our results indicate that Sirt3 functions as a downstream target gene of PGC-1alpha and mediates the PGC-1alpha effects on cellular ROS production and mitochondrial biogenesis. Thus, SIRT3 integrates cellular energy metabolism and ROS generation.”

Calorie restriction results in increased expression of SIRT3 which produces a stronger mitochondrial defense against free radicals resulting in mammals living longer 

A November 2010 publication  Sirt3 Mediates Reduction of Oxidative Damage and Prevention of Age-Related Hearing Loss under Caloric Restriction links SIRT3 to the longevity benefits of calorie restriction.  “Here, we report that CR reduces oxidative DNA damage in multiple tissues and prevents AHL in wild-type mice but fails to modify these phenotypes in mice lacking the mitochondrial deacetylase Sirt3, a member of the sirtuin family. In response to CR, Sirt3 directly deacetylates and activates mitochondrial isocitrate dehydrogenase 2 (Idh2), leading to increased NADPH levels and an increased ratio of reduced-to-oxidized glutathione in mitochondria. In cultured cells, overexpression of Sirt3 and/or Idh2 increases NADPH levels and protects from oxidative stress-induced cell death. Therefore, our findings identify Sirt3 as an essential player in enhancing the mitochondrial glutathione antioxidant defense system during CR and suggest that Sirt3-dependent mitochondrial adaptations may be a central mechanism of aging retardation in mammals.” 

Thus, actions of SIRT3 link up the Oxidative Damageand the Mitochondrial Damagetheories of aging.  The research strongly suggests that minimizing mitochondrial oxidative damage can extend life spans.  Further, three practical sirtuin-related ways of extending life spans appear to be 1. increasing expression of SIRT3 via exercise, 2.  Promotion of expression of SIRT1 via resveratrol and  3.  calorie restriction which appears to affect both SIRT1 and SIRT3. 

According to the November 2010 Science Daily report on this research Scientists Ferret out a Key Pathway for Aging, “It has been well documented in species ranging from spiders to monkeys that a diet with consistently fewer calories can dramatically slow the process of aging and improve health in old age. But how a reduced diet acts at the most basic level to influence metabolism and physiology to blunt the age-related decline of tissues and cells has remained, for the most part, a mystery. — Now, writing in the Nov. 18 online issue of the journal Cell, a team of scientists from the University of Wisconsin-Madison and their colleagues describe a molecular pathway that is a key determinant of the aging process. The finding not only helps explain the cascade of events that contributes to aging, but also provides a rational basis for devising interventions, drugs that may retard aging and contribute to better health in old age. — “We’re getting closer and closer to a good understanding of how caloric restriction works,” says Tomas A. Prolla, a UW-Madison professor of genetics and a senior author of the new Cell study. “This study is the first direct proof for a mechanism underlying the anti-aging effects we observe under caloric restriction.” — The Wisconsin study focuses on an enzyme known as Sirt3, one of a family of enzymes known as sirtuins, which have been implicated in previous studies in the aging process, gene transcription, programmed cell death and stress resistance under reduced calorie conditions. In mammals, including humans, there are seven sirtuins that seem to have wide-ranging influence on cell fate and physiology. — Sirt3 has been less studied than other members of the sirtuin family, but the new study provides “the first clear evidence that sirtuins have anti-aging effects in mammals,” according to John M. Denu of UW-Madison’s Wisconsin Institute for Discovery and a senior author of the report. — The Sirt3 enzyme, Denu explains, acts on mitochondria, structures inside cells that produce energy and that are the sources of highly reactive forms of oxygen known as free radicals, which damage cells and promote the effects of aging. Under reduced-calorie conditions, levels of Sirt3 amp up, altering metabolism and resulting in fewer free radicals produced by mitochondria. — “This is the strongest and most direct link that caloric restriction acts through mitochondria,” says Prolla, who has studied the effects of reduced calorie diets on aging and health for more than a decade. “Sirt3 is playing a surprisingly important role in reprogramming mitochondria to deal with an altered metabolic state under caloric restriction.”

SIRT3 regulates P53-induced cell senescence

The 2010 publication p53-induced growth arrest is regulated by the mitochondrial SirT3 deacetylasereports “A hallmark of p53 function is to regulate a transcriptional program in response to extracellular and intracellular stress that directs cell cycle arrest, apoptosis, and cellular senescence. Independent of the role of p53 in the nucleus, some of the anti-proliferative functions of p53 reside within the mitochondria [1].  p53 can arrest cell growth in response to mitochondrial p53 in an EJ bladder carcinoma cell environment that is naïve of p53 function until induced to express p53 [2]. TP53 can independently partition with endogenous nuclear and mitochondrial proteins consistent with the ability of p53 to enact senescence.In order to address the role of p53 in navigating cellular senescence through the mitochondria, we identified SirT3 to rescue EJ/p53 cells from induced p53-mediated growth arrest. Human SirT3 function appears coupled with p53 early during the initiation of p53 expression in the mitochondria by biochemical and cellular localization analysis. Our evidence suggests that SirT3 partially abrogates p53 activity to enact growth arrest and senescence.  Additionally, we identified the chaperone protein BAG-2 in averting SirT3 targeting of p53 -mediated senescence. These studies identify a complex relationship between p53, SirT3, and chaperoning factor BAG-2 that may link the salvaging and quality assurance of the p53 protein for control of cellular fate independent of transcriptional activity.”

SIRT3 protects in-vitro fertilized embryos against P53-mediated developmental arrest induced by oxidative stress

According to the 2010 publication Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest, “When Sirt3-knockdown embryos were transferred to pseudopregnant mice after long-term culture, implantation and fetal growth rates were decreased, indicating that Sirt3-knockdown embryos were sensitive to in vitro conditions and that the effect was long lasting. Further experiments revealed that maternally derived Sirt3 was critical. Sirt3 inactivation increased mitochondrial ROS production, leading to p53 upregulation and changes in downstream gene expression. The inactivation of p53 improved the developmental outcome of Sirt3-knockdown embryos, indicating that the ROS-p53 pathway was responsible for the developmental defects. These results indicate that Sirt3 plays a protective role in preimplantation embryos against stress conditions during in vitro fertilization and culture.”

SIRT3 affects gene expression in two longevity-related gene families: the sirtuins and FOXO

The 2008 publication SIRT3 interacts with the daf-16 homolog FOXO3a in the mitochondria, as well as increases FOXO3a dependent gene expressionrelates “Cellular longevity is a complex process relevant to age-related diseases including but not limited to chronic illness such as diabetes and metabolic syndromes. Two gene families have been shown to play a role in the genetic regulation of longevity; the Sirtuin and FOXO families. It is also established that nuclear Sirtuins interact with and under specific cellular conditions regulate the activity of FOXO gene family proteins. Thus, we hypothesize that a mitochondrial Sirtuin (SIRT3) might also interact with and regulate the activity of the FOXO proteins. To address this we used HCT116 cells overexpressing either wild-type or a catalytically inactive dominant negative SIRT3. For the first time we establish that FOXO3a is also a mitochondrial protein and forms a physical interaction with SIRT3 in mitochondria. Overexpression of a wild-type SIRT3 gene increase FOXO3a DNA-binding activity as well as FOXO3a dependent gene expression. Biochemical analysis of HCT116 cells over expressing the deacetylation mutant, as compared to wild-type SIRT3 gene, demonstrated an overall oxidized intracellular environment, as monitored by increase in intracellular superoxide and oxidized glutathione levels. As such, we propose that SIRT3 and FOXO3a comprise a potential mitochondrial signaling cascade response pathway.”

SIRT3 blocks the cardiac hypertrophic response in mice via a FOXO-related pathway

The 2009 paper Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice states “Sirtuin 3 (SIRT3) is a member of the sirtuin family of proteins that promote longevity in many organisms. Increased expression of SIRT3 has been linked to an extended life span in humans. Here, we have shown that Sirt3 protects the mouse heart by blocking the cardiac hypertrophic response. Although Sirt3-deficient mice appeared to have normal activity, they showed signs of cardiac hypertrophy and interstitial fibrosis at 8 weeks of age. Application of hypertrophic stimuli to these mice produced a severe cardiac hypertrophic response, whereas Sirt3-expressing Tg mice were protected from similar stimuli. In primary cultures of cardiomyocytes, Sirt3 blocked cardiac hypertrophy by activating the forkhead box O3a-dependent (Foxo3a-dependent), antioxidant-encoding genes manganese superoxide dismutase (MnSOD) and catalase (Cat), thereby decreasing cellular levels of ROS. Reduced ROS levels suppressed Ras activation and downstream signaling through the MAPK/ERK and PI3K/Akt pathways. This resulted in repressed activity of transcription factors, specifically GATA4 and NFAT, and translation factors, specifically eukaryotic initiation factor 4E (elf4E) and S6 ribosomal protein (S6P), which are involved in the development of cardiac hypertrophy. These results demonstrate that SIRT3 is an endogenous negative regulator of cardiac hypertrophy, which protects hearts by suppressing cellular levels of ROS.”

SIRT3 is a tumor suppressor

The 2010 comment in the Journal Cancer Cell A tumor suppressor SIRTainty outlines the story: “Sirtuin deacetylases are linked to longevity, aging, and stress responses. In this issue of Cancer Cell, Kim et al. show that SIRT3 functions as a tumor suppressor by enhancing the expression of mitochondrial MnSOD. Loss of SIRT3 leads to increased mitochondrial ROS, which then enhances cellular transformation and tumor growth.”  The Kim et al. publication is SIRT3 is a mitochondria-localized tumor suppressor required for maintenance of mitochondrial integrity and metabolism during stress.  “This work demonstrates that SIRT3(-/-) mouse embryonic fibroblasts (MEFs) exhibit abnormal mitochondrial physiology as well as increases in stress-induced superoxide levels and genomic instability. Expression of a single oncogene (Myc or Ras) in SIRT3(-/-) MEFs results in in vitro transformation and altered intracellular metabolism. Superoxide dismutase prevents transformation by a single oncogene in SIRT3(-/-) MEFs and reverses the tumor-permissive phenotype as well as stress-induced genomic instability. In addition, SIRT3(-/-) mice develop ER/PR-positive mammary tumors. Finally, human breast and other human cancer specimens exhibit reduced SIRT3 levels. These results identify SIRT3 as a genomically expressed, mitochondria-localized tumor suppressor.”

I comment that this work is coincident with a rising focus on mitochondrial ROS damage as a possible and even probable cause for cancer as outlined in the 2010 publicationThe causes of cancer revisited: “mitochondrial malignancy” and ROS-induced oncogenic transformation – why mitochondria are targets for cancer therapy.  “— more recent evidence indicates the importance of two additional key factors imposed on proliferating cells that are involved in transformation to malignancy and these are hypoxia and/or stressful conditions of nutrient deprivation (e.g. lack of glucose). These two additional triggers can initiate and promote the process of malignant transformation when a low percentage of cells overcome and escape cellular senescence. It is becoming apparent that hypoxia causes the progressive elevation in mitochondrial ROS production (chronic ROS) which over time leads to stabilization of cells via increased HIF-2alpha expression, enabling cells to survive with sustained levels of elevated ROS. –. Recent evidence also indicates that the resulting mutated cancer-causing proteins feedback to amplify the process by directly affecting mitochondrial function in combinatorial ways that intersect to play a major role in promoting a vicious spiral of malignant cell transformation. Consequently, many malignant processes involve periods of increased mitochondrial ROS production when a few cells survive the more common process of oxidative damage induced cell senescence and death. The few cells escaping elimination emerge with oncogenic mutations and survive to become immortalized tumors. — ”

SIRT3 plays an important role in cardioprotection

The 2008 publication SIRT3 Is a Stress-Responsive Deacetylase in Cardiomyocytes That Protects Cells from Stress-Mediated Cell Death by Deacetylation of Ku70 relates “We show that, like human SIRT3, mouse SIRT3 is expressed in two forms, a 44-kDa long form and a 28-kDa short form. Whereas the long form is localized in the mitochondria, nucleus, and cytoplasm, the short form is localized exclusively in the mitochondria of cardiomyocytes. During stress, SIRT3 levels are increased not only in mitochondria but also in the nuclei of cardiomyocytes. We also identified Ku70 as a new target of SIRT3. SIRT3 physically binds to Ku70 and deacetylates it, and this promotes interaction of Ku70 with the proapoptotic protein Bax. Thus, under stress conditions, increased expression of SIRT3 protects cardiomyocytes, in part by hindering the translocation of Bax to mitochondria.”

The 2010 publication Mitochondrial SIRT3 and heart disease reports “Although the role of SIRT3 in cell biology is only beginning to be understood, initial studies have shown that SIRT3 plays a major role in free fatty acid oxidation and maintenance of cellular ATP levels. In the heart SIRT3 has been found to block development of cardiac hypertrophy and protect cardiomyocytes from oxidative stress-mediated cell death. Similarly, SIRT3 has been reported to have tumour-suppressive characteristics. In this article, we review the known effects of SIRT3 in different tissues and relate them to the protection of cardiomyocytes under stress.”

Pharmaceutical researchers are investigating interventions for disease conditions based on promoting or inhibiting SIRT3 activity

The October 2010 Harvard University Office of Technology Development  posting Regulation of hypoxia and glycolysis through modulation of SIRT3 activity: SIRT3 activators for cancer metabolism and SIRT3 inhibitors for vascular diseaserelates“Pharmaceutical targeting of the SIRT3 activity may provide a novel therapeutic strategy for the treatment and/or prevention of cancer and vascular disease – SIRT3 has tumor suppressive function and acts by destabilizing HIF1? and reducing the glycolytic metabolism. A small molecule, protein or gene therapeutic that upregulates SIRT3 would reduce the level of glycolytic metabolism and deprive solid tumors of energy, without significantly impacting healthy cells that rely mostly on the TCA cycle. — “

Concluding: 

One impact on me of reviewing these documents is observing that the classical Oxidative damage theory of aging, the first one covered in my treatise, is far from dead.  The SIRT3 research is giving this theory new life and connecting it to newer ones. 

I have set out here to cover some of the high points of SIRT3 research.  However, the field is moving very fast.  I have possibly left important material out and, without doubt, soon there will be much more to report.

By Brendan Hussey

(I invited Brendan Hussey to generate this blog post after reading his comments to the post Closing the loop in the stem cell supply chain – presented graphically. It was clear that he had something fundamental to say about an important theory of aging and can write clearly and concisely. The only editing modifications I have made are minor corrections and implementing hyperlinks to referenced documents. Otherwise, the post is as written by Brendan. Also, I have posted a comment following the post.  Vince)

In its simplest form this theory states that stochastic DNA damage (mutations) accumulate throughout life, progressively disabling cells, tissues and organs until they can no longer function. This is sensible when one considers “In principle, all other macromolecules are renewable, whereas nuclear DNA, the blueprint of virtually all cellular RNA and proteins, is irreplaceable; any acquired error is permanent and may have irreversible consequences².” The primary sources of DNA damage are largely endogenous; metabolism and replication. Damaged differentiated cells are destroyed naturally through apoptosis (programmed cell death) or they may be frozen in a state of senescence. This is often not a problem in many tissues as these lost cells are replaced via stem cell populations (eg. gut epithelium) or through division of neighboring differentiated cells (eg. liver). It is commonly assumed that accumulation of DNA mutations in stem/progenitor cells is what leads to eventual tissue failure due to lack of tissue homeostasis. While this theory has strong support, the role that DNA damage plays is this homeostatic decline is less well established. As is often the case in biology, the issue is complex and reconciling the data inevitably incorporates arguments from many of the various treaties in this Anti-Aging Firewalls blog..

Chronological & Replicative Damage

An important point of a lecture on mutation from my undergrad was to emphasize “Contrary to popular belief…Most DNA damage is caused by endogenous mutagens.” Consider that it often takes 20-30 years of heavy smoking to succumb to cancer; that is dying of exogenous influence. Endogenously generated mutations result from the most basic and essential of all cellular processes, metabolism and replication. Generally, metabolism as well as exogenous insults can be grouped into the category of chronological damage and replication can be grouped into replicative damage, which results in chronological ageing and replicative ageing ³, respectively (Figure 1). This distinction is a useful one as it allows us to categorize tissues based on their rate of replication (which is tied directly to cell turnover) and level of environmental exposure as well as rate and type of metabolism. This then allows us to predict the types of damage to which each tissue is likely to be vulnerable. As will be discussed in more detail later, tissue homeostasis stratagems depend on these variables and their interactions. For example, replication dilutes insults generated by chronological ageing, such as damaged proteins and lipids, which is important for highly metabolic cells or those with high levels of environmental exposure. However, the trade off is that replication increases mutations to DNA, which accumulate in dividing stem cells.

Metabolism continuously generates toxic byproducts such as reactive oxygen species (ROS) and advanced glycation end products (AGEs) which damage macromolecules such as DNA. ROS and AGEs are major candidates for aging and discussed in more detail in the Oxidative Damage and Tissue Glycation sections of the firewalls treatise. Such metabolic stress generates ~70,000 mutations per day in the form of single strand breaks (SSBs), double strand breaks (DSBs), depurinations, oxidations, etc (ref. lecture from my undergrad). Other estimates are even higher at about 100,000 DNA damage events per cell per day¹². Environmental insults such as radiation, carcinogenic small molecules and viruses also contribute to chronological aging. As such, chronological ageing affects all cell types, differing in intensity depending on the metabolic rate and type of various cells as well as the level environmental exposure and repair fidelity. For example, a highly metabolic tissue with high environmental exposure such as the gut epithelium would be expected to generate a large amount of chronological damage and hence age chronologically more quickly than other tissues. The gut epithelium has a very high cell turnover rate to compensate for such damage, as will be discussed in more detail later.

Replication is another large source of mutagenesis for cells, particularly for the DNA, and is especially a problem for high turnover tissues. A simple calculation from the same lecture illustrates this fact; “Consider just errors made during DNA replication….You each have 46 chromosomes = 6 X 10⁹ bp DNA/cell. On average, a mistake is made once every in 10⁹ bp of DNA copied. So, you have 6 mistakes/cell/division. You have ~10¹⁴ cells in your body that divide a minimum of once per year, so, a very conservative estimate is ~ 6 X 10¹⁴ mistakes per year…Or, at least 60 billion mistakes while in class for MBG*4270 today!!!” These are errors generated just by DNA polymerase, the DNA copying machine, and do not include the large mutations generated by reactivation of retroviruses and similar elements due to replication or the loss of telomere length (see the Telomere Shortening and Damage discussion). The actual error rate of DNA polymerase is 10⁴ bp of DNA copied, but proofreading on the polymerase corrects many of these. Such DNA damage sensing and repair pathways are essential to cell survival. Not surprisingly these pathways are central to the DNA damage theory of aging.

Cellular Response to Mutation and DNA Damage

Before delving into mechanisms, a distinction must be made between two terms which are often used interchangeably; DNA damage and mutation¹¹. DNA damage is a change to the DNA that compromises its functionality such as DSBs, SSBs, interstrand cross-links (ICLs) and telomere shortening, which are difficult to repair and are often cytotoxic resulting in cell death. Mutation, on the other hand, is a change to the coding of the DNA resulting in different functional products such as mutated proteins and can lead to numerous forms of cellular dysfunction such as cancer. Mutations result from polymerase errors in replication and from repair to DNA damage and are not inherently cytotoxic to the same extent as DNA damage. This distinction is central to cell survival and can predict cell turnover rates as well as susceptibility to cancer and aging.

A large amount of evidence for the DNA damage theory of aging comes from studies of humans and mice that have mutations in some aspect of the DNA sensing and repair pathways¹. Mice have proved valuable models for disease in humans “In some cases, a mouse model even paved the way for identifying the parallel human syndrome… leaving no doubt that mouse models and human syndromes constitute valid ageing mutants.¹” Mutations in genes which encode components of the sensing and repair pathways generate either, progeroid (pro-aging-like), cancerous, or both phenotypes depending on the cellular machinery affected.

The molecular mechanisms underlying these phenotypes hint at an interesting pattern and involve one of the most important DNA damage sensing and repair systems, the nucleotide excision repair (NER) pathway. Generally, damage to components which repair DNA damage often results in progeria, whereas damage to components which detect and repair mutations result in cancer. For example, defects in transcription joined NER (TC-NER), which repair damage in transcribed regions as transcription is happening, tend to cause progeria. Whereas, defects in global genome NER (GG-NER), which detect mutations anywhere in the genome at any time, often result in cancer. Defects to core NER components which are shared by both TC and GG-NER and other core cellular components such as DSB repair can cause both progeria and cancer. Progeria seems to be caused by defective repair machinery, that while able to detect mutation, cannot fix it defaulting to an apoptotic response to cytotoxicity via cellular mechanisms independent of NER. This results in increased tissue turnover as damaged cells are quickly discarded and replaced. Incidentally, progeroid syndromes of this type result in decreased incidence of cancer, as it is thought that cancerous cells cannot survive long enough to become malignant.

Cancer, on the other hand, seems to be the result of defective mutation detection machinery (not necessarily part of NER), which allows mutations to accumulate undetected, not triggering apoptosis. As aforementioned, tissue turnover initially helps prevent cancer development as differentiated cells which are harboring mutations are recycled before they can become malignant. Additionally, “…when oncogenic mutations induce pathological cell proliferation, rapid tissue expansion is originally limited by a corresponding increase in cell death. It is only when this natural compensatory mechanism is overcome by additional mutations that cancer progresses…⁵” Progeria and cancer can occur simultaneously depending on the type of DNA damage, the specifics of the pathways involved and importantly, their particular cell turnover rates.

The Role of Cell Turnover

Additional evidence for the link between progeria, cancer and cell turnover is evidenced in experiments involving p53, which promotes apoptosis. “The balance between deleting and preserving damaged cells appears to be particularly important for optimizing the trade-off between aging and cancer. This was elegantly demonstrated in a study by Tyner et al (2002) of which a mutant form of p53 showed constitutive activation. Heterozygotes between mutant and wild-type p53 showed increased p53 activity and had greatly reduced cancer incidence. However, they also showed faster aging. Their shortened life spans were accompanied by accelerated age-related reduction in mass and cellularity of various tissues, including spleen, liver, kidney, and testis. Accelerated age-related losses were also noted in skin thickness, hair growth, wound healing, and stress resistance (to anesthesia and to 5-fluorouracil treatment in hematopoietic precursor cells).²”

Another example comes serendipitously from what began as an interest in chronologically controlled knockout of the DNA damage response gene Atr. “Loss of Atr is toxic to proliferating cells… and when Atr was somatically excised in adult mice in a widespread manner by conditional inactivation, the vast majority of proliferating cells rapidly disappeared, producing marked intestinal atrophy and bone marrow hypoplasia 2 weeks after conditional activation. However, the animals survived this transient period of cell loss because rare stem cells that had not recombined the Atr allele replaced the lost cells. By 1 month after conditional activation, the mice appeared largely normal, with rapidly proliferating tissues that had been fully reconstituted by sporadic Atr-competent cells. Surprisingly though, these reconstituted mice then developed a marked progeroid phenotype a few months later, with osteopaenia, graying and loss of lymphoid and haematopoietic progenitors.¹⁵”

The same pattern is observed in aged mice, when challenged with radiation: “In the case of gut epithelial stem cells, even a very low dose (0.1 Gy) [of radiation] is sufficient to initiate apoptosis… It may be significant that, in aged mice, these stem cells, which exhibit some functional deterioration already, show increased levels of apoptosis in response to low-dose genotoxic stress…In general, in tissues where apoptosis is used to delete damaged cells, increased levels of apoptosis in aged organisms are likely to reflect higher background levels of accumulated cellular damage.²” Humans who have undergone chemo – radiotherapy again tie cell death back to ageing: “Indeed, long-term survivors of chemo- or radiotherapy show evidence of premature ageing.¹”

The idea of a tradeoff between aging and cancer is compelling. The above examples suggest that increased cell turnover protects against cancer, with the cost of aging. At least for the cases in which apoptosis is a response to damage and mutation. While cell turnover rates can be modulated by DNA damage, tissues have inherent turnover rates ranging from very frequent to practically non-existent. For example the intestinal epithelium turns-over as a whole in about 5 days⁴, whereas most CNS neurons never turn over in the lifetime of the organism. Regarding cell turnover, “In humans, the magnitude of this flux is truly astounding—it has been estimated that each of us eradicates and, in parallel, generates a mass of cells equal to almost our entire body weight each year⁵.” Hence, one may expect than that tissues with the highest rates of cell turnover, such as the gut epithelium, to be protected from cancer and very low turnover tissues, such as cardiac muscle and the CNS, to be leading cancer incidence rates. However, what is actually observed is the opposite. In terms of cell division alone, a clear correlation can be found between tissues with the highest cell turnover rates and most cancers, when lung cancer is omitted due to smoking^3,5,13. For example, prostate, breast and colorectal cancers lead incidence rates for combined men and women, which are among tissues with the highest cell turnover rates^3,5. The trend continues with other leading cell turnover tissues such as lymphoid, uterine and skin as cancer leaders.

This correlation may at first seem to be at odds with the aforementioned experimental evidence. However, sensitivity to genomic stress resulting in increased apoptosis is not the same thing as natural cell turnover. As mentioned, co-occurring with progeria is decreased organ cellularity; decreased tissue homeostasis. Stem cells supply the reservoir of replacement cells is most tissues⁵, and the commonly observed increase in apoptosis of stem cell progeny in these models explains the decrease in tissue homeostasis. This is dissimilar from what we consider regular tissue homeostasis in which stem cell progeny maintain tissue homeostasis. It is this difference that can explain why increasing sensitivity to DNA damage and hence increasing apoptosis can prevent cancer at the cost of aging but why normal, homeostatic tissue turnover causes cancer. The sensitivity of progeria models to DNA damage and mutations prevents their progeny from surviving and manifesting cancer, but this is not the case in normal tissue turnover in which the progeny are less sensitive to damage and survive long enough to become cancerous. This is consistent with a model of ageing and mutation showing that “If mutations occur as a result of errors during cell division, the model suggests that a low cellular turnover rate protects both against aging and the development of cancer. On the other hand, if mutations occur independent from cell division (e.g. if DNA is hit by damaging agents), I find that a high cellular turnover rate protects against aging, while it promotes the development of cancer.¹⁰” This model can also help explain why tissues have different cell turnover rates. Tissues such as the gut epithelium, blood cells and skin have high environmental exposure (independent of cell division) and hence a high turnover rate protects them from ageing, at least initially as tissue cellularity is highly maintained. These are also the same tissues that lead the incidence rates of cancer. This tradeoff between growth /tissue homeostasis and repair/maintenance appears to be a case of antagonistic pleiotropy, the evolutionary genetics theory of selection for traits which benefit the young and fertile but compromise the heath of the old. Evolutionary theories are often evoked for explaining differences in cell maintenance as not much information currently exists as to why such differences arose.

The “Disposable Soma” Theory

Due to the genotoxic nature of cell division, the question arises as to why cells take the risk of dividing so often in so many tissues given that examples exist in which cell turnover is not required for tissue fidelity over the lifetime of the organism. Clearly, cellular maintenance can be such that error production is so low, or so well repaired, that cells need not turnover as is the case for CNS neurons. Furthermore, CNS related disorders are far from the leading causes of disability and death in old age¹⁴, demonstrating the functional efficacy of their maintenance. The efficacy of cellular maintenance mechanisms is further exemplified in germ cells, which are immortal and divide massive amounts, especially in mammalian males. This difference in germ cell and somatic cell maintenance is central to the “disposable soma” hypothesis. The disposable soma is an evolutionary theory that seeks to explain the disparity in cellular maintenance between germ and somatic cells by means of limited resource allocation between the two energetically intensive processes of reproduction/growth and repair/maintenance.

Kirkwood says it best; “Somatic maintenance needs only to be good enough to keep the organism in sound physiological condition for as long as it has a reasonable chance of survival in the wild. For example, since more than 90% of wild mice die in their first year…any investment of energy in mechanism for survival beyond this age benefits 10% of the population…Energy is scarce, as shown by the fact that the major cause of mortality for wild mice is cold, due to failure to maintain thermogenesis…The mouse will therefore benefit by investing any spare energy into thermogenesis or reproduction, rather than into better capacity for somatic maintenance and repair, even though this means that damage will eventually accumulate to cause aging…The idea that intrinsic longetivity is tuned to the prevailing level of extrinsic mortality is supported by extensive observation on natural populations…Evolutionary adaptations such as flight, protective shells, and large brains all of which reduce extrinsic mortality, are associated with increased longetvity”². There are numerous lines of supporting evidence for the disposable soma theory and as such it is the most widely supported (see refs 1-3, 6, 7). Some examples include the highly conserved IGF-1/insulin pathway as well as mTOR signaling and the negative correlation between lifespan and fecundity.

The disposable soma theory can help explain (in an evolutionary sense) why some tissues, such as the CNS, do not turn over yet remain highly functional throughout life. The brain, as a defense from extrinsic mortality, is essential to reproductive success and as such is maintained with as much efficacy. This is part of the reason that the human brain is a huge metabolic expense; “Although the brain represents only 2% of the body weight, it receives 15% of the cardiac output, 20% of total body oxygen consumption, and 25% of total body glucose utilization.⁸” However, not dividing helps by removing a large source of mutagenesis as does being isolated from the external environment. Physiological constraints involved in path finding and wiring also help explain why most CNS neurons do not divide in adulthood. Few other tissues benefit from such metabolically expensive cellular maintenance, notably heart muscle, the retina, lung parenchyma and kidneys³. Notice that these organs are also very infrequently the cause of death or incidence of cancer in old age^{13, 14}, suggesting immediate reproductive importance. Additionally, this suggests that highly proliferative tissues are the limiting factors in ageing and culprits of cancer, agreeing with aforementioned evidence relating cellular turnover to the tradeoffs between cancer and progeria. Inevitably, stem cells are suspect as they represent the reservoir for tissue homeostasis. As replication is one of the dominant forces introducing mutations in cells it is only a matter of time before progenitor cells accumulate enough mutations to become cancerous. This is the basis for the widely accepted, but not uncontested, stem cell theory of ageing.

Stem cells

The mere existence of post-mitotic tissues that are maintained for an organism’s lifespan seems to be in sharp contrast to the commonly held belief that aging is a result of stem cell depletion and subsequent lack of tissue turnover. Indeed, this issue is far from resolved. While “ [a]lmost every tissue studied has shown age-related decrements in the rate and/or efficacy of normal cellular turnover and regeneration in response to injury³”, “… there is no evidence that the maximal lifespan of any species is determined by declining stem-cell function or, conversely, that increasing the number or functionality of any single stem-cell population would extend lifespan.³” As reviewed in ref. 3, experiments with haematopoietic stem (HS) cells and skeletal muscle satellite cells show little intrinsic limitations in stem cell functionality that cannot be corrected with transplantation to young, stimulating microenvironments. “When HS cells have been tested in serial-transplantation experiments, complete reconstitution of the blood occurs over several lifespans in mice, and old HS cells are as effective as young HS cells at reconstituting the blood lineages after transplantation” and “…regeneration mediated by aged satellite cells was highly effective when the cells were transplanted into young animals as whole-muscle grafts. In fact, the results were indistinguishable from the regeneration mediated by grafting of young muscle.” This seems to be in contrast to the aging models presented earlier where increased cell turnover and stem cell depletion specifically, as in the Atr mouse, caused aged phenotypes.

However this does not disqualify stem cells as the cause of aging. It merely suggests that cell-autonomous properties of stem cells, such as DNA damage, may not be the driving force for ageing. This brings to light an important detail that needs to be stressed from the stem cell studies; the stem cells were fully functional when transplanted into young hosts BUT they were not acting youthful initially in their aged donors. They were in fact acting quite impaired in many measures from delayed and inefficacious migration to aberrant transcription profiles, all characteristics in aged individuals. This has been shown quite extensively elsewhere as well¹⁵, and also see Stem Cell Supply Chain Breakdown theory of aging. What this suggests is that it is not the stem cells which are damaged per se, it is their environment. In fact, it appears that any measure in which stem cells initially seemed to be impaired can be fixed with the right microenvironment.

In depth discussion of systemic messengers that influence stem cells to cause ageing is beyond the scope of this article but there are numerous candidates worth noting. One source of messengers are senescent stem cells. “Senescent cells increase with age in mice, non-human primates and humans…[and] secrete inflammatory cytokines and other molecules that alter tissue microenvironments.⁶” The role of cytokines suggests a potential role of immune cells, which is provocative given the number of age related autoimmune diseases, most prominently atherosclerosis, the leading cause of death in old age¹⁴ (also see the discussions on Chronic Inflammation, Immune System Compromise and Susceptibility to Cardiovascular Disease). Additionally, senescent cells may alter stem cell proliferation by competing for soluble small molecules such as members of the Transforming Growth Factor-β (TGF- β)and Wnt families, as is seen in drosophila models (see ref 5). Additional evidence comes from the fact that, “In mammals, circulating levels of Wnt signal proteins increase with age, and this increase triggers muscle stem-cell ageing.⁷” TGF- β family members also play a role in mammals “with the identification of myostatin, a mammalian member of the TGF-β superfamily… This molecule is produced by adult skeletal muscle, circulates in the blood, and limits muscle fiber growth.⁵” Along the same lines of evidence for endocrine regulation is liver homeostasis. “A recent study suggests that adult liver homeostasis and regeneration is controlled by a similar mechanism of growth regulation. Bile acids are synthesized from cholesterol in the liver, secreted into the intestine to aid in lipid digestion, and then returned to the liver via the circulatory system. When liver function is compromised, circulating bile acid levels become elevated.

Intriguingly, Huang et al. recently found that bile acid triggers liver regeneration in mice through activation of a nuclear receptor signaling pathway… This observation leads to a simple model in which nuclear receptors regulate liver size by sensing its functional capacity. When liver injury or damage leads to a bile acid buildup, these receptors promote liver growth until normal hepatic function is restored and bile acid levels return to normal.⁵” This example is particularly intriguing as it ties in with the leading causes of death in old age, specifically the role of cholesterol and lipoproteins in the various forms of cardiovascular disease¹⁴. Furthermore, “[w]ith regard to the effects that the accumulation of senescent cells may have, there is evidence that the accumulation of senescent cells plays a role in liver fibrosis…, in immune dysfunction …, osteoarthritis … and in the development of atheroma.¹⁶” Finally, one cannot speak of systemic messengers without at least mentioning the IGF-1/insulin pathway as it is foundational to all ageing research and discussed in detail elsewhere^{1-3,6,7,12,15,16}.

Implications for Ageing Interventions

As a whole, the DNA damage theory of ageing has implications for stem cell treatments, especially those intending to utilize induced plutipotent stem cells (iPSCs) as described in the Stem Cell Supply Chain Breakdown theory of aging. One issue is most iPSCs are derived from differentiated somatic cells such as the commonly used fibroblast. Fibroblasts are highly replicative and unsurprisingly display exponential increases in DNA damage markers with age resulting in senescent cells constituting as many as 35% of fibroblast populations in aged primates¹⁷. This accumulated DNA damage and increased sensitivity to senescence may be one reason why iPSC induction efficiencies are so low and iPSCs show impaired proliferative capacity and early senescence¹⁸. Furthermore, fibroblasts are one of the better donors for iPSC induction besides more pluripotent progenitors¹⁹, suggesting that many other tissues may have even more accumulated DNA damage that hinders their survival. This is supported by the study that generated the first all iPSC mice, which not only showed very low survival rates past early embryonic stages but more importantly that iPSC lines derived from the youngest donor cells had the highest survival rates²⁰.  When one considers these experiments in light of the DNA damage theory of ageing, it appears that supplementing dwindling stem cell populations with iPSCs derived from aged differentiated somatic cells as a form of treatment for longevity promotion would be counterproductive. In order for this type of therapy to be effective, cells with very little DNA damage should be stored and expanded selectively for therapeutic needs for individuals throughout their life. One option is embryonic-like cells harvested very early in life as these cells would harbor the least amount of DNA damage and hence offer the most consistent and safe regenerative potential.

Concluding Statement

To summarize, “…current theoretical understanding suggests that, as cells age, they tend to accumulate damage. The rate at which damage arises is dictated, on the average, by genetically determined energy investments in cellular maintenance and repair, at levels optimized to take into account of evolutionary trade-offs. Long lived organisms make greater investments in cellular maintenance and repair than short lived organisms, resulting in slower accumulation of damage. In order to manage the risk presented by damaged cells, particularly the risk of malignancy, organisms have additionally evolved mechanisms, such as tumor suppressor functions, to deal with damaged cells. The actions of such ‘coping’ mechanisms will frequently involve second tier trade-offs.²” A primary example is cellular turnover, which results in such trade-offs between cancer and ageing. As such, high cell turnover tissues are the limiting factors in the DNA damage theory of aging, as exemplified by the leading causes of death from cancer. Dysregulated stem cells form the heart of failing tissue homeostasis but debate remains on the role of DNA damage in these stem cells. That the local niche environment or other soluble messengers have such a strong influence on stem cells suggests endocrine dysregulation and systemic decline as prime candidates for ageing. Additionally, leading causes of natural, “old age”, death such as atherosclerosis and other forms of cardiovascular disease do not clearly involve DNA damage. It is also important to note issues with applications in this area. From what we understand about the accumulation of DNA damage in differentiated somatic cells, care should be taken when considering the use of iPSCs derived from such cells for regenerative purposes.

References

1.    Garinis GA, van der Horst GT, Vijg J, Hoeijmakers JH. DNA damage and ageing: new-age ideas for an age-old problem. Nat Cell Biol. 2008 Nov;10(11):1241-7. Review.

2.    Kirkwood TB. Understanding the odd science of aging. Cell. 2005 Feb 25;120(4):437-47. Review

3.    Rando TA. Stem cells, ageing and the quest for immortality. Nature. 2006 Jun 29;441(7097):1080-6. Review

4.    Blanpain C, Horsley V, Fuchs E. 2007. Epithelial stem cells: turning over new leaves. Cell 128:445–58

5.    Pellettieri J, Sánchez Alvarado A. Cell turnover and adult tissue homeostasis: from humans to planarians. Annu Rev Genet. 2007;41:83-105. Review

6.    Vijg J, Campisi J. Puzzles, promises and a cure for ageing. Nature. 2008 Aug 28;454(7208):1065-71. Review.

7.    Kenyon CJ. The genetics of ageing.Nature. 2010 Mar 25;464(7288):504-12. Review.

8.    Book Chapter. Brain Energy Metabolism. An Integrated Cellular Perspective. Pierre J. Magistretti, Luc Pellerin, and Jean-Luc Martin. From Psychopharmacology – 4th Generation of Progress, Floyd E. Bloom, MD & David J. Kupfer, MD

9.    Yang J, Benyamin B, McEvoy BP, Gordon S, Henders AK, Nyholt DR, Madden PA, Heath AC, Martin NG, Montgomery GW, Goddard ME, Visscher PM. Common SNPs explain a large proportion of the heritability for human height. Nat Genet. 2010 Jul;42(7):565-9

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13.                       U.S. Cancer Statistics Working Group. United States Cancer Statistics: 1999–2007 Incidence and Mortality Web-based Report. Atlanta: U.S. Department of Health and Human Services, Centers for Disease Control and Prevention and National Cancer Institute; 2010. Available at: www.cdc.gov/uscs.

14.                       World Health Organization (2004). “Annex Table 2: Deaths by cause, sex and mortality stratum in WHO regions, estimates for 2002” (pdf). The world health report 2004 – changing history.

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Brendan Hussey is a B.Sc in Molecular Biology and Genetics, minor Neuroscience, University of Guelph. His e-mail is bjohnhussey@gmail.com.

In the previous blog entry Nitrates and nitrites – Part 1: bad for you I described  how there are two current contradictory views about the value of human consumption of nitrates/nitrites.  One view is that consumption of these substances, perhaps through drinking fertalizer-contaminated water, perhaps by eating processed meats, is dangerous and should be avoided.  The result could be incidences of cancer, Alzheimer’s disease, diabetes or premature aging.  The arguments for this “bad for you” view and research justification for this view are laid out in the previous blog entry.  This current blog entry lays out an opposite and contradictory view – that consumption of nitrates can be “good for you.”  I also comment on some critical differences between the “bad for you” and “good for you” studies and say where I am on the issue.

The question of whether dietary nitrates are friends or foes goes back at least ten years

For many decades nitrates in drinking water were seen to be serious threats to public health, as illustrated by several publication s quoted in the previous “nitrates and nitrites are bad for you” blog entry.  The 1999 review article Dietary nitrate in man: friend or foe? Was one of the first to throw this view into question.  “Based on the premise that dietary nitrate is detrimental to human health, increasingly stringent regulations are being instituted to lower nitrate levels in food and water. Not only does this pose a financial challenge to water boards and a threat to vegetable production in Northern Europe, but also may be eliminating an important non-immune mechanism for host defense. Until recently nitrate was perceived as a purely harmful dietary component which causes infantile methaemoglobinaemia, carcinogenesis and possibly even teratogenesis. Epidemiological studies have failed to substantiate this. It has been shown that dietary nitrate undergoes enterosalivary circulation. It is recirculated in the blood, concentrated by the salivary glands, secreted in the saliva and reduced to nitrite by facultative Gram-positive anaerobes (Staphylococcus sciuri and S. intermedius) on the tongue. Salivary nitrite is swallowed into the acidic stomach where it is reduced to large quantities of NO and other oxides of N and, conceivably, also contributes to the formation of systemic S-nitrosothiols. NO and solutions of acidified nitrite, mimicking gastric conditions, have been shown to have antimicrobial activity against a wide range of organisms. In particular, acidified nitrite is bactericidal for a variety of gastrointestinal pathogens such as Yersinia and Salmonella. NO is known to have vasodilator properties and to modulate platelet function, as are S-nitrosothiols. Thus, nitrate in the diet, which determines reactive nitrogen oxide species production in the stomach (McKnight et al. 1997), is emerging as an effective host defense against gastrointestinal pathogens, as a modulator of platelet activity and possibly even of gastrointestinal motility and microcirculation. Therefore dietary nitrate may have an important therapeutic role to play, not least in the immunocompromised and in refugees who are at particular risk of contracting gastroenteritides.”

A 2007 article with the same theme is Dietary nitrate increases gastric mucosal blood flow and mucosal defense. “Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria. In the acidic stomach, nitrite is further reduced to bioactive nitrogen oxides, including nitric oxide (NO). In this study, we investigated the gastroprotective role of nitrate intake and of luminally applied nitrite against provocation with diclofenac and taurocholate. Mucosal permeability ((51)Cr-EDTA clearance) and gastric mucosal blood flow (laser-Doppler flowmetry) were measured in anesthetized rats, either pretreated with nitrate in the drinking water or given acidified nitrite luminally. Diclofenac was given intravenously and taurocholate luminally to challenge the gastric mucosa. Luminal NO content and nitrite content in the gastric mucus were determined by chemiluminescence. The effect of luminal administration of acidified nitrite on the mucosal blood flow was also investigated in endothelial nitric oxide synthase-deficient mice. Rats pretreated with nitrate or given nitrite luminally had higher gastric mucosal blood flow than controls. Permeability increased more during the provocation in the controls than in the nitrate- and nitrite-treated animals. Dietary nitrate increased luminal NO levels 50 times compared with controls. Nitrate intake also resulted in nitrite accumulation in the loosely adherent mucous layer; after removal of this mucous layer, blood flow was reduced. Nitrite administrated luminally in endothelial nitric oxide synthase-deficient mice increased mucosal blood flow. We conclude that dietary nitrate and direct luminal application of acidified nitrite decrease diclofenac- and taurocholate-induced mucosal damage. The gastroprotective effect likely involves a higher mucosal blood flow caused by nonenzymatic NO production. These data suggest an important physiological role of nitrate in the diet.”

Oral bacteria appears to play an important role in converting ingested nitrates into nitrites

This point is made in the 2008 publication The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash.  “Recent studies surprisingly show that dietary inorganic nitrate, abundant in vegetables, can be metabolized in vivo to form nitrite and then bioactive nitric oxide. A reduction in blood pressure was recently noted in healthy volunteers after dietary supplementation with nitrate; an effect consistent with formation of vasodilatory nitric oxide. Oral bacteria have been suggested to play a role in bioactivation of nitrate by first reducing it to the more reactive anion nitrite. In a cross-over designed study in seven healthy volunteers we examined the effects of a commercially available chlorhexidine-containing antibacterial mouthwash on salivary and plasma levels of nitrite measured after an oral intake of sodium nitrate (10mg/kg dissolved in water). In the control situation the salivary and plasma levels of nitrate and nitrite increased greatly after the nitrate load. Rinsing the mouth with the antibacterial mouthwash prior to the nitrate load had no effect on nitrate accumulation in saliva or plasma but abolished its conversion to nitrite in saliva and markedly attenuated the rise in plasma nitrite. We conclude that the acute increase in plasma nitrite seen after a nitrate load is critically dependent on nitrate reduction in the oral cavity by commensal bacteria. The removal of these bacteria with an antibacterial mouthwash will very likely attenuate the NO-dependent biological effects of dietary nitrate.”

The salutatory effects of consuming nitrates seem to revolve around release of nitric oxide which improves blood flow.  Many other health benefits are reported in addition to protection against gastrointesinal pathogens.

Dietary nitrates are protective of the heart and other organs

The 2009 publication Dietary nitrate and nitrite modulate blood and organ nitrite and the cellular ischemic stress response relates “Dietary nitrate, found in abundance in green vegetables, can be converted to the cytoprotective molecule nitrite by oral bacteria, suggesting that nitrate and nitrite may represent active cardioprotective constituents of the Mediterranean diet. We therefore tested the hypothesis that dietary nitrate and nitrite levels modulate tissue damage and ischemic gene expression in a mouse liver ischemia-reperfusion model. We found that stomach content, plasma, heart, and liver nitrite levels were significantly reduced after dietary nitrate and nitrite depletion and could be restored to normal levels with nitrite supplementation in water. Remarkably, we confirmed that basal nitrite levels significantly reduced liver injury after ischemia-reperfusion. Consistent with an effect of nitrite on the posttranslational modification of complex I of the mitochondrial electron transport chain, the severity of liver infarction was inversely proportional to complex I activity after nitrite repletion in the diet. The transcriptional response of dietary nitrite after ischemia was more robust than after normoxia, suggesting a hypoxic potentiation of nitrite-dependent transcriptional signaling. Our studies indicate that normal dietary nitrate and nitrite levels modulate ischemic stress responses and hypoxic gene expression programs, supporting the hypothesis that dietary nitrate and nitrite are cytoprotective components of the diet.”

Nitrates and brain functioning

The 2010 publication Acute effect of a high nitrate diet on brain perfusion in older adults reports “AIMS  Poor blood flow and hypoxia/ischemia contribute to many disease states and may also be a factor in the decline of physical and cognitive function in aging. Nitrite has been discovered to be a vasodilator that is preferentially harnessed in hypoxia. Thus, both infused and inhaled nitrite are being studied as therapeutic agents for a variety of diseases. In addition, nitrite derived from nitrate in the diet has been shown to decrease blood pressure and improve exercise performance. Thus, dietary nitrate may also be important when increased blood flow in hypoxic or ischemic areas is indicated. These conditions could include age-associated dementia and cognitive decline. The goal of this study was to determine if dietary nitrate would increase cerebral blood flow in older adults. METHODS AND RESULTS In this investigation we administered a high vs. low nitrate diet to older adults (74.7 ± 6.9 years) and measured cerebral perfusion using arterial spin labeling magnetic resonance imaging. We found that the high nitrate diet did not alter global cerebral perfusion, but did lead to increased regional cerebral perfusion in frontal lobe white matter, especially between the dorsolateral prefrontal cortex and anterior cingulate cortex. CONCLUSION These results suggest that dietary nitrate may be useful in improving regional brain perfusion in older adults in critical brain areas known to be involved in executive functioning.”

Dietary nitrates, metabolic syndrome and visceral fat accumulation

The 2010 publication Dietary inorganic nitrate reverses features of metabolic syndrome in endothelial nitric oxide synthase-deficient mice states “The metabolic syndrome is a clustering of risk factors of metabolic origin that increase the risk for cardiovascular disease and type 2 diabetes. A proposed central event in metabolic syndrome is a decrease in the amount of bioavailable nitric oxide (NO) from endothelial NO synthase (eNOS). Recently, an alternative pathway for NO formation in mammals was described where inorganic nitrate, a supposedly inert NO oxidation product and unwanted dietary constituent, is serially reduced to nitrite and then NO and other bioactive nitrogen oxides. Here we show that several features of metabolic syndrome that develop in eNOS-deficient mice can be reversed by dietary supplementation with sodium nitrate, in amounts similar to those derived from eNOS under normal conditions. In humans, this dose corresponds to a rich intake of vegetables, the dominant dietary nitrate source. Nitrate administration increased tissue and plasma levels of bioactive nitrogen oxides. Moreover, chronic nitrate treatment reduced visceral fat accumulation and circulating levels of triglycerides and reversed the prediabetic phenotype in these animals. In rats, chronic nitrate treatment reduced blood pressure and this effect was also present during NOS inhibition. Our results show that dietary nitrate fuels a nitrate-nitrite-NO pathway that can partly compensate for disturbances in endogenous NO generation from eNOS. These findings may have implications for novel nutrition-based preventive and therapeutic strategies against cardiovascular disease and type 2 diabetes.”

The amazing properties of beetroot juice

A number of recent publications have been concerned with the positive circulatory effects of consuming beetroot juice, a juice high in nitrates.  The nitrates convert in the mouth into nitrites which produce nitric oxide which dilates blood vessels reducing blood pressure and increasing circulation.

Nitrates reduce blood pressure

The 2010 publication Inorganic nitrate supplementation lowers blood pressure in humans: role for nitrite-derived NO reports “Ingestion of dietary (inorganic) nitrate elevates circulating and tissue levels of nitrite via bioconversion in the entero-salivary circulation. In addition, nitrite is a potent vasodilator in humans, an effect thought to underlie the blood pressure-lowering effects of dietary nitrate (in the form of beetroot juice) ingestion. Whether inorganic nitrate underlies these effects and whether the effects of either naturally occurring dietary nitrate or inorganic nitrate supplementation are dose dependent remain uncertain. Using a randomized crossover study design, we show that nitrate supplementation (KNO(3) capsules: 4 versus 12 mmol [n=6] or 24 mmol of KNO(3) (1488 mg of nitrate) versus 24 mmol of KCl [n=20]) or vegetable intake (250 mL of beetroot juice [5.5 mmol nitrate] versus 250 mL of water [n=9]) causes dose-dependent elevation in plasma nitrite concentration and elevation of cGMP concentration with a consequent decrease in blood pressure in healthy volunteers. In addition, post hoc analysis demonstrates a sex difference in sensitivity to nitrate supplementation dependent on resting baseline blood pressure and plasma nitrite concentration, whereby blood pressure is decreased in male volunteers, with higher baseline blood pressure and lower plasma nitrite concentration but not in female volunteers. Our findings demonstrate dose-dependent decreases in blood pressure and vasoprotection after inorganic nitrate ingestion in the form of either supplementation or by dietary elevation. In addition, our post hoc analyses intimate sex differences in nitrate processing involving the entero-salivary circulation that are likely to be major contributing factors to the lower blood pressures and the vasoprotective phenotype of premenopausal women.”

Nitrites are cardioprotective

The 2009 publication Myocardial protection by nitrite explains: “Nitrite has long been considered to be an inert oxidative metabolite of nitric oxide (NO). Recent work, however, has demonstrated that nitrite represents an important tissue storage form of NO that can be reduced to NO during ischaemic or hypoxic events. This exciting series of discoveries has created an entirely new field of research that involves the investigation of the molecular, biochemical, and physiological activities of nitrite under a variety of physiological and pathophysiological states. This has also led to a re-evaluation of the role that nitrite plays in health and disease. As a result there has been an interest in the use of nitrite as a therapeutic strategy for the treatment of acute myocardial infarction. Nitrite therapy has now been studied in several animal models and has proven to be an effective means to reduce myocardial ischaemia-reperfusion injury. This review article will provide a brief summary of the key findings that have led to the re-evaluation of nitrite and highlight the evidence supporting the cardioprotective actions of nitrite and also highlight the potential clinical application of nitrite therapy to cardiovascular diseases.”

The 2009 publication Emerging role of nitrite in myocardial protection relates “Nitrite has long been considered an inert oxidative metabolite of nitric oxide (NO). However, recent experimental findings strongly suggest that nitrite is a critical storage form of NO that is converted back into NO during ischemic or hypoxic events as well as under physiological conditions. Thus, the conversion of nitrite into NO during cellular stress may be an evolutionarily conserved and redundant means for NO generation at a time when endothelial nitric oxide synthase is non-functional. As a result of the recent revelation that the nitrite anion serves an important biological function a large number of studies have been performed to characterize both the physiological actions and therapeutic potential of nitrite under diverse conditions. While the earliest experiments characterized the vasodilatory effects of nitrite in both animal models and humans, more recent research efforts have focused on the potential benefits of nitrite in a number of pathological states. Nitrite therapy has now been studied in numerous animal models and has proven to be an effective means to ameliorate myocardial ischemia-reperfusion (I/R) injury. This review will focus on recent experimental findings related to the cytoprotective actions of nitrite therapy in the setting of myocardial I/R injury.”

Dietary nitrates make exercise more efficient

A very-recent (December 2010) report A toast to health and performance! Beetroot juice lowers blood pressure and the O2 cost of exercise relates “Dietary nitrate administered in the form of beetroot juice decreases resting systolic blood pressure (SBP) and O(2) consumption during walking and running. The effects of dietary nitrate are thought to be mediated via reduction to biologically active nitrite and nitric oxide (NO) molecules. Potential mechanisms for dietary nitrate effects on O(2) cost of exercise are improved matching of O(2) delivery and consumption of active motor units, increased efficiency of mitochondrial oxidative phosphorylation, and stoichiometry of calcium transport to ATP hydrolysis by the sarcoplasmic reticulum calcium-ATPase.”

The 2010 study Acute and chronic effects of dietary nitrate supplementation on blood pressure and the physiological responses to moderate-intensity and incremental exercise relates “Dietary nitrate (NO(3)(-)) supplementation with beetroot juice (BR) over 4-6 days has been shown to reduce the O(2) cost of submaximal exercise and to improve exercise tolerance. However, it is not known whether shorter (or longer) periods of supplementation have similar (or greater) effects. We therefore investigated the effects of acute and chronic NO(3)(-) supplementation on resting blood pressure (BP) and the physiological responses to moderate-intensity exercise and ramp incremental cycle exercise in eight healthy subjects. Following baseline tests, the subjects were assigned in a balanced crossover design to receive BR (0.5 l/day; 5.2 mmol of NO(3)(-)/day) and placebo (PL; 0.5 l/day low-calorie juice cordial) treatments. The exercise protocol (two moderate-intensity step tests followed by a ramp test) was repeated 2.5 h following first ingestion (0.5 liter) and after 5 and 15 days of BR and PL. Plasma nitrite concentration (baseline: 454 ± 81 nM) was significantly elevated (+39% at 2.5 h postingestion; +25% at 5 days; +46% at 15 days; P < 0.05) and systolic and diastolic BP (baseline: 127 ± 6 and 72 ± 5 mmHg, respectively) were reduced by ∼4% throughout the BR supplementation period (P < 0.05). Compared with PL, the steady-state Vo(2) during moderate exercise was reduced by ∼4% after 2.5 h and remained similarly reduced after 5 and 15 days of BR (P < 0.05). The ramp test peak power and the work rate at the gas exchange threshold (baseline: 322 ± 67 W and 89 ± 15 W, respectively) were elevated after 15 days of BR (331 ± 68 W and 105 ± 28 W; P < 0.05) but not PL (323 ± 68 W and 84 ± 18 W). These results indicate that dietary NO(3)(-) supplementation acutely reduces BP and the O(2) cost of submaximal exercise and that these effects are maintained for at least 15 days if supplementation is continued.” 

The 2009 publication Dietary nitrate supplementation reduces the O2 cost of low-intensity exercise and enhances tolerance to high-intensity exercise in humans reports “Pharmacological sodium nitrate supplementation has been reported to reduce the O2 cost of submaximal exercise in humans. In this study, we hypothesized that dietary supplementation with inorganic nitrate in the form of beetroot juice (BR) would reduce the O2 cost of submaximal exercise and enhance the tolerance to high-intensity exercise. In a double-blind, placebo (PL)-controlled, crossover study, eight men (aged 19-38 yr) consumed 500 ml/day of either BR (containing 11.2 +/- 0.6 mM of nitrate) or blackcurrant cordial (as a PL, with negligible nitrate content) for 6 consecutive days and completed a series of “step” moderate-intensity and severe-intensity exercise tests on the last 3 days. On days 4-6, plasma nitrite concentration was significantly greater following dietary nitrate supplementation compared with PL (BR: 273 +/- 44 vs. PL: 140 +/- 50 nM; P < 0.05), and systolic blood pressure was significantly reduced (BR: 124 +/- 2 vs. PL: 132 +/- 5 mmHg; P < 0.01). During moderate exercise, nitrate supplementation reduced muscle fractional O2 extraction (as estimated using near-infrared spectroscopy). The gain of the increase in pulmonary O2 uptake following the onset of moderate exercise was reduced by 19% in the BR condition (BR: 8.6 +/- 0.7 vs. PL: 10.8 +/- 1.6 ml.min(-1).W(-1); P < 0.05). During severe exercise, the O2 uptake slow component was reduced (BR: 0.57 +/- 0.20 vs. PL: 0.74 +/- 0.24 l/min; P < 0.05), and the time-to-exhaustion was extended (BR: 675 +/- 203 vs. PL: 583 +/- 145 s; P < 0.05). The reduced O2 cost of exercise following increased dietary nitrate intake has important implications for our understanding of the factors that regulate mitochondrial respiration and muscle contractile energetics in humans.”

The November 2010 publication Dietary nitrate supplementation reduces the O2 cost of walking and running: a placebo-controlled study reports “Dietary supplementation with beetroot juice (BR) has been shown to reduce resting blood pressure and the O(2) cost of sub-maximal exercise and to increase the tolerance to high-intensity cycling. We tested the hypothesis that the physiological effects of BR were consequent to its high nitrate content, per se, and not to the presence of other potentially bioactive compounds. We investigated changes in blood pressure, mitochondrial oxidative capacity (Q(max)), and the physiological responses to walking, moderate-intensity running and severe-intensity running following dietary supplementation with BR and nitrate-depleted beetroot juice (PL). Following control (non-supplemented) tests, nine healthy, physically-active male subjects were assigned in a randomized, double-blind, cross-over design to receive BR (0.5 L(.)d(-1); containing ~6.2 mmol of nitrate) and PL (0.5 L(.)d(-1); containing ~0.003 mmol of nitrate) for six days. Subjects completed treadmill exercise tests on days four and five, and knee-extension exercise tests for the estimation of Q(max) (using (31)P-MRS) on day six of the supplementation periods. Relative to PL, BR elevated plasma [nitrite] (PL: 183±119 vs. BR: 373±211 nM, P<0.05) and reduced systolic blood pressure (PL: 129±9 vs. BR: 124±10 mmHg; P<0.01). Q(max) was not different between PL and BR (PL: 0.93±0.05 vs. BR: 1.05±0.22 mM(.)s(-1)). The O(2) cost of walking (PL: 0.87±0.12 vs. BR: 0.70±0.10 L(.)min(-1); P<0.01), moderate-intensity running (PL: 2.26±0.27 vs. BR: 2.10±0.28 L(.)min(-1); P<0.01), and severe-intensity running (End-exercise V(O2); PL: 3.77±0.57 vs. BR: 3.50±0.62 L(.)min(-1); P<0.01) was reduced by BR, and time-to-exhaustion during severe-intensity running was increased by 15% (PL: 7.6±1.5 vs. BR: 8.7±1.8 min; P<0.01). In contrast, relative to control, nitrate-depleted beetroot juice did not alter plasma [nitrite], blood pressure or the physiological responses to exercise. These results indicate that the positive effects of 6 days of BR supplementation on the physiological responses to exercise can be ascribed to the high nitrate content per se.”

Perhaps the vasoprotective effects of eating certain vegetables is due to nitrates/nitrites and the production of NO.   

This theory is put forward in the 2008 publication  Acute blood pressure lowering, vasoprotective, and antiplatelet properties of dietary nitrate via bioconversion to nitrite (2008).   “Diets rich in fruits and vegetables reduce blood pressure (BP) and the risk of adverse cardiovascular events. However, the mechanisms of this effect have not been elucidated. Certain vegetables possess a high nitrate content, and we hypothesized that this might represent a source of vasoprotective nitric oxide via bioactivation. In healthy volunteers, approximately 3 hours after ingestion of a dietary nitrate load (beetroot juice 500 mL), BP was substantially reduced (Delta(max) -10.4/8 mm Hg); an effect that correlated with peak increases in plasma nitrite concentration. The dietary nitrate load also prevented endothelial dysfunction induced by an acute ischemic insult in the human forearm and significantly attenuated ex vivo platelet aggregation in response to collagen and ADP. Interruption of the enterosalivary conversion of nitrate to nitrite (facilitated by bacterial anaerobes situated on the surface of the tongue) prevented the rise in plasma nitrite, blocked the decrease in BP, and abolished the inhibitory effects on platelet aggregation, confirming that these vasoprotective effects were attributable to the activity of nitrite converted from the ingested nitrate. These findings suggest that dietary nitrate underlies the beneficial effects of a vegetable-rich diet and highlights the potential of a “natural” low cost approach for the treatment of cardiovascular disease.”By now, you should be getting the picture that nitrates and nitrites and substances that contain these like beetroot juice are definitely beneficial – the opposite message of that conveyed in the previous blog entry. 

Beetroot juice could possibly be useful in the treatment of obesity

The 2009 study In vitro effects of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals  relates “Oxidative stress and inflammation are involved in the development of obesity. Beetroot (Beta vulgaris var. rubra) is a food ingredient containing betalain pigments that show antioxidant activity. The in vitro effect of beetroot juice and chips on oxidative metabolism and apoptosis in neutrophils from obese individuals has been investigated. Fifteen obese women (aged 45 +/- 9 years, BMI >30 kg/m2) and nine healthy controls (women, aged 29 +/- 11 years, BMI = 22.2 +/- 1.6 kg/m2) were examined. –. Neutrophils from obese individuals had a significantly higher ROS production compared with the controls (p < 0.05). Beetroot products inhibited neutrophil oxidative metabolism in a concentration-dependent manner. Also observed were the pro-apoptotic effects of beetroot at a concentration range of 0.1-10% in 24 h culture of stimulated neutrophils. These natural products (in both the liquid and solid state) have antioxidant and antiinflammatory capacity, and could be an important adjunct in the treatment of obesity.”

Nitrites play a role in hypoxic signaling

 One if the alternative theories of aging described in my treatise is Declining hypoxic response.  I speculate there that keeping the hypoxic response turned on can possibly contribute to longevity. 

The 2009 paper Nitrite as regulator of hypoxic signaling in mammalian physiology relates: “In this review we consider the effects of endogenous and pharmacological levels of nitrite under conditions of hypoxia. In humans, the nitrite anion has long been considered as metastable intermediate in the oxidation of nitric oxide radicals to the stable metabolite nitrate. This oxidation cascade was thought to be irreversible under physiological conditions. However, a growing body of experimental observations attests that the presence of endogenous nitrite regulates a number of signaling events along the physiological and pathophysiological oxygen gradient. Hypoxic signaling events include vasodilation, modulation of mitochondrial respiration, and cytoprotection following ischemic insult. These phenomena are attributed to the reduction of nitrite anions to nitric oxide if local oxygen levels in tissues decrease. Recent research identified a growing list of enzymatic and nonenzymatic pathways for this endogenous reduction of nitrite. Additional direct signaling events not involving free nitric oxide are proposed. We here discuss the mechanisms and properties of these various pathways and the role played by the local concentration of free oxygen in the affected tissue.”

I note that VIAGRA® (sildenafil citrate), CIALIS® (tadalafil) and other drugs for erectile dysfunction work through release of nitric oxide (NO) from nerve terminals and endothelial cells and consequent dilation of arteries and increase of blood flow.  These drugs appear to be useful for treating a number of other conditions where enhancing circulation is important, for example relieving pulmonary arterial hypertension and symptoms of scleroderma. While not nitrates or nitrites in themselves, their circulatory functions appear to be quite similar.  In fact, some blogs have suggested that beetroot juice might be an alternative to VIAGRA or CIALIS, though i have seen no research to that effect. 

There is an explosion of research in the areas of nitrites-based signaling, physiology and medical applications.  Some of the many additional relevant publications are:

Nitrite as a physiological source of nitric oxide and a signalling molecule in the regulation of the cardiovascular system in both mammalian and non-mammalian vertebrates, The emerging role of nitrite as an endogenous modulator and therapeutic agent of cardiovascular function,The role of nitrite in nitric oxide homeostasis: a comparative perspective, Protective effect of red beetroot against carbon tetrachloride- and N-nitrosodiethylamine-induced oxidative stress in rats, Clinical translation of nitrite therapy for cardiovascular diseases, Mechanisms of nitrite reduction to nitric oxide in the heart and vessel wall, A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis, Nitrite as a vascular endocrine nitric oxide reservoir that contributes to hypoxic signaling, cytoprotection, and vasodilation, Nitrite in nitric oxide biology: cause or consequence?, A systems-based review, Nitrite modulates contractility of teleost (Anguilla anguilla and Chionodraco hamatus, i.e. the Antarctic hemoglobinless icefish) and frog (Rana esculenta) hearts, Beta-adrenergic receptors and nitric oxide generation in the cardiovascular system, .

, Role of the anion nitrite in ischemia-reperfusion cytoprotection and therapeutics, ,Nitrite exerts potent negative inotropy in the isolated heart via eNOS-independent nitric oxide generation and cGMP-PKG pathway activation, Nitrite reductase activity of cytochrome c, Nitrite is a signaling molecule and regulator of gene expression in mammalian tissues,  Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice,  and Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities.

My take on dietary nitrates and nitrites

Reviewing the research described in this blog entry and in the previous blog entry, I frankly am unable at this time to come down on one side or the other on the question of health benefits of ingesting nitrates/nitrites or supplements containing them like beetroot juice.  The arguments on both sides of the ledger seem compelling.  Hopefully it has been valuable to readers to review both sides of the issue.  I can make a few comments, however.

1.     The cell-level and body-level research relating to the beneficial effects of nitrates/nitrites and NO is generally newer and there seems to be much more of it at the moment than corresponding research on the negative effects. Yet, publications on both sides of the good/bad ledger are continuing to appear.

2.     Some of the arguments relating to the negative health effects of nitrates and nitrites seems to be circumstantial, e.g., incidences of diabetes increasing concurrently with drinking water nitrites levels.  Other arguments seem to be well-founded.  E.g. under certain circumstances nitrites are converted to nitrosamines, and these are definitely carcinogenic. 

3.     Some of the “bad for you” research reports are based on very large multi-year population studies involving hundreds or thousands of people, while the “good for you” studies are typically based on theoretical knowledge and experimental studies with relatively small numbers of subjects.

4.     The two categories of studies tend to measure very different things and could possibly be both essentially valid.  E.g. it could be the case that consuming beetroot juice could improve circulation, exercise, cardiovascular and cognitive performance and at the same time increase susceptibility to cancers and neurodegenerative diseases and accelerate biomarkers of aging.   The situation could be similar to that of taking HGH shots where there may be immediate improvements in body morphology and exercise capability but the long-term effects are likely to be life-shortening(ref).   

5.     The “good for you” and the “bad for you” researchers seem to be pursuing different agendas and largely ignore each other’s work. 

6.     Some of the “good for you” researchers point out that the levels of consumption of nitrates and nitrites or even beetroot juice associated with eating plenty of vegetables is too small to be concerned with negative health effects.

7.     The “good for you” researchers have focused on nitrate/nitrite-inducing supplements like beetroot juice and, insofar as I have seen, have avoided talking about processed meat which contains nitrites.  They have also avoided discussing nitrosamines.

I am waiting to see how this seeming-conflict in an important area of health and aging plays out.  Particularly I am awaiting more studies of relating NO levels to the hypoxic and other longevity-related gene-activation pathways.  Meanwhile I intend to consume generous amounts of vegetables but for now I am holding off on the beetroot juice as a regular supplement.

Every great once in a while mainline medical advice reverses itself about something important – what was bad becomes good or what was good becomes bad.  That happened with Vitamin D in recent years when stern warnings never to exceed 400IU a day gave way to recommendations that it is good for older people to take anywhere up to 4,000IU per day.  A reversal may now be taking place having to do with whether nitrates and nitrites lead to cancers and Alzheimer’s Disease and are therefore bad for you, or whether they have great cardiovascular effects and are therefore good for you.  There is controversy about this, clashing and clanging of gears as contradictory health advice is commonly given. 

The issue at hand is whether it is good or bad for your health to consume foods containing high amount of nitrates and nitrites, foods like processed meats and certain supplements like beet root juice.  The controversy extends to whether the large quantities of nitrates used in mass-scale agriculture constitutes a public health hazard because of contamination of public water supplies with nitrates.  Is this good or bad for public health?  This Part 1 blog entry presents the research case against consumption of nitrites and nitrites.  A following Part 2 blog entry presents the research case in favor of consumption of nitrites and nitrites. I added some additional citations here on January 10, 2010.

Nitrates/nitrites and nitrosamines

A nitrate is a polyatomic ion with  the molecular formula NO₃ while nitrites are salts of nitrous acid (HNO₂) containing the nitrite ion NO₂.  “Nitrates can be reduced to nitrites by certain microorganisms present in foods and in the gastrointestinal tract(ref).”  “Sodium nitrite is used for the curing of meat because it prevents bacterial growth and, in a reaction with the meat’s myoglobin, gives the product a desirable dark red color. Because of the toxicity of nitrite (the lethal dose of nitrite for humans is about 22 mg per kg body weight(ref)), the maximum allowed nitrite concentration in meat products is 200 ppm. Under certain conditions, especially during cooking, nitrites in meat can react with degradation products of amino acids, forming nitrosamines, which are known carcinogens(ref)” 

“Under certain conditions not yet fully understood, the natural breakdown products of proteins known as amines can combine with nitrites to form compounds known as nitrosamines. There are many different types of nitrosamines, most of which are known carcinogens in test animals(ref).”  Cooking a meat containing nitrites may generate greater or fewer nitrosamines depending on how the cooking takes place.  “Thus, well done or burned bacon probably is potentially more hazardous than less well done bacon. Bacon cooked by microwave has less nitrosamine than fried bacon(ref).”

“Nitrosamines are formed by a chemical reaction between nitrites or other proteins. Sodium nitrite is deliberately added to meat and fish to prevent toxin production; it is also used to preserve, color and flavor meats. Ground beef, cured meats and bacon in particular contain abundant amounts of amines due to their high protein content. Because of the significant levels of added nitrates and nitrites, nitrosamines are nearly always detectable in these foods. Nitrosamines are also easily generated under strong acid conditions, such as in the stomach, or at high temperatures associated with frying or flame broiling. Reducing sodium nitrite content reduces nitrosamine formation in foods. — Nitrosamines basically become highly reactive at the cellular level, which then alters gene expression and causes DNA damage(ref).” 

“As established by the U.S. Department of Agriculture (USDA) in the Meat Inspection Regulations —  the use of nitrites, nitrates, or combinations of them cannot result in more than 200 parts per million (ppm), calculated as sodium nitrite, in the finished product(ref).”  People may have significant exposure to nitrites and nitrates from eating vegetable as well as from eating processed meats.  Green lettuce, spinach, celery and beets tend to have the greatest concentrations of nitrites and concentrations can be particularly high due to excessive pre-harvest use of fertilizers.

The health case against consumption of nitrates and nitrites

The arguments against consumption of substantial amounts of nitrates and nitrites are both old and new and are fairly convincing:

·        Nitrites are converted into nitrosamines under a number of conditions, and nitrosamines are known to be toxic or carcinogenic.

·        Exposure to nitrates, nitrites and nitrosamines is correlated with higher incidences of Alzheimer’s Disease, Parkinson’s disease and diabetes.

·        Consumption of foods containing large amounts of nitrates or nitrites lead to higher incidences of several cancers.

·        Shorter telomere lengths, biomarkers of aging,  are associated with consumption of processed meats containing nitrates/nitrites/nitrosamines, but not with consumption of unprocessed meats.  

Nitrates/nitrites/nitrosamines and Alzheimer’s Disease, Parkinson’s disease and diabetes 

Consumption of excess nitrates/nitrites/nitrosamines seems to play a role in creating or worsening these disease processes. 

The professionally-worded title of this 2009 publication conceals a strong underlying message:  Epidemilogical trends strongly suggest exposures as etiologic agents in the pathogenesis of sporadic Alzheimer’s disease, diabetes mellitus, and non-alcoholic steatohepatitis:  “Nitrosamines mediate their mutagenic effects by causing DNA damage, oxidative stress, lipid peroxidation, and pro-inflammatory cytokine activation, which lead to increased cellular degeneration and death. However, the very same pathophysiological processes comprise the “unbuilding” blocks of aging and insulin-resistance diseases including, neurodegeneration, diabetes mellitus (DM), and non-alcoholic steatohepatitis (NASH). Previous studies demonstrated that experimental exposure to streptozotocin, a nitrosamine-related compound, causes NASH, and diabetes mellitus Types 1, 2 and 3 (Alzheimer (AD)-type neurodegeneration). Herein, we review evidence that the upwardly spiraling trends in mortality rates due to DM, AD, and Parkinson’s disease typify exposure rather than genetic-based disease models, and parallel the progressive increases in human exposure to nitrates, nitrites, and nitrosamines via processed/preserved foods. We propose that such chronic exposures have critical roles in the pathogenesis of our insulin resistance disease pandemic. Potential solutions include: 1) eliminating the use of nitrites in food; 2) reducing nitrate levels in fertilizer and water used to irrigate crops; and 3) employing safe and effective measures to detoxify food and water prior to human consumption. Future research efforts should focus on refining our ability to detect and monitor human exposures to nitrosamines and assess early evidence of nitrosamine-mediated tissue injury and insulin resistance.”

As further reported in a 2009 Science Daily article Nitrates May Be Environmental Trigger For Alzheimer’s, Diabetes And Parkinson’s Disease “A new study by researchers at Rhode Island Hospital have found a substantial link between increased levels of nitrates in our environment and food with increased deaths from diseases, including Alzheimer’s, diabetes mellitus and Parkinson’s. The study was published in the Journal of Alzheimer’s Disease. — Led by Suzanne de la Monte, MD, MPH, of Rhode Island Hospital, researchers studied the trends in mortality rates due to diseases that are associated with aging, such as diabetes, Alzheimer’s, Parkinson’s, diabetes and cerebrovascular disease, as well as HIV. They found strong parallels between age adjusted increases in death rate from Alzheimer’s, Parkinson’s, and diabetes and the progressive increases in human exposure to nitrates, nitrites and nitrosamines through processed and preserved foods as well as fertilizers. — De la Monte and the authors propose that the increase in exposure plays a critical role in the cause, development and effects of the pandemic of these insulin-resistant diseases. — De la Monte — says, “We have become a ‘nitrosamine generation.’ In essence, we have moved to a diet that is rich in amines and nitrates, which lead to increased nitrosamine production. We receive increased exposure through the abundant use of nitrate-containing fertilizers for agriculture.” She continues, “Not only do we consume them in processed foods, but they get into our food supply by leeching from the soil and contaminating water supplies used for crop irrigation, food processing and drinking.” — Nitrites and nitrates belong to a class of chemical compounds that have been found to be harmful to humans and animals. More than 90 percent of these compounds that have been tested have been determined to be carcinogenic in various organs. They are found in many food products, including fried bacon, cured meats and cheese products as well as beer and water. Exposure also occurs through manufacturing and processing of rubber and latex products, as well as fertilizers, pesticides and cosmetics.  — The researchers note that the role of nitrosamines has been well-studied, and their role as a carcinogen has been fully documented. The investigators propose that the cellular alterations that occur as a result of nitrosamine exposure are fundamentally similar to those that occur with aging, as well as Alzheimer’s, Parkinson’s and Type 2 diabetes mellitus. — De la Monte comments, “All of these diseases are associated with increased insulin resistance and DNA damage. Their prevalence rates have all increased radically over the past several decades and show no sign of plateau. Because there has been a relatively short time interval associated with the dramatic shift in disease incidence and prevalence rates, we believe this is due to exposure-related rather than genetic etiologies.”

A 2009 publication reinforces the message, at least insofar as nitrosamines are concerned, Nitrosamine exposure causes insulin resistance diseases: relevance to type 2 diabetes mellitus, non-alcoholic steatohepatitis, and Alzheimer’s disease. “The current epidemics of type 2 diabetes mellitus (T2DM), non-alcoholic steatohepatitis (NASH), and Alzheimer’s disease (AD) all represent insulin-resistance diseases. Previous studies showed that streptozotocin, a nitrosamine-related com-pound, causes insulin resistance diseases including, T2DM, NASH, and AD-type neurodegeneration. We hypothesize that chronic human exposure to nitrosamine compounds, which are widely present in processed foods, contributes to the pathogenesis of T2DM, NASH, and AD. Long Evans rat pups were treated with N-nitrosodiethylamine (NDEA) by i.p. (x3) or i.c. (x1) injection, and 2-4 weeks later, they were evaluated for cognitive-motor dysfunction, insulin resistance, and neurodegeneration using behavioral, biochemical, and molecular approaches. NDEA treatment caused T2DM, NASH, deficits in motor function and spatial learning, and neurodegeneration characterized by insulin resistance and deficiency, lipid peroxidation, cell loss, increased levels of amyloid-beta protein precursor/amyloid-beta, phospho-tau, and ubiquitin immunoreactivities, and upregulated expression of pro-inflammatory cytokine and pro-ceramide genes, which together promote insulin resistance. In conclusion, environmental and food contaminant exposures to nitrosamines play critical roles in the pathogenesis of major insulin resistance diseases including T2DM, NASH, and AD. Improved detection and prevention of human exposures to nitrosamines will lead to earlier treatments and eventual quelling of these costly and devastating epidemics.”

Another 2009 publication continues the theme Mechanisms of nitrosamine-mediated neurodegeneration: potential relevance to sporadic Alzheimer’s disease. “Streptozotocin (STZ) is a nitrosamine-related compound that causes Alzheimer’s disease (AD)-type neurodegeneration with cognitive impairment, brain insulin resistance, and brain insulin deficiency. Nitrosamines and STZ mediate their adverse effects by causing DNA damage, oxidative stress, lipid peroxidation, pro-inflammatory cytokine activation, and cell death, all of which occur in AD. We tested the hypothesis that exposure to N-nitrosodiethylamine (NDEA), which is widely present in processed/preserved foods, causes AD-type molecular and biochemical abnormalities in central nervous system (CNS) neurons. NDEA treatment of cultured post-mitotic rat CNS neurons (48 h) produced dose-dependent impairments in ATP production and mitochondrial function, and increased levels of 8-hydroxy-2′-deoxyguanosine, 4-hydroxy-2-nonenal, phospho-tau, amyloid-beta protein precursor-amyloid-beta (A beta PP-A beta), and ubiquitin immunoreactivity. These effects were associated with decreased expression of insulin, insulin-like growth factor (IGF)-I, and IGF-II receptors, and choline acetyltransferase. Nitrosamine exposure causes neurodegeneration with a number of molecular and biochemical features of AD including impairments in energy metabolism, insulin/IGF signaling mechanisms, and acetylcholine homeostasis, together with increased levels of oxidative stress, DNA damage, and A beta PP-A beta immunoreactivity. These results suggest that environmental exposures and food contaminants may play critical roles in the pathogenesis of sporadic AD.” 

Nitrates, nitrites and nitrosamines and cancer

The 2008 publication Processed meat and colorectal cancer: a review of epidemiologic and experimental evidence implicates nitrites and nitrosamines “Processed meat intake may be involved in the etiology of colorectal cancer, a major cause of death in affluent countries. The epidemiologic studies published to date conclude that the excess risk in the highest category of processed meat-eaters is comprised between 20% and 50% compared with non-eaters. In addition, the excess risk per gram of intake is clearly higher than that of fresh red meat. Several hypotheses, which are mainly based on studies carried out on red meat, may explain why processed meat intake is linked to cancer risk. Those that have been tested experimentally are (i) that high-fat diets could promote carcinogenesis via insulin resistance or fecal bile acids; (ii) that cooking meat at a high temperature forms carcinogenic heterocyclic amines and polycyclic aromatic hydrocarbons; (iii) that carcinogenic N-nitroso compounds are formed in meat and endogenously; (iv) that heme iron in red meat can promote carcinogenesis because it increases cell proliferation in the mucosa, through lipoperoxidation and/or cytotoxicity of fecal water. Nitrosation might increase the toxicity of heme in cured products.”

The 2008 publication Nutrition and gastric cancer risk: an update relates “Data from epidemiologic, experimental, and animal studies indicate that diet plays an important role in the etiology of gastric cancer. High intake of fresh fruits and vegetables, lycopene and lycopene-containing food products, and potentially vitamin C and selenium may reduce the risk for gastric cancer. Data also suggest that high intake of nitrosamines, processed meat products, salt and salted foods, and overweight and obesity are associated with increased risk for gastric cancer. However, current data provide little support for an association of beta-carotene, vitamin E, and alcohol consumption with risk for gastric cancer.”

The 2010 publication Nitrate intake and the risk of thyroid cancer and thyroid disease relates “BACKGROUND: Nitrate is a contaminant of drinking water in agricultural areas and is found at high levels in some vegetables. Nitrate competes with uptake of iodide by the thyroid, thus potentially affecting thyroid function.– METHODS: We investigated the association of nitrate intake from public water supplies and diet with the risk of thyroid cancer and self-reported hypothyroidism and hyperthyroidism in a cohort of 21,977 older women in Iowa who were enrolled in 1986 and who had used the same water supply for >10 years. — We observed no association with prevalence of hypothyroidism or hyperthyroidism. Increasing intake of dietary nitrate was associated with an increased risk of thyroid cancer (highest vs. lowest quartile, RR = 2.9 [1.0-8.1]; P for trend = 0.046) and with the prevalence of hypothyroidism (odds ratio = 1.2 [95% CI = 1.1-1.4]), but not hyperthyroidism.  — CONCLUSIONS: Nitrate may play a role in the etiology of thyroid cancer and warrants further study.” 

The 2007 report Nitrate intake relative to antioxidant vitamin intake affects gastric cancer risk: a case-control study in Korea had some surprising conclusions: “The objective of this study was to determine whether the intake of nitrate relative to antioxidant vitamin rather than absolute intake of nitrate affects the risk of gastric cancer (GC). In a case-control study in Korea using a food frequency questionnaire, trained dietitians interviewed 136 GC cases and an equal number of controls matched by sex and age. As an index of nitrate intake relative to antioxidant vitamins intake, we calculated the nitrate:antioxidant vitamin consumption ratio. The mean daily nitrate intake from foods was very high in our subjects. Higher absolute intake of nitrate was not associated with GC risk [odds ratios (OR) = 1.13; 95% confidence interval (CI) = 0.42-3.06]. However, the GC risk distinctly increased as the nitrate:antioxidant vitamin consumption ratio increased, particularly with higher nitrate:vitamin E (OR = 2.78; 95% CI = 1.01-7.67) and nitrate:folate ratios (OR = 3.37; 95% CI = 1.28-8.87). Therefore, GC risk was influenced by the intake of nitrate relative to antioxidant vitamins. Our results suggest that a decrease in the intake of nitrate relative to antioxidant vitamins is considerably more effective in reducing GC risk than either a lower absolute intake of nitrate or a higher intake of antioxidant vitamins alone.”

Note that there is a theme here that shows up frequently in the literature: cancer and other risks associated with ingestion of nitrites or nitrites or nitrosamines can be reduced by consumption of antioxidants.

 A number of other recent publications link nitric oxide to cancers.  The 2007 publication An emerging role for endothelial nitric oxide synthase in chronic inflammation and cancer relates “Nitric oxide (NO) is a free radical that is involved in carcinogenesis. Recent literature indicates that endothelial NO synthase (eNOS) can modulate cancer-related events (angiogenesis, apoptosis, cell cycle, invasion, and metastasis). We review the literature linking eNOS to carcinogenesis to encourage future research assessing the role of eNOS in cancer prevention and treatment.” 

The 2010 publication Nitric oxide and cancer relates “Nitric oxide (NO) is a lipophilic, highly diffusible and short-lived physiological messenger which regulates a variety of important physiological responses including vasodilation, respiration, cell migration, immune response and apoptosis. NO is synthesized by three differentially gene-encoded NO synthase (NOS) in mammals: neuronal NOS (nNOS or NOS-1), inducible NOS (iNOS or NOS-2) and endothelial NOS (eNOS or NOS-3). All isoforms of NOS catalyze the reaction of L-arginine, NADPH and oxygen to NO, L-citrulline and NADP. NO may exert its cellular action by cGMP-dependent as well as by cGMP-independent pathways including postranslational modifications in cysteine (S-nitrosylation or S-nitrosation) and tyrosine (nitration) residues, mixed disulfide formation (S-nitrosoglutathione or GSNO) or promoting further oxidation protein stages which have been related to altered protein function and gene transcription, genotoxic lesions, alteration of cell-cycle check points, apoptosis and DNA repair. NO sensitizes tumor cells to chemotherapeutic compounds. The expression of NOS-2 and NOS-3 has been found to be increased in a variety of human cancers. The multiple actions of NO in the tumor environment is related to heterogeneous cell responses with particular attention in the regulation of the stress response mediated by the hypoxia inducible factor-1 and p53 generally leading to growth arrest, apoptosis or adaptation.”  Several earlier publications also link NO to cancers, for example the 2003 publication Nitric oxide-mediated promotion of mammary tumour cell migration requires sequential activation of nitric oxide synthase, guanylate cyclase and mitogen-activated protein kinase. “ Together, these results indicate sequential activation of NOS, GC and MAPK pathways in mediating signals for C3L5 cell migration, an essential step in invasion and metastasis. Since NOS activity is positively associated with human breast cancer progression, the present results are relevant for development of therapeutic modalities for this disease.”  

Going back to 2001 we have the publication Municipal drinking water nitrate level and cancer risk in older women: the Iowa Women’s Health Study.  This study was based on a cohort of 21,977 Iowa women who were 55-69 years of age at baseline in 1986 and had used the same water supply more than 10 years.  The study showed a positive association with increasing nitrate in drinking water with bladder cancer but not for other cancers.

The 2009 publication Too much of a good thing? Nitrate from nitrogen fertilizers and cancer points to a possible public health problem.  “Nitrate levels in water supplies have been increasing in many areas of the world; therefore, additional studies of populations with well-characterized exposures are urgently needed to further our understanding of cancer risk associated with nitrate ingestion. Future studies should assess exposure for individuals (e.g., case-control, cohort studies) in a time frame relevant to disease development, and evaluate factors affecting nitrosation. Estimating N-nitroso compounds formation via nitrate ingestion requires information on dietary and drinking water sources of nitrate, inhibitors of nitrosation (e.g., vitamin C), nitrosation precursors (e.g., red meat, nitrosatable drugs), and medical conditions that may increase nitrosation (e.g., inflammatory bowel disease). Studies should account for the potentially different effects of dietary and water sources of nitrate and should include the population using private wells for whom exposure levels are often higher than public supplies.”

The 2010 study Estimation of incidence and social cost of colon cancer due to nitrate in drinking water in the EU: a tentative cost-benefit assessment  concludes “RESULTS: For above median meat consumption the risk of colon cancer doubles when exposed to drinking water exceeding 25 mg/L of nitrate (NO3) for more than ten years. — CONCLUSIONS: Our cost estimates indicate that current measures to prevent exceedance of 50 mg/L NO3 are probably beneficial for society and that a stricter nitrate limit and additional measures may be justified. –”

Eating processed meat and telomere lengths

The blog entry Telomere lengths, Part 2: Lifestyle, dietary, and other factors associated with telomere shortening and lengthening contains a long passage related to the different impacts of eating processed meats and unprocessed meats on telomere lengths. Because the difference between processed and unprocessed meats is largely associated with inclusion of nitrates/nitrites and nitrosamines, the passage is worth reproducing here:

Telomere lengths and processed meats

It is possible to couple the results of two studies related to processed meats to see some interesting relationships.  The first such study is described in a 2010 publication published in Circulation, a journal of the American Heart Association Red and Processed Meat Consumption and Risk of Incident Coronary Heart Disease, Stroke, and Diabetes Mellitus.  This study is a meta-analysis of studies relating red and processed meat to CHD (coronary heart disease), stroke, and diabetes mellitus. “Background— Meat consumption is inconsistently associated with development of coronary heart disease (CHD), stroke, and diabetes mellitus, limiting quantitative recommendations for consumption levels. Effects of meat intake on these different outcomes, as well as of red versus processed meat, may also vary. ^— Methods and Results— We performed a systematic review and meta-analysis of evidence for relationships of red (unprocessed), processed, and total meat consumption with incident CHD, stroke, and diabetes mellitus. We searched for any cohort study, case-control study, or randomized trial that assessed these exposures and outcomes in generally healthy adults. Of 1598 identified abstracts, 20 studies met inclusion criteria, including 17 prospective cohorts and 3 case-control studies. All data were abstracted independently in duplicate. Random-effects generalized least squares models for trend estimation were used to derive pooled dose-response estimates. The 20 studies included 1 218 380 individuals and 23 889 CHD, 2280 stroke, and 10 797 diabetes mellitus cases. Red meat intake was not associated with CHD (n=4 studies; relative risk per 100-g serving per day=1.00; 95% confidence interval, 0.81 to 1.23; P for heterogeneity=0.36) or diabetes mellitus (n=5; relative risk=1.16; 95% confidence interval, 0.92 to 1.46; P=0.25). Conversely, processed meat intake was associated with 42% higher risk of CHD (n=5; relative risk per 50-g serving per day=1.42; 95% confidence interval, 1.07 to 1.89; P=0.04) and 19% higher risk of diabetes mellitus (n=7; relative risk=1.19; 95% confidence interval, 1.11 to 1.27; P<0.001). Associations were intermediate for total meat intake. Consumption of red and processed meat were not associated with stroke, but only 3 studies evaluated these relationships. ^— Conclusions— Consumption of processed meats, but not red meats, is associated with higher incidence of CHD and diabetes mellitus. These results highlight the need for better understanding of potential mechanisms of effects and for particular focus on processed meats for dietary and policy recommendations.”

The second study (2008) looks at telomere lengths as related to kinds of food intake Dietary patterns, food groups, and telomere length in the Multi-Ethnic Study of Atherosclerosis (MESA).  “Objective: With data from 840 white, black, and Hispanic adults from the Multi-Ethnic Study of Atherosclerosis, we studied cross-sectional associations between telomere length and dietary patterns and foods and beverages that were associated with markers of inflammation. ^— Design: Leukocyte telomere length was measured by quantitative polymerase chain reaction. Length was calculated as the amount of telomeric DNA (T) divided by the amount of a single-copy control DNA (S) (T/S ratio). Intake of whole grains, fruit and vegetables, low-fat dairy, nuts or seeds, nonfried fish, coffee, refined grains, fried foods, red meat, processed meat, and sugar-sweetened soda were computed with responses to a 120-item food-frequency questionnaire completed at baseline. Scores on 2 previously defined empirical dietary patterns were also computed for each participant.   Results: After adjustment for age, other demographics, lifestyle factors, and intakes of other foods or beverages, only processed meat intake was associated with telomere length. For every 1 serving/d greater intake of processed meat, the T/S ratio was 0.07 smaller (β ± SE: –0.07 ± 0.03, P = 0.006). Categorical analysis showed that participants consuming 1 serving of processed meat each week had 0.017 smaller T/S ratios than did nonconsumers. Other foods or beverages and the 2 dietary patterns were not associated with telomere length.  ^– Conclusions: Processed meat intake showed an expected inverse association with telomere length, but other diet features did not show their expected associations.

So, together the two studies say:

·        Consumption of processed meat correlates with both shorter telomere lengths and increased susceptibility to CHD and diabetes mellitus.  Neither of these correlations exist for consumption of red meat.

·        Of a number of possibly not-good-for-you foods like sugar-sweetened soda, only consumption of processed meats was correlated with shorter telomeres.

·        Causal chain is unclear, e.g. whether eating processed meats leads to shorter telomeres which leads to increased disease susceptibilities or whether eating processed meats leads to disease susceptibilities which lead to shorter telomeres, or both or neither.

       From a health and longevity perspective the two studies combine fairly powerfully to contraindicate eating processed meats, foods which have long been suspected to be carcinogenic because they tend to be infused with nitrites(ref).

I resist the temptation to go on quoting more and more publications that are negative about nitrates/nitrites/nitrosamines.  A few central points seem to be made:

1.      A number of both laboratory and epidemiological studies suggest strong associations between consumption of nitrates/nitrites/nitrosamines with many diseases including Alzheimer’s disease, Parkinson’s disease, diabetes and multiple cancers.  The chain of causality is fairly well understood in terms of actions of nitrates/nitrites/nitrosamines.

2.     Telomere lengths, a proxy measure for aging, are definitely shorter among those eating processed meat but not unprocessed meat, again implicating nitrates/nitrites/nitrosamines.  Likewise eating processed meat but not unprocessed meat is associated with greater susceptibility to coronary heart disease and diabetes mellitus.

3.     The major sources of nitrates/nitrites/nitrosamines are drinking water contaminated with fertilizer runoff, certain vegetables and processed meat.

4.     The health risks associated with consumption of  nitrates/nitrites/nitrosamines can be significantly reduced by consumption of antioxidants and plant-based polyphenols which interfere with nitrosation.

One would think that with the collective evidence presented above, no respectable health scientists would advocate consuming nitrates or nitrites for health reasons.  However, in the past few years a case is being built up for exactly that.  In fact certain health supplements like beet root juice are being sold precisely because they are concentrated sources of nitrates.  The case for nitrates/nitrites seems to be that they lead to increased nitric oxide expression in the body and offer a number of positive cardiovascular benefits.  I will present this case in the following blog entry Nitrates and nitrites –Part 2: good for you.

Why the pursuit of anti-aging science?  I take a short break from science in this blog entry and look at myths about aging and how aging is viewed in popular folklore – ancient and current.  These myths are important because aging science exists in our more-general culture and efforts to suggest an impending possibility for life-extension are often met with misplaced negative reactions. 

The reputation of aging in our culture is generally not a good one.  Facts about aging are often misperceived.   Aging is usually ignored, viewed in a context of resignation, or felt to be irrelevant until it is imminent.  Diseases and accidents are seen to be the main causes of death even though those diseases and accidents are ones of old age.  Myths about aging, old ones and modern ones, provide insights into where our culture has been and where it is now with respect to aging.

In ancient Greece aging was seen as ugly and tragic – except in Sparta

The paper Old Age in Ancient Greece: Narratives of desire, narratives of disgust illustrates how some of the current ambivalences about aging have very early roots.  “The Greek habit of dividing the world into mutually exclusive categories was a hallmark of their culture. One such division, between youth and old age, formed a persistent theme in Greek myth, poetry and theatre. Youth – neotas – was sweet, beautiful and heroic. To leave youth meant one quickly passed the threshold to old age – gems. Old age was ugly, mean and tragic. There was no middle ground, no third age. Sparta, the city state least inclined toward literature, litigation, art and trade provides an instructive contrast. Here an unchanging politics engendered an unending respect for those older than oneself. This was institutionalized in the powers of the Gerousia or Council of Elders.”

In Greek mythology “GERAS was the spirit (daimon) of old age, one of the malevolent spirits spawned by the goddess Nyx (Night). — He was depicted as a tiny shrivelled up old man. Geras’ opposite number was the goddess of youth, Hebe(ref).”

Old people were very rare in ancient Rome and seen with a mixture of disdain and respect

From Old age in ancient Rome: Mary Harlow and Ray Laurence look at what it meant to become a senior citizen in ancient Rome – “Rome, was the first ever metropolis, containing one million people and an urban culture that included architectural achievements unsurpassed until the modern period. This picture of an almost modern nation masks another of massive inequality, alongside sickness and disease that have not been experienced in the West for generations. Life expectancy at birth was short: on average roughly twenty-five to thirty years, with 50 per cent of those born not passing the age of ten. In other words, the demographic regime was not unlike that experienced in countries today such as Botswana through the causes of AIDS, international debt, poverty and inequality–a far cry from the modern Western world where average life expectancy becomes ever-higher and runs well into the seventies. A key question for understanding Rome is how society viewed those few people who survived into old age and experienced a life-span not unlike our own today.  — In short, once a man had reached the chronological age of sixty, he could step down from his formal obligations as a citizen and lead a life of leisure. This departure from public life was double-edged: it could be seen as a lifestyle that was characterized as productive (or indulgent) leisure but it could also mean social marginalization. Moving out of public life in effect led to a loss of social power and status in the eyes of those still in power. Retiring from public life was no easier for individuals in the Roman period than it is for some today–many of whom continue to work after their sixty-fifth birthday. Despite this, and the fact that there was no social marker, no rite of transition to mark this phase, there was pressure for older men to stand down in favour of younger. “   This sounds a lot like it is today.

Saturn was the god of Roman mythology who ruled over old age, but he was a very multi-faceted god with numerous other duties(ref). 

In the good-old-days, aging was generally seen as a crummy deal

Although we like to think that aging was seen in romantic terms in the good-old-days, the opposite seems to be true, at least in Western societies.  From Aging and Death in Folklore:   “For most pre-industrial cultures, life’s last chapter has been a bitter one. Surviving folklore reflects widespread resignation as to the inevitability of impoverishment, sexual impotence, failing health and vitality, and the loss of family and community status. No one expected the impossible. Such euphemisms as “golden years” and “senior citizens” did not exist.

• You cannot teach an old dog new tricks.
• There is no fool like an old fool.
• An old man who takes a young wife invites Death to the wedding.
• Nothing good will come from an old man who still wants to dance.
• For an old man to marry is like wanting to harvest in the wintertime.
• Old people can dye their hair, but they can’t change their backs.
• Age is poverty.
• Age is a troublesome guest.
• Age is a sickness from which everyone must die.
• Youth rises, age falls.
• A young wife is an old man’s dispatch horse to the grave.
• A young woman with an old husband is a wife by day and a widow by night.
• A woman’s beauty, an echo in the forest, and a rainbow all quickly disappear.
• When the old cow dances, her claws rattle.
• When the wolf grows old, the crows ride him.

Source: Wander, Deutsches Sprichwörter-Lexikon, vol. 1, cols. 55, 58-60; Simrock, Die deutschen Sprichwörter, pp. 281, 614; Jente, Proverbia Communia, nos. 28, 102.

“These proverbs reflect a chapter of life that most of us would prefer to ignore. We do not like to be reminded of our own mortality, and in today’s world, institutions such as hospitals, hospices, retirement centers, and funeral homes (euphemisms abound in the language of death!) shield us from the worst of the Grim Reaper’s ravages. We cope, or so it might seem, by pretending that death does not exist(ref).”

Old women were particularly distrusted in folklore.

From the same source, Aging and Death in Folklore:   “In spite of the numerous tales and proverbs celebrating the wisdom of old people and promoting their care, folklore is replete with reflections of a basic distrust of age. Various demonic personages, notably changelings and the devil himself, can be rendered powerless by tricking them into revealing their age. More significantly, in pre-industrial Europe superstitions abound that cast suspicion at old people, especially women. Proverbs and popular superstitions state the claim succinctly:

• If the devil can’t come himself, he sends an old woman.
• It is not good if one goes out in the morning and encounters an old woman.
• He who walks between two old women early in the morning shall have only bad luck the rest of the day.
• To meet old women first thing in the morning means bad luck; young people, good luck.
• Many men would rather let themselves be beaten to death, than to pass between two old women.
• A person on his way to an important undertaking will have bad luck if he encounters an old woman. Encountering a young girl will bring him good luck.

Source: Wander, Deutsches Sprichwörter-Lexikon, vol. 4, col. 1105. Simrock, Die deutschen Sprichwörter, p. 554; Grimm, Deutsche Mythologie, vol. 3, items 58, 380, 791, 938, 1015.”

“Further, the sinister nature of old women is reflected in numerous folktales, for example: An old woman, promised a pair of shoes by the devil if she could bring discord to a happily married couple, told the wife that she could increase her husband’s love by cutting a few hairs from his chin. She then told the husband that his wife was plotting to cut his throat while he slept. The man pretended to sleep. Seeing his wife silently approaching with a razor, he struck her dead with a stick.”

“Source: Retold from “An Old Woman Sows Discord,” Ranke, Folktales of Germany, no. 66. Type 1353.

Such tales help explain the widespread superstition, documented above, that if the first person you saw in the morning was an old woman, you would have bad luck.”

There are many current myths about aging

Many websites elaborate and refute current myths about what aging is like.  One set of examples is from The Five Myths of Aging By  Lauri M. Aesoph N.D.The myths discussed and refuted there are:

“Myth #1: When I get old, I’ll become senile. ”

“Myth #2: Old age means losing all my teeth. ”

“Myth #3: The older I get, the sicker I’ll get. ”

“Myth #4: Lifestyle changes won’t help me when I get old. ”

“Myth #5: As long as I maintain the eating habits I had when I was younger, I’ll stay healthy.”

Another set of myths is treated in the website 5 Common Myths About Aging By Deborah Kotz.  They are:

1.     Losing those few extra pounds will extend your life.

2.     You ‘ll need a hearing aid.

3.     You’re bound to get crotchety and withdrawn.

4.     Senility is inevitable.

5.     You won’t have the energy to exercise well in your 80s 

Common myths relate to the conditions people expect when aging

A list of more-subtle myths is in the site COMMON MYTHS OF AGING by DeLee Lantz, Ph.D.  The examples and comments are drawn from a NIH questionnaire.

“1.  Baby boomers are the faster growing segment of the population.  False. Fact:   There are more than 3 million Americans over the age of 85. That number is expected to quadruple by the year 2040, when there will be more than 12 million people in that age group. The population age 85 and older is the fastest growing age group in the U.S. 

2.  Families don’t usually bother with older relatives.  False. Fact: Most older people live close to their    children and see them often. Many live with their    spouses. An estimated 80% of men and 60% of    women live in family settings. Only 5% of the older    populations lives in nursing homes.    

3.  Everyone becomes confused or forgetful if they live long enough.  Fact:  Confusion and serious forgetfulness in old age can be caused by Alzheimer’s disease or other conditions that result in irreversible damage to the brain. But at least 100 other problems can bring on the same symptoms. A minor head injury, high fever, poor nutrition, adverse drug reactions, and depression also can lead to confusion. These conditions are treatable, however, and the confusion they cause can be eliminated.    

4.  You can become too old to exercise.  False.  Fact:   Exercise at any age can help strengthen the heart and lungs and lower blood pressure. It also can improve muscle strength, and, if carefully chosen, lessen bone loss with age. 

5.  Heart disease is a much bigger problem for older men than for older women. False.   Fact:  The risk of heart disease increases dramatically for women after menopause. By age 65, both men and women have a one in three chance of showing symptoms. But risks can    be significantly reduced by following a healthy  diet and exercising.  

6  You can become too old to exercise. False.  Fact:   Exercise at any age can help strengthen the heart and lungs and lower blood pressure. It also can improve muscle strength, and, if carefully chosen, lessen bone loss with age.

7.  Heart disease is a much bigger problem for older men than for older women. False Fact:The risk of heart disease increases dramatically for women after menopause. By age 65, both men and women have a one in    three chance of showing symptoms. But risks can    be significantly reduced by following a healthy diet and exercising.  

8.  The older you get, the less you sleep.  False.  Fact: In later life, it’s the quality of sleep that declines, not total sleep time. Researchers found that sleep tends to become more fragmented as people age. A number of reports suggest that older people are less likely than younger people  to stay awake throughout the day and that older people tend to take more naps than younger people.      

9.   Most older people are depressed. Why shouldn’t they be? False.    Fact:  Most older people are not depressed. When  it does occur, depression is treatable throughout    the life cycle using a variety of approaches, such   family support, psychotherapy, or antidepressant medications.  A physician can determine whether    the depression is caused by medication an older   person might be taking, by physical illness, stress,    or other factors.  

10.  There’s no point in screening older people for cancer because they can’t be treated.  False.   Fact:  Many older people can beat cancer,  especially if it’s found early. Over half of all cancers occur in people 65 and older, which  means that screening for cancer in this age group is especially important.  

11.  If your parent had Alzheimer’s DIsease, you  will most likely get it.  False.  Fact: The overwhelming number of people with Alzheimer’s disease have not inherited the disorder. In a few families, scientists have seen an extremely high incidence of the disease and have identified genes in these families which they think may be responsible.  

12.  As your body changes with age, so does your personality.   False.Fact:  Research has found that, except for the changes that can result from Alzheimer’s disease and other forms of dementia, personality is one of the few constants of life. That is, you are likely to age much as you’ve lived.

13.  Older people might as well accept urinary accidents as a fact of life. False. Fact:  Urinary incontinence is a symptom, not a disease.  Usually, it is caused by specific changes in body function that can result from infection, diseases, pregnancy, or the use of certain medications. A variety of treatment options are available for people who seek medical attention.

14.  Falls and injuries just naturally happen to older people.  False.Fact: Falls are the most common cause of injuries among people over age 65. But many of these injuries, which result in broken bones, can be avoided. Regular vision and hearing tests and good safety habits can help prevent accidents. Knowing whether your medications affect balance and coordination is also a good idea.    

15.  Everyone eventually gets cataracts.   False.  Fact: Not everyone gets cataracts, although a great many older people do. Some 18 percent of    people between the ages of 65 and 74 have cataracts, while more than 40 percent of those  between 75 and 85 have the problem. Cataracts  can be treated very successfully with surgery; more than 90 percent of people say they can see better after the procedure. 

16.  “You can’t teach an old dog new tricks.“  False.  Fact:  People at any age can learn new information and skills. Research indicates that older people can obtain new skills and improve old ones, including how to use a computer. 

There are many current myths and half-myths about aging science and the impact of life extension on society

This list is my own.

1.     Aging is part of the natural order Facts:  True and true also for death.  But lifespans vary greatly by species and human lifespans have varied significantly and can probably be extended significantly.

2.     Aging is inevitable; nothing can be done about it. Again, the inevitability is historically true but not necessarily true in the future.  Things can be done today to modulate the rate of aging within limits.  Lifestyle and dietary interventions can significantly delay physiological aging as can many poor habits accelerate it.  Further, it is my opinion that by 2017 we will see available interventions that will extend average lifespans by about 10 years in advanced countries.  Further, I project that by 2022 we will see interventions that will extend average human lifespans by more than 100 years.

3.     All the talk about social consequences of extending lifespans is irrelevant because life extension techniques are in the future and may never happen.  Out and out false!  First of all, independent of any special interventions our average lifespans are getting longer at a furious rate due to changes in our epigenomes, by about four hours every day.  See the blog entries , The Social ethics of longevity and Average US life expectancy is up 73 days in one year.  Increasing  knowledge of healthy lifestyles and dietary patterns and health-promoting supplements is leading millions of people to live longer, and the first wave of powerful science-based anti-aging interventions is probably only 5 years away.

4.     Social consequences of increased longevity are possibly things to think about in the future, but are not relevant now.  Again, false.  Some initial social consequences are being felt now.  It is in the news that the first wave of baby boomers is turning 65 this year, 75 million people who will be retiring, and drawing on federal health care and social security programs, programs predicated and funded on the basis of shorter projected life spans.

5.     Extending lifespans is not a good idea because it will overwhelm our healthcare system and send healthcare costs over the top.  While such a consequence on our healthcare system may be true in the short term, in the longer term the opposite is true.  All experiments that extend the lives of animals as well as theory points to the conclusion that extension of lifespan and extension of healthspan go hand-in-hand.  The diseases of old age still occur with the extension of lives but they are postponed proportionally .  So, if we could extend everyone’s lifespan in the US by 10 years, the result would be a precipitous drop in age-related cancers, dementia, cases of diabetes, fall injuries, etc.  As more people are becoming eligible for Medicare now, they are generally healthier than were people entering Medicare a dozen years ago.

6.     Extending lifespans is not a good idea because adding many unproductive older people would be an unbearable burden on the working and productive young.  Again, the opposite is true.  Many tens of millions of educated and skilled working adults representing trillions of dollars in human capital would stay as productive contributors in the workforce, utilizing their accumulated human capital to produce innovations and wealth.  Of course many things like raising retirement ages will have to happen to take advantage of that human capital.  We will also have to change a number of views about older people for them to be employable and paid attention to, and views about working in new careers among the older people themselves.  See the blog entries Getting the world ready for radical life extension, Social ethics of longevity, and Social evolution and biological evolution – another dialog with Marios Kyriazis.

7.     Old people tend to be rigid and set in their ways, so having a society with lots of old people due to life extension will lead to things becoming fixed and rigid.  The stereotype is not correct though many older people do tend to shrivel up and become rigid, particularly people who retire and seek “the good life” in a warmer climate but find nothing particularly generative or relevant.  Speaking personally , I was a forward-looking chance-taker in my youth, at the age of 22 in 1952 throwing myself into a completely undeveloped path called computers hoping that would lead to a good and very long career.  It did.  Fifty five years later in 2007 at the age of 77, I decided to take another chance and throw myself into another undeveloped path, longevity science with an eye to life extension, hoping that will lead to another good and very long career.  And that shows initial promise of happening.  I see myself contributing to society as much as I ever have in my life.  I have made several new close friends and colleagues during the last year and see my life as branching out in multiple new directions.  I think I am not unique.  Men and women in their 60s, 70s and 80s in good health and with good mental capabilities have a great capacity to innovate, to do things new and to create.  In fact we can take chances we might have hesitated to take in our youths because we have already lived a normal successful life.  We have little to loose and everything to gain. 

Average lifespan has nearly tripled since the time of the Romans and we have accommodated.  We will probably have the scientific possibility of tripling it again within a couple of decades.   This time the accommodation in our thinking and institutions will have to happen a lot faster for the possibility to become real – in decades rather than in millennia.   

 Enough about myths for now.  The next post will be back on the science of aging. 

A group of well-intentioned people, mostly men, take human growth hormone (HGH) or an HGH promoter to stay young and fit and, they think, to live longer.  They are wrong in one important respect: injection of HGH or use of a promoter of HGH, if anything, shortens lives.  Very recent research indicates that inhibition of growth hormone may in fact be an approach to life extension.  This blog reviews key past and current research related to growth hormone, its health effects and longevity/shortivity.

What is HGH?

“Growth hormone (GH) is a protein-based peptide hormone. It stimulates growth, cell reproduction and regeneration in humans and other animals. Growth hormone is a 191-amino acid, single-chain polypeptide that is synthesized, stored, and secreted by the somatotroph cells within the lateral wings of the anterior pituitary gland. Somatotropin refers to the growth hormone 1 produced naturally in animals, whereas the term somatropin refers to growth hormone produced by recombinant DNA technology,^[1] and is abbreviated “HGH” in humans. — Growth hormone is used as prescription drug in medicine to treat children’s growth disorders and adult growth hormone deficiency. In the United States, it is only available legally from pharmacies, by prescription from a doctor(ref).”

“In recent years in the United States, some doctors have started to prescribe growth hormone in GH-deficient older patients (but not on healthy people) to increase vitality. While legal, the efficacy and safety of this use for HGH has not been tested in a clinical trial. At this time, HGH is still considered a very complex hormone, and many of its functions are still unknown(ref).^[2]

”Starting back in the late 1990s a number of publications appeared suggesting that HGH treatment may produce a number of positive effects in individuals with GH deficiency and may even help with aging, such as the 1997 publication Growth hormone-releasing hormone and growth hormone-releasing peptide as therapeutic agents to enhance growth hormone secretion in disease and aging.  “These GH secretagogues may have a therapeutic role in short stature and adult GH deficiency. In addition, the use of GH secretagogues in normal aging merits investigation, as growth hormone may regulate body composition in older adults.”  The results of those early studies are sometimes cited out of context today to help sell such secretagogues as anti-aging supplements. 

Marketing of HGH and HGH promoters for anti-aging

Normal production of HGH, like many other hormones, declines precipitously with advancing age.  Today HGH and HGH secretagogues are shamelessly marketed by many companies as anti-aging substances despite lack of supporting research evidence.  “Claims for GH as an anti-aging treatment date back to 1990 when the New England Journal of Medicine published a study wherein GH was used to treat 12 men over 60 (Effects of human growth hormone in men over 60 years old). At the conclusion of the study, all the men showed statistically significant increases in lean body mass and bone mineral, while the control group did not. The authors of the study noted that these improvements were the opposite of the changes that would normally occur over a 10- to 20-year aging period. Despite the fact the authors at no time claimed that GH had reversed the aging process itself, their results were misinterpreted as indicating that GH is an effective anti-aging agent(ref)”  This has led to organizations such as the controversial American Academy of Anti-Aging Medicine promoting the use of this hormone as an “anti-aging agent”.^[32]”

The 2008 report Systematic review: the safety and efficacy of growth hormone in the healthy elderly relates “The literature published on randomized, controlled trials evaluating GH therapy in the healthy elderly is limited but suggests that it is associated with small changes in body composition and increased rates of adverse events. On the basis of this evidence, GH cannot be recommended as an antiaging therapy.” 

The 2007 article No proof that growth hormone therapy makes you live longer, study finds relates “Surveyors of anti-aging elixirs tout human growth hormone as a remedy for all things sagging-from skin to libidos – and claim it can even prevent or reverse aging. But researchers at the Stanford University School of Medicine say there’s no evidence to suggest that this purported fountain of youth has any more effect than a trickle of tap water when it comes to fending off Father Time. — “There is certainly no data out there to suggest that giving growth hormone to an otherwise healthy person will make him or her live longer,” said Hau Liu, MD, a research fellow in the division of endocrinology and in the Center for Primary Care and Outcomes Research, and first author of a review study to be published in the Jan. 16 issue of Annals of Internal Medicine. “We did find, however, that there was substantial potential for adverse side effects.” — Those negative side effects included joint swelling and pain, carpal tunnel syndrome and a trend toward increased new diagnoses of diabetes or pre-diabetes. “You’re paying a lot of money for a therapy that may have minimal or no benefit and yet has a potential for some serious side effects,” Liu said. “You’ve got to really think about what this drug is doing for you.” — Growth hormone is widely promoted on the Internet and its use as a purported anti-aging drug has caught the attention of the popular media, ranging from the “Today Show” to Business Week. — Between 20,000 and 30,000 people in the United States used growth hormone as an anti-aging therapy in 2004, a tenfold increase since the mid-1990s, according to the authors of an unrelated study published in the Journal of the American Medical Association in 2005. This increase comes despite both the high cost of such therapy – often more than $1,000 a month – and the illegality of distributing growth hormone for anti-aging therapy in this country. Those numbers prompted Liu and some colleagues to see if the medical literature provided any support for such therapy.”

The Quackwatch article Growth Hormone Schemes and Scams provides a history of how marketing has trumped science in promoting HGH as an anti-aging substance. “Human growth hormone (HGH) is a substance secreted by the pituitary gland that promotes growth during childhood and adolescence. Growth hormone acts on the liver and other tissues to stimulate production of insulin-like growth factor I (IGF-I), which is responsible for the growth-promoting effects of growth hormone and also reflects the amount produced. Blood levels of circulating IGF-I tend to decrease as people age or become obese [1]. Many marketers would like you to believe that boosting HGH blood levels can reduce body fat; build muscle; improve sex life, sleep quality, vision and memory; restore hair growth and color; strengthen the immune system; normalize blood sugar; increase energy; and “turn back your body’s biological clock.” This article traces the history of these claims and why you should disregard them. — Marketing “Milestones” — The drive to popularize growth hormone began about 20 years ago with publication of the book Life Extension: A Practical Scientific Approach, by Durk Pearson and Sandy Shaw [2]. The book’s central premise was large amounts of vitamins, minerals, amino acids, and other substances would cause people to add muscle, burn fat, and live much longer. Although their advice had no scientific basis [3,4], Pearson and Shaw made hundreds of talk-show appearances that boosted sales of the substances they recommended. — Soon after the book’s publication, many amino acid products were claimed to cause overnight weight loss by increasing the release of growth hormone. So called “growth-hormone releasers” were also marketed to bodybuilders with claims that they would help build muscle. Such claims are unfounded because amino acids taken by mouth do not stimulate growth hormone release. These formulations are based mainly on misinterpreted studies of intravenous arginine, which can increase HGH blood levels for an hour or so. Taking it by mouth has no such effect. The FTC [5-9], and the New York City Department of Consumer Affairs [10] attacked some companies making “growth-hormone release” claims, but these actions had very little effect on the overall marketplace. — In 1990, The New England Journal of Medicine published a study that attracted mainstream media attention. The study involved 12 men, aged 61 to 81, who were apparently healthy but had IGF-I levels below those found in normal young men. The 12 men were given growth hormone injections three times a week for six months and compared with 9 men who received no treatment. The treatment resulted in a decrease in adipose (fatty) tissue and increases in lean body (muscle) mass and lumbar spine density [11]. An accompanying editorial warned that some of the subjects had experienced side effects and that the long-range effects of administering HGH to healthy adults were unknown. It also warned that the hormone shots were expensive and that the study had not examined whether the men who received the hormone had substantially improved their muscle strength, mobility, or quality of life [1]. — Despite the warning, the study inspired many offbeat physicians to market themselves as “anti-aging specialists.” Many such physicians offer expensive tests that supposedly determine the patient’s “biological age,” which they promise to lower with expensive hormone shots and dietary supplements.”

Continuing the Quackwatch quote, “In 2001, NBC’s Dateline showed what happened when a 57-year-old woman visited a Cenegenics clinic in Las Vegas, Nevada, where she underwent $1,500 worth of tests and was offered a hormone and 40-pill-a-day supplement program that would cost $1,500 a month. She was told that although she tested at “age 54,”her hormone levels were “sub-optimal” and that optimal would be the level of a 30-year -old [12]. — — The Internet has added another dimension to the HGH marketplace. Thousands of Web sites and spam e-mailers are hawking the actual hormone; alleged HGH releasers; alleged oral hormone products (which can’t work because any HGH would be digested); and/or “homeopathic HGH” products. – The bottom line: Although growth hormone levels decline with age, it has not been proven that trying to maintain the levels that exist in young persons is beneficial. Considering the high cost, significant side effects, and lack of proven effectiveness, HGH shots appear to be a very poor investment. So called “growth-hormone releasers,” oral “growth hormone,” and “homeopathic HGH” products are fakes.” (The writer might be going a bit too far here, in that while some products are fakes, HGH secretagogues may to some extent work.)

Substances marketed mainly to men for strength and sexual vitality include include testosterone and strength-promoting steroids in addition to HGH.  See the recent well-researched three-part press exposition in the Star-Ledger Strong at any cost, especially Part 3:  Booming anti-aging business relies on risky mix of steroids, growth hormone.

Molecular biology of GH and aging

A number of publications point to the importance of IGF-1 axis signaling in regulating healthspan and lifespan, for example the 2008 publication  Role of the GH/IGF-1 axis in lifespan and healthspan: lessons from animal models.  Growth hormone administration normally stimulates IGF production in tissues whereas greater longevity is normally associated with downregulation of IGF activity.  The 2004 article The GH/IGF-I axis and longevity reports “These and other results suggest that in mammals too, lifespan can be increased by continuous, long-term downregulation of IGF signaling. Since growth hormone administration normally stimulates IGF production in tissues, the question arises whether the beneficial effects of GH, as reported by others, could be IGF independent.”  So, besides producing nasty side effects, giving HGH to healthy elderly people may in fact shorten their lives.  As pointed out in the 2010 article [IGF and insulin signaling pathways in longevity]: “The role of the somatotropic hormone axis in mammalian longevity has been studied in diverse experimental models in vivo. This endocrine axis allows regulation of lifespan via metabolism modifications and oxidative stress defense mechanisms.”  Interventions like rapamycin which affect this axis via suppression of the mTOR gene or promotion of SIRT1 activity via resveratrol which also impacts on this axis appear indeed to be life-extending.  The evidence suggests that affecting this axis via exogenous GH administration could well produce the opposite effect and be life-shortening. 

 Inhibiting growth hormone and cancer

Starting in the mid-late 1990s, inhibition of growth hormone has been seen as a possible anti-cancer therapy.  The 2001 paper Antagonists of GHRH Decrease Production of GH and IGF-I in MXT Mouse Mammary Cancers and Inhibit Tumor Growth.  “The goal of our study was to investigate whether antagonists of GHRH can interfere with the effects of GH and IGF-I in MXT mouse mammary cancers. GHRH antagonists JV-1-36 and JV-1-38 inhibited growth of estrogen-independent MXT mouse mammary cancers in vivo, producing about 50% reduction in tumor volume (P < 0.05). This growth inhibition was associated with a decrease in cell proliferation and an increase in apoptosis in MXT cancers.– Our results demonstrate that GHRH antagonists decrease the local production of both GH and IGF-I in MXT mouse mammary cancers, the resulting growth inhibition being the consequence of reduced cell proliferation and increased apoptosis.” 

Other publications on the anti-cancer effects of inhibiting growth hormone go as far back as 1997.  They include Antagonists of growth hormone-releasing hormone and somatostatin analog RC-160 inhibit the growth of the OV-1063 human epithelial ovarian cancer cell line xenografted into nude mice, Antagonists of growth hormone-releasing hormone inhibit the proliferation of experimental non-small cell lung carcinoma, Suppression of tumor growth by growth hormone-releasing hormone antagonist JV-1-36 does not involve the inhibition of autocrine production of insulin-like growth factor II in H-69 small cell lung carcinoma, Review Endocrine and antineoplastic actions of growth hormone-releasing hormone antagonists,  Inhibition of proliferation of PC-3 human prostate cancer by antagonists of growth hormone-releasing hormone: lack of correlation with the levels of serum IGF-I and expression of tumoral IGF-II and vascular endothelial growth factor, Antagonists of growth hormone-releasing hormone arrest the growth of MDA-MB-468 estrogen-independent human breast cancers in nude mice, Antagonists of growth hormone-releasing hormone (GH-RH) inhibit in vivo proliferation of experimental pancreatic cancers and decrease IGF-II levels in tumours, Inhibition of growth and metastases of MDA-MB-435 human estrogen-independent breast cancers by an antagonist of growth hormone-releasing hormone, Antagonists of growth hormone-releasing hormone (GH-RH) inhibit IGF-II production and growth of HT-29 human colon cancers, Inhibition of PC-3 human prostate cancers by analogs of growth hormone-releasing hormone (GH-RH) endowed with vasoactive intestinal peptide (VIP) antagonistic activity, Inhibition of proliferation in human MNNG/HOS osteosarcoma and SK-ES-1 Ewing sarcoma cell lines in vitro and in vivo by antagonists of growth hormone-releasing hormone: effects on insulin-like growth factor II, Inhibition of growth, production of insulin-like growth factor-II (IGF-II), and expression of IGF-II mRNA of human cancer cell lines by antagonistic analogs of growth hormone-releasing hormone in vitro, Inhibition of growth and reduction in tumorigenicity of UCI-107 ovarian cancer by antagonists of growth hormone-releasing hormone and vasoactive intestinal peptide, Growth hormone-releasing hormone (GHRH) antagonists inhibit the proliferation of androgen-dependent and -independent prostate cancers, Growth hormone-releasing hormone: an autocrine growth factor for small cell lung carcinoma, , Growth hormone-releasing hormone antagonist MZ-5-156 inhibits growth of DU-145 human androgen-independent prostate carcinoma in nude mice and suppresses the levels and mRNA expression of insulin-like growth factor II in tumors, Growth hormone-releasing hormone antagonist MZ-4-71 inhibits in vivo proliferation of Caki-I renal adenocarcinoma, Growth inhibition of estrogen-dependent and estrogen-independent MXT mammary cancers in mice by the bombesin and gastrin-releasing peptide antagonist RC-3095. 

Inhibition of growth hormone may be an approach to life extension

A December 2010 research publication suggests that life extension could possibly result from the opposite of what the HGH hucksters are promoting: Effects of a growth hormone-releasing hormone antagonist on telomerase activity, oxidative stress, longevity, and aging in mice.  “Here, we determined the effects of treatment with the GH-releasing hormone (GHRH) receptor antagonist MZ-5-156 on aging in SAMP8 mice, a strain that develops with aging cognitive deficits and has a shortened life expectancy. Starting at age 10 mo, mice received daily s.c. injections of 10 μg/mouse of MZ-5-156. Mice treated for 4 mo with MZ-5-156 showed increased telomerase activity, improvement in some measures of oxidative stress in brain, and improved pole balance, but no change in muscle strength. MZ-5-156 improved cognition after 2 mo and 4 mo, but not after 7 mo of treatment (ages 12, 14 mo, and 17 mo, respectively). Mean life expectancy increased by 8 wk with no increase in maximal life span, and tumor incidence decreased from 10 to 1.7%. These results show that treatment with a GHRH antagonist has positive effects on some aspects of aging, including an increase in telomerase activity.” 

I caution that the SAMP8 mice used in the experiment were relatively short-lived to start with and it is not clear that the same experiment applied to normal wild-type mice would also result in healthspan extension and lifespan extension.  Nonetheless, the popular press has speculated that the research results probably apply to humans as well.  According a press report that appeared in multiple publications “ Scientists found blocking growth hormone with a compound called MZ-5-156, might actually help people live longer and reverse signs of aging, contrary to current thinking. — The researchers say the study is important because many older adults use growth hormone, thinking it is the fountain of youth, when instead it may be just the opposite and hazardous. — The study, published online in the Proceedings of the National Academy of Sciences, showed that blocking growth hormone in mice with a compound called MZ-5-156 improved cognition and activity of telomerase that protects DNA from damage that could increase lifespan. — They also found decreased tumor activity in the mice that are genetically engineered for studying the aging process. John E. Morley, M.D., study co-investigator and director of the divisions of geriatric medicine and endocrinology at Saint Louis University School of Medicine says sometime people take growth hormone because they think it will be the fountain of youth. — MZ-5-156 that is a “growth hormone-releasing hormone (GHRH) antagonist”, inhibited a variety of cancers, including prostate, breast, brain and lung cancers.” The ability of many GHRH antagonists for curbing cancer has been noted in the past. — In the aging mice, MZ-5-156 improved short-term memory and reversed oxidative stress in the brain, in turn reversing memory loss. — William A. Banks, M.D., lead study author and professor of internal medicine and geriatrics at the University of Washington School of Medicine in Seattle, said the findings from the research team, “determine that antagonists of growth hormone-releasing hormone have beneficial effects on aging.” — Contrary to the popular belief that growth hormone may be the “fountain-of-youth”, the new findings show that blocking the effect of growth hormone with the growth hormone releasing compound MZ-5-156, reversed signs of aging in mice and inhibited several types of cancer.

As a personal note, I took an HGH secretagogue for a short period about 15 years ago and then stopped.  As long-stated in my treatise “I do not take HGH or HGH promoters because they can have serious side effects – I tried once and got a serious case of arthritis.” 

The central guiding principle of this blog is scientific integrity.  For that reason I do not accept advertisements or commercial sponsorships and I do not associate myself with anti-aging practitioners who provide therapies based on faulty or misrepresented science.

The 14^th theory of aging described in my treatise is Programmed Epigenomic Changes.  But exactly what are the epigenomic changes and how do they work?  Much is still to be learned in this area but recent research is going a long way to increase our understanding.  A great deal of this research has focused on epigenetic mechanisms in certain cancers but many of the lessons being learned also relate to aging  This blog entry reviews selected research publications on topics related to the epigenetics of certain cancers and aging. 

The two most- studied forms of epigenetic changes are:

·        DNA methylation of the promoter regions of certain genes, generally resulting in the silencing of the affected genes. If a pro-apoptic gene like P53 is silenced, for example, the result can be tumor formation.  If a longevity-related gene like SIRT1 is silenced, the result could be susceptibility to an age-related disease like diabetes and shortened lifespan.  Or, silencing SIRT1 in a cancer cell might lead that cell to die.  If certain DNA repair genes like WRN are methylated and silenced, the result can be premature aging. “DNA methylation also affects the expression of genes involved in maintaining the integrity of the genome through DNA repair and detoxification of reactive oxygen species(ref).”  Of particular interest from the viewpoint of DNA methylation in mammals are the so-called CpG islands.  “CpG islands typically occur at or near the transcription start site of genes, particularly housekeeping genes, in vertebrates.^[2]  – “Unlike CpG sites in the coding region of a gene, in most instances, the CpG sites in the CpG islands of promoters are unmethylated if genes are expressed(ref).”

·        Histone deacetylation and acetylation, generally having to do respectively with silencing or unsilencing of genes.   Histones are spindles in a cell’s nucleus around which DNA is wrapped; they play important roles in gene activation.  Histone acetylation is a chemical modification of a portion of a histone which leads to selective unwrapping of the DNA making the exposed genes amenable to activation and expression.  Histone deacetylation is the opposite.  

For further background on what is covered here you can review some of my previous blog posts including Epigenetics, epigenomics and aging, DNA methylation, personalized medicine and longevity, Histone acetylase and deacetylase inhibitors, Homicide by DNA methylation, Epigenomic complexity, Epigenetics going mainstream, DNA repair cleanup failure – a root cause for cancers?   and Genomic stability, DNA repair and the sirtuin SIRT6. The May 2010 blog entry Epigenetics, inflammation, cancer, immune system, neurological and cardiovascular disease and aging quotes from publications dealing with practical applications of epigenetics in a variety of biological situations.  This current blog post focuses on cancers and aging.

Overview

The 2010 publication DNA methylation and cancer provides an overview on DNA methylation.  “DNA methylation is one of the most intensely studied epigenetic modifications in mammals. In normal cells, it assures the proper regulation of gene expression and stable gene silencing. DNA methylation is associated with histone modifications and the interplay of these epigenetic modifications is crucial to regulate the functioning of the genome by changing chromatin architecture. The covalent addition of a methyl group occurs generally in cytosine within CpG dinucleotides which are concentrated in large clusters called CpG islands. DNA methyltransferases are responsible for establishing and maintenance of methylation pattern. It is commonly known that inactivation of certain tumor-suppressor genes occurs as a consequence of hypermethylation within the promoter regions and a numerous studies have demonstrated a broad range of genes silenced by DNA methylation in different cancer types. On the other hand, global hypomethylation, inducing genomic instability, also contributes to cell transformation. Apart from DNA methylation alterations in promoter regions and repetitive DNA sequences, this phenomenon is associated also with regulation of expression of noncoding RNAs such as microRNAs that may play role in tumor suppression. DNA methylation seems to be promising in putative translational use in patients and hypermethylated promoters may serve as biomarkers. Moreover, unlike genetic alterations, DNA methylation is reversible what makes it extremely interesting for therapy approaches. The importance of DNA methylation alterations in tumorigenesis encourages us to decode the human epigenome. Different DNA methylome mapping techniques are indispensable to realize this project in the future.” 

Research findings

Typical patterns of GPC island methylation together with certain mutations appear to be associated with specific cancers

Colorectal cancer is one of the most-studied in this regard.  Going back to 2007, the publication TGFBR2 mutation is correlated with CpG island methylator phenotype in microsatellite instability-high colorectal cancer reports “The transforming growth factor-beta receptor type 2 gene (TGFBR2) is mutated in most microsatellite instability-high (MSI-H) colorectal cancers. Promoter methylation of RUNX3 (runt-related transcription factor 3; encoding a transcription factor downstream of the TGF-beta pathway) is observed in colorectal cancer with CpG island methylator phenotype (CIMP), which is characterized by extensive promoter methylation and is associated with MSI-H and BRAF mutations. —  Using 144 MSI-H colorectal cancers derived from 2 large prospective cohort studies, we analyzed a mononucleotide repeat of TGFBR2 and quantified DNA methylation (by MethyLight technology) in 8 CIMP-specific promoters  –.  After stratification by sex, location, tumor differentiation, RUNX3 status, KRAS/BRAF status, or p53 status, CIMP-high was persistently correlated with TGFBR2 mutation. In contrast, RUNX3, KRAS, or BRAF status was no longer correlated with TGFBR2 mutation after stratification by CIMP status. In conclusion, TGFBR2 mutation is associated with CIMP-high and indirectly with RUNX3 methylation. Our findings emphasize the importance of analyzing global epigenomic status (for which CIMP status is a surrogate marker) when correlating a single epigenetic event (eg, RUNX3 methylation) with any other molecular or clinicopathologic variables.” 

[“Microsatellites are repeated sequences of DNA. Although the length of these microsatellites is highly variable from person to person, each individual has microsatellites of a set length. — These repeated sequences are common, and normal. — The appearance of abnormally long or short microsatellites in an individual’s DNA is referred to as microsatellite instability. Microsatellite instability (MSI) is a condition manifested by damaged DNA due to defects in the normal DNA repair process.^[1] Sections of DNA called microsatellites, which consist of a sequence of repeating units of 1-6 base pairs in length, become unstable and can shorten or lengthen, –(ref)”]

The 2008 publication Comprehensive biostatistical analysis of CpG island methylator phenotype in colorectal cancer using a large population-based sample reports “The CpG island methylator phenotype (CIMP) is a distinct phenotype associated with microsatellite instability (MSI) and BRAF mutation in colon cancer. — DNA methylation at 16 CpG islands [CACNA1G, IGF2, NEUROG1, RUNX3 and SOCS1 plus CDKN2A (p16), CHFR, CRABP1, HIC1, IGFBP3, MGMT, MINT1, MINT31, MLH1, p14 (CDKN2A/ARF) and WRN] was quantified in 904 colorectal cancers by real-time PCR (MethyLight). — multivariate logistic regression demonstrated that CIMP-high was independently associated with older age, proximal location, poor differentiation, MSI-high, BRAF mutation, and inversely with LINE-1 hypomethylation and beta-catenin (CTNNB1) activation. — CONCLUSIONS: Our study provides valuable data for standardization of the use of CIMP-high-specific methylation markers. CIMP-high is independently associated with clinical and key molecular features in colorectal cancer. Our data also suggest that KRAS mutation is related with a random CpG island methylation pattern which may lead to CIMP-low tumors.”

The 2010 review publication DNA methylation markers in colorectal cancer reports: “Colorectal cancer (CRC) arises as a consequence of the accumulation of genetic and epigenetic alterations in colonic epithelial cells during neoplastic transformation. Epigenetic modifications, particularly DNA methylation in selected gene promoters, are recognized as common molecular alterations in human tumors. Substantial efforts have been made to determine the cause and role of aberrant DNA methylation (“epigenomic instability”) in colon carcinogenesis. In the colon, aberrant DNA methylation arises in tumor-adjacent, normal-appearing mucosa. Aberrant methylation also contributes to later stages of colon carcinogenesis through simultaneous methylation in key specific genes that alter specific oncogenic pathways. Hypermethylation of several gene clusters has been termed CpG island methylator phenotype and appears to define a subgroup of colon cancer distinctly characterized by pathological, clinical, and molecular features. DNA methylation of multiple promoters may serve as a biomarker for early detection in stool and blood DNA and as a tool for monitoring patients with CRC. DNA methylation patterns may also be predictors of metastatic or aggressive CRC. Therefore, the aim of this review is to understand DNA methylation as a driving force in colorectal neoplasia and its emerging value as a molecular marker in the clinic.”

The 2010 publication [Promoter hypermethylation and CpG island methylator phenotype in colorectal carcinogenesis] summarizes the situation.  “Amino acid alterations or insufficient protein synthesis caused by the mutation on genes has long been recognized as the main mechanism of silencing of suppressor genes leading to carcinogenesis. However, epigenetic silencing of the cancer related genes induced by hyper-methylation of promoter is recognized as an additional important molecular mechanism for carcinogenesis. Differing molecular mechanisms of colorectal carcinogenesis have become known after advanced understanding of genes silenced by promoter methylation.” 

Hypomethylation as well as hypermethylation can play roles in cancer susceptibility and ill-health

Some genes can best remain methylated.  The 2010 publication Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors reports “BACKGROUND: Genome-wide DNA hypomethylation plays a role in genomic instability and carcinogenesis. LINE-1 (L1 retrotransposon) constitutes a substantial portion of the human genome, and LINE-1 methylation correlates with global DNA methylation status. LINE-1 hypomethylation in colon cancer has been strongly associated with poor prognosis. However, whether LINE-1 hypomethylators constitute a distinct cancer subtype remains uncertain. Recent evidence for concordant LINE-1 hypomethylation within synchronous colorectal cancer pairs suggests the presence of a non-stochastic mechanism influencing tumor LINE-1 methylation level. Thus, it is of particular interest to examine whether its wide variation can be attributed to clinical, pathologic or molecular features. — DESIGN: Utilizing a database of 869 colorectal cancers in two prospective cohort studies, we constructed multivariate linear and logistic regression models for LINE-1 methylation (quantified by Pyrosequencing). Variables included age, sex, body mass index, family history of colorectal cancer, smoking status, tumor location, stage, grade, mucinous component, signet ring cells, tumor infiltrating lymphocytes, CpG island methylator phenotype (CIMP), microsatellite instability, expression of TP53 (p53), CDKN1A (p21), CTNNB1 (beta-catenin), PTGS2 (cyclooxygenase-2), and FASN, and mutations in KRAS, BRAF, and PIK3CA. — CONCLUSIONS: LINE-1 extreme hypomethylators appear to constitute a previously-unrecognized, distinct subtype of colorectal cancers, which needs to be confirmed by additional studies. Our tumor LINE-1 methylation data indicate enormous epigenomic diversity of individual colorectal cancers.”

Hypermethylation of microRNA genes can play roles in cancers

The 2008 publication Inactivation of miR-34a by aberrant CpG methylation in multiple types of cancer reports “Recently, we and others identified the microRNA miR-34a as a target of the tumor suppressor gene product p53. Ectopic miR-34a induces a G(1) cell cycle arrest, senescence and apoptosis. Here we report that miR-34a expression is silenced in several types of cancer due to aberrant CpG methylation of its promoter. 19 out of 24 (79.1%) primary prostate carcinomas displayed CpG methylation of the miR-34a promoter and concomitant loss of miR-34a expression. CpG methylation of the miR-34a promoter was also detected in breast (6/24; 25%), lung (7/24; 29.1%), colon (3/23; 13%), kidney (3/14; 21.4%), bladder (2/6; 33.3%) and pancreatic (3/19; 15.7%) carcinoma cell lines, as well as in melanoma cell lines (19/44; 43.2%) and primary melanoma (20/32 samples; 62.5%). Silencing of miR-34a was dominant over its transactivation by p53 after DNA damage. Re-expression of miR-34a in prostate and pancreas carcinoma cell lines induced senescence and cell cycle arrest at least in part by targeting CDK6. These results show that miR-34a represents a tumor suppressor gene which is inactivated by CpG methylation and subsequent transcriptional silencing in a broad range of tumors. 

The 2010 publication Epigenetic silencing of miR-137 is an early event in colorectal carcinogenesis reports “Global downregulation of microRNAs (miRNA) is a common feature in colorectal cancer (CRC). Whereas CpG island hypermethylation constitutes a mechanism for miRNA silencing, this field largely remains unexplored. Herein, we describe the epigenetic regulation of miR-137 and its contribution to colorectal carcinogenesis. We determined the methylation status of miR-137 CpG island in a panel of six CRC cell lines and 409 colorectal tissues [21 normal colonic mucosa from healthy individuals (N-N), 160 primary CRC tissues and their corresponding normal mucosa (N-C), and 68 adenomas]. TaqMan reverse transcription-PCR and in situ hybridization were used to analyze miR-137 expression. In vitro functional analysis of miR-137 was performed. Gene targets of miR-137 were identified using a combination of bioinformatic and transcriptomic approaches. We experimentally validated the miRNA:mRNA interactions. Methylation of the miR-137 CpG island was a cancer-specific event and was frequently observed in CRC cell lines (100%), adenomas (82.3%), and CRC (81.4%), but not in N-C (14.4%; P < 0.0001 for CRC) and N-N (4.7%; P < 0.0001 for CRC). Expression of miR-137 was restricted to the colonocytes in normal mucosa and inversely correlated with the level of methylation. Transfection of miR-137 precursor in CRC cells significantly inhibited cell proliferation. Gene expression profiling after miR-137 transfection discovered novel potential mRNA targets. We validated the interaction between miR-137 and LSD-1. Our data indicate that miR-137 acts as a tumor suppressor in the colon and is frequently silenced by promoter hypermethylation. Methylation silencing of miR-137 in colorectal adenomas suggests it to be an early event, which has prognostic and therapeutic implications.” 

Aberrant DNA methylation and histone modifications can work together to induce silencing of miRNA genes in cancers 

The 2009 publication Epigenetic regulation of microRNA expression in colorectal cancer reports “In the last years, microRNAs (miRNA) have emerged as new molecular players involved in carcinogenesis. Deregulation of miRNAs expression has been shown in different human cancer but the molecular mechanism underlying the alteration of miRNA expression is unknown. To identify tumor-supressor miRNAs silenced through aberrant epigenetic events in colorectal cancer (CRC), we used a sequential approach. We first identified 5 miRNAs down-regulated in patient with colorectal cancer samples and located around/on a CpG island. Treatment with a DNA methyltransferase inhibitor and a HDAC inhibitor restored expression of 3 of the 5 microRNAs (hsa-miR-9, hsa-miR-129 and hsa-miR-137) in 3 CRC cell lines. Expression of hsa-miR-9 was inversely correlated with methylation of their promoter regions as measure by MSP and bisulphate sequencing. Further, methylation of the hsa-miR-9-1, hsa-miR-129-2 and hsa-miR-137 CpG islands were frequently observed in CRC cell lines and in primary CRC tumors, but not in normal colonic mucosa. Finally, methylation of hsa-miR-9-1 was associated with the presence of lymph node metastasis. In summary, our results aid in the understanding of miRNA gene regulation showing that aberrant DNA methylation and histone modifications work together to induce silencing of miRNAs in CRC.” 

P53 apoptotic protection in cancers can be subverted by promoter methylation and silencing of its microRNA components microRNA-34b/c

The 2008 publication Epigenetic silencing of microRNA-34b/c and B-cell translocation gene 4 is associated with CpG island methylation in colorectal cancer relates “Altered expression of microRNA (miRNA) is strongly implicated in cancer, and recent studies have shown that, in cancer, expression of some miRNAs cells is silenced in association with CpG island hypermethylation. To identify epigenetically silenced miRNAs in colorectal cancer (CRC), we screened for miRNAs induced in CRC cells by 5-aza-2′-deoxycytidine (DAC) treatment or DNA methyltransferase knockout. We found that miRNA-34b (miR-34b) and miR-34c, two components of the p53 network, are epigenetically silenced in CRC; that this down-regulation of miR-34b/c is associated with hypermethylation of the neighboring CpG island; and that DAC treatment rapidly restores miR-34b/c expression. Methylation of the miR-34b/c CpG island was frequently observed in CRC cell lines (nine of nine, 100%) and in primary CRC tumors (101 of 111, 90%), but not in normal colonic mucosa. Transfection of precursor miR-34b or miR-34c into CRC cells induced dramatic changes in the gene expression profile, and there was significant overlap between the genes down-regulated by miR-34b/c and those down-regulated by DAC. We also found that the miR-34b/c CpG island is a bidirectional promoter which drives expression of both miR-34b/c and B-cell translocation gene 4 (BTG4); that methylation of the CpG island is also associated with transcriptional silencing of BTG4; and that ectopic expression of BTG4 suppresses colony formation by CRC cells. Our results suggest that miR-34b/c and BTG4 are novel tumor suppressors in CRC and that the miR-34b/c CpG island, which bidirectionally regulates miR-34b/c and BTG4, is a frequent target of epigenetic silencing in CRC.”

This document also points out how a hypermethylation problem in the miR-34b/c CpG island can be cleared up using a demethylation agent DAC.  This agent, 5-Aza-2′-deoxycytidine, has been known for some time to inhibit promoter methylation and to suppress the growth of certain tumor cell lines. “We exposed seven human tumor cell lines and two human fibroblast cell strains to the demethylating agent, 5-aza-2′-deoxycytidine (5-Aza-CdR), to determine whether the silencing of growth-regulatory genes by de novo methylation in immortalized cell lines could be reversed, possibly restoring growth control. After recovery from the immediate cytotoxic effects of 5-Aza-CdR, this agent suppressed cellular growth in all seven tumor lines but not in either fibroblast strain(ref).”

The 2010 publication The miR-34 family in cancer and apoptosis is a review paper confirming the role of hypermethylation in miR-34a/b/c as inactivating P53 protection in a variety of tumor types including neuroblastomas: “Recently, the transcription factor encoded by tumor suppressor gene p53 was shown to regulate the expression of microRNAs. The most significant induction by p53 was observed for the microRNAs miR-34a and miR-34b/c, which turned out to be direct p53 target genes. Ectopic miR-34 expression induces apoptosis, cell-cycle arrest or senescence. In many tumor types the promoters of the miR-34a and the miR-34b/c genes are subject to inactivation by CpG methylation. MiR-34a resides on 1p36 and is commonly deleted in neuroblastomas. Furthermore, the loss of miR-34 expression has been linked to resistance against apoptosis induced by p53 activating agents used in chemotherapy. In this review, the evidence for a role of miR-34a and miR-34b/c in the apoptotic response of normal and tumor cells is surveyed.”  This knowledge could conceivably lead to treatments for otherwise untreatable and rapid-killer diseases like gliablastoma. 

Additional interesting publications relating CpG Island methylator phenotype (CIMP) to cancers are (2010) NGX6 gene mediated by promoter methylation as a potential molecular marker in colorectal cancer,  (2009) Colon tumor mutations and epigenetic changes associated with genetic polymorphism: insight into disease pathways and the 2006 report Association of smoking, CpG island methylator phenotype, and V600E BRAF mutations in colon cancer. 

Histone deacetylase inhibition is being investigated as an epigenetic treatment for cancers

For example, the 2009 publication Epigenetic Targeting of Transforming Growth Factor beta Receptor II and Implications for Cancer Therapy reports “The transforming growth factor (TGF) beta signaling pathway is involved in many cellular processes including proliferation, differentiation, adhesion, motility and apoptosis. The loss of TGFbeta signaling occurs early in carcinogenesis and its loss contributes to tumor progression. The loss of TGFbeta responsiveness frequently occurs at the level of the TGFbeta type II receptor (TGFbetaRII) which has been identified as a tumor suppressor gene (TSG). In keeping with its TSG role, the loss of TGFbetaRII expression is frequently associated with high tumor grade and poor patient prognosis.   Reintroduction of TGFbetaRII into tumor cell lines results in growth suppression. Mutational loss of TGFbetaRII has been characterized, particularly in a subset of colon cancers with DNA repair enzyme defects. However, the most frequent cause of TGFbetaRII silencing is through epigenetic mechanisms. Therefore, re-expression of TGFbetaRII by use of epigenetic therapies represents a potential therapeutic approach to utilizing the growth suppressive effects of the TGFbeta signaling pathway. However, the restoration of TGFbeta signaling in cancer treatment is challenging because in late stage disease, TGFbeta is a pro-metastatic factor. This effect is associated with increased expression of the TGFbeta ligand. In this Review, we discuss the mechanisms associated with TGFbetaRII silencing in cancer and the potential usefulness of histone deacetylase (HDAC) inhibitors in reversing this effect. The use of HDAC inhibitors may provide a unique opportunity to restore TGFbetaRII expression in tumors as their pleiotropic effects antagonize many of the cellular processes, which mediate the pro-metastatic effects associated with increased TGFbeta expression.” 

The SIRT1 gene is activated in cancers – whoops!

In the course of this discussion we find that another familiar gene SIRT1 is involved in a whole new context.  In previous blog entries and in the aging-science community, SIRT1 has been mainly discussed as a longevity gene, the one involved in calorie restriction.  And activation of SIRT1 via substances such as resveratrol has been seen as a very good thing for longevity(ref)(ref)(ref).  However, the flip side is that the SIRT1 gene is activated in many cancers and it has been suggested that SIRT1 inhibition may provide an approach to shrinking tumors.

The 2009 publication SIRT1 histone deacetylase expression is associated with microsatellite instability and CpG island methylator phenotype in colorectal cancer speaks to this issue.  “The class III histone deacetylase SIRT1 (sir2) is important in epigenetic gene silencing. Inhibition of SIRT1 reactivates silenced genes, suggesting a possible therapeutic approach of targeted reversal of aberrantly silenced genes. In addition, SIRT1 may be involved in the well-known link between obesity, cellular energy balance and cancer. However, a comprehensive study of SIRT1 using human cancer tissue with clinical outcome data is currently lacking, and its prognostic significance is uncertain. Using the database of 485 colorectal cancers in two independent prospective cohort studies, we detected SIRT1 overexpression in 180 (37%) tumors by immunohistochemistry. We examined its relationship to the CpG island methylator phenotype (CIMP), related molecular events, clinical features including body mass index, and patient survival. We quantified DNA methylation in eight CIMP-specific promoters (CACNA1G, CDKN2A, CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1) and eight other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN) by MethyLight. SIRT1 overexpression was associated with CIMP-high (> or =6 of 8 methylated CIMP-specific promoters, P=0.002) and microsatellite instability (MSI)-high phenotype (P<0.0001).  — In both univariate and multivariate analyses, SIRT1 overexpression was significantly associated with the CIMP-high MSI-high phenotype (multivariate odds ratio, 3.20; 95% confidence interval, 1.35-7.59; P=0.008). In addition, mucinous component (P=0.01), high tumor grade (P=0.02), and fatty acid synthase overexpression (P=0.04) were significantly associated with SIRT positivity in multivariate analysis. SIRT1 was not significantly related with age, sex, tumor location, stage, signet ring cells, cyclooxygenase-2 (COX-2), LINE-1 hypomethylation, KRAS, BRAF, BMI, PIK3CA, HDAC, p53, beta-catenin, COX-2, or patient prognosis. In conclusion, SIRT1 expression is associated with CIMP-high MSI-high colon cancer, suggesting involvement of SIRT1 in gene silencing in this unique tumor subtype.”

P53 in the absence of hypomethylation activates the microRNA miR-34a resulting in reduced SIRT1 and tumor suppression, at least in glioma cells    

Going further, the 2010 publication MicroRNA-34a: a novel tumor suppressor in p53-mutant glioma cell line U25 relates “BACKGROUND AND AIMS: Previous studies showed that microRNA-34 (miR-34a) family was found to be a direct target of p53, functioning downstream of the p53 pathway as tumor suppressors. MiR-34a was identified to represent the status of p53 and participate in initiation and progress of cancers. We undertook this study to investigate the role of miR-34a in glioma cells. — METHODS: Expression levels of miR-34a in glioma cell lines and normal brains were detected using qRT-PCR. Human U251 glioma cells were transfected with miR-34a mimics, and the effects of miR-34a restoration were assessed by MTT assays, cell cycle analysis, caspase-3 activation, and in vitro migration and invasion assays. A computational search revealed a conserved target site of miR-34a within the 3′-untranslated region of SIRT1. Luciferase reporter assay was performed to examine the effects of miR-34a on expression of potential target gene SIRT1, and mRNA and protein expression of SIRT1 after miR-34a transfection were detected by qRT-PCR and Western blot analysis. — RESULTS: MiR-34a expression was markedly reduced in p53-mutant cells U251 compared with A172 and SHG-44 cells expressing wild-type p53 and normal brains. Overexpression of miR-34a in U251 cells resulted in inhibition of cell growth and arrest in G0-G1 phase and induced apoptosis. Also, restoration of miR-34a significantly reduced in vitro migration and invasion capabilities. Reporter assays indicated that SIRT1 was a direct target of miR-34a. In U251 cells, overexpression of miR-34a decreased SIRT1 protein levels but not mRNA expressions, which demonstrated miR-34a-induced SIRT1 inhibition occurred at the posttranscriptional level. — CONCLUSIONS: Our results demonstrate that miR-34a acts as a tumor suppressor in p53-mutant glioma cells U251, partially through regulating SIRT1.”

There has been controversy about the positive or negative roles SIRT1 plays in cancers.  The probable bottom line is that SIRT1 and SIRT1 activators including resveratrol can play positive roles in both preventing/treating cancers and extending lifespans

To delve further into the role of SIRT1 in cancers and aging and its relationship to epigenetics I quote rather extensively from the 2009 publication SIRT1, Is It a Tumor Promoter or Tumor Suppressor? “SIRT1 has been considered as a tumor promoter because of its increased expression in some types of cancers and its role in inactivating proteins that are involved in tumor suppression and DNA damage repair. However, recent studies demonstrated that SIRT1 levels are reduced in some other types of cancers, and that SIRT1 deficiency results in genetic instability and tumorigenesis, while overexpression of SIRT1 attenuates cancer formation in mice heterozygous for tumor suppressor p53 or APC. Here, I review these recent findings and discuss the possibility that activation of SIRT1 both extends lifespan and inhibits cancer formation. — SIRT1, a proto member of the sirtuin family, modifies histones through deacetylation of K26 in histone H1 (H1K26), K9 in histone H3 (H3K9) and K16 in histone H4 (H4K16). It also deacetylates many non-histone proteins that are involved in cell growth, apoptosis, neuronal protection, adaptation to calorie restriction, organ metabolism and function, cell senescence, and tumorigenesis [1, 3–5]. However, it remains controversial whether SIRT1 acts as a tumor promoter or tumor suppressor due to recent controversy over SIRT1 regarding: 1) its expression level in human cancers; 2) its activity on tumor suppressors and oncoproteins; 3) its effect on growth arrest, cell death, and DNA damage repair; and, finally, 4) its long-term impact on lifespan and cancer risk.”

Going on “It has been shown that SIRT1 is significantly elevated in human prostate cancer [6], acute myeloid leukemia [7], and primary colon cancer [8]. Hida et al. examined SIRT1 protein levels in several different types of skin cancer by immunohistochemical analysis [9]. Overexpression of SIRT1 was frequently observed in all kinds of non-melanoma skin cancers including squamous cell carcinoma, basal cell carcinoma, Bowen’s disease, and actinic keratosis. Based on the elevated levels of SIRT1 in cancers, it was hypothesized that SIRT1 serves as a tumor promoter [10]. However it does not rule out a possibility that increased expression of SIRT1 is a consequence, rather than a cause, of tumorigenesis. In contrast, Wang et al. analyzed a public database and found that SIRT1 expression was reduced in many other types of cancers, including glioblastoma, bladder carcinoma, prostate carcinoma and ovarian cancers as compared to the corresponding normal tissues [11]. Their further analysis of 44 breast cancer and 263 hepatic carcinoma cases also revealed reduced expression of SIRT1 in these tumors [11]. These data suggest that SIRT1 acts as a tumor suppressor rather than a promoter in these tissues.”

The author of this paper presents arguments on both sides of the issue.  On the one hand, there are the surface factors that suggest that SIRT1 activation in older people are likely to increase the risk of carcinogenesis, such as high expression of SIRT1 in certain cancers and inhibition of P53 apoptosis of cancer cells by SIRT1.  On the other hand are the factors that suggest that SIRT1 activation is likely to be both protective against cancers and enhance longevity such as feedback loops through which SIRT1 expression indirectly triggers cancer cell death. “To illustrate the molecular mechanism underlying how activated SIRT1 triggers cell death, the researchers demonstrated that SIRT1 negatively regulates expression of Survivin, which encodes an anti-apoptotic protein, by deacetylating H3K9 within the promoter of Survivin [40]. Altogether, these data suggest that SIRT1 mediates BRCA1 signaling and inhibits tumor growth through repressing transcription of oncogenes or activity of oncoproteins.”  Further SIRT1 plays an important rold in DNA damage repair.  “Sirt1-/- cells displayed chromosome aneuploidy and structural aberrations, conceivably originated from the continuous division of abnormal mitosis. SIRT1 deficiency also causes reduced ability to repair DNA-double strand breaks (DSBs), radiation sensitivity, and impaired DDRs characterized by diminished γH2AX, BRCA1, RAD51 and NBS1 foci formation upon γ-irradiation. Thus, SIRT1 may play a role in recruiting these proteins to DNA damage sites. — In response to oxidative DNA damage, SIRT1 dissociates from its original loci and relocalizes to DSBs to promote repair and maintain genome integrity. Their data indicated that the efficient recruitment of SIRT1 to DSBs requires DNA damage signalling through ATM and H2AX. Without SIRT1, recruitment of RAD51 and NBS1 to DSBs was delayed and strongly reduced, thus highlighting a key role of SIRT1 in the DNA damage repair process. The researchers further showed that SIRT1 inhibits a functionally diverse set of genes that are dereprssed by oxidative stress.”

The author come down on the side of SIRT1 playing a highly positive role both for possibly treating cancers and even for possible life extension.  “Aging has been considered as the most potent carcinogen for cancer, as cancer incidence is quickly elevated in the aging population [46]. It is of great interest to define the conditions, in which the activation of SIRT1 can both extend lifespan and inhibits tumor formation. In C. elegans, mutations that increase the lifespan can also inhibit tumor growth [47]. In animal models, caloric restriction, which activates SIRT1, extends lifespan and is also highly protective against cancer [19, 48–50]. — Then, can direct activation of SIRT1 both extend lifespan and reduce cancer risk? Several lines of evidence suggest that it is possible. First, it has been shown that activation of SIRT1 by a low dose of dietary resveratrol partially mimics caloric restriction and retards aging parameters in mice [51]. Similarly, SRT1720, a more potent and specific agonist in activating SIRT1 than resveratrol [52], mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation [49].”

The article concludes “Finally, as illustrated above that SIRT1 overexpression suppresses the age-related transcriptional changes and reduces formation of colon cancer in APC+/min mice [38], BRCA1-associated mammary cancer [40], spontaneous cancers in multiple tissues of Sirt1+/-;p53+/- mice [11], and γ-irradiation induced lymphoma in p53+/- mice [42]. Thus, through improving metabolic conditions by increasing SIRT1 activity, it is possible to both extend lifespan and reduce cancer risk in humans in the foreseeable future.” 

I am inclined to support the author’s optimistic viewpoint.  Like many SIRT1 researchers, I regularly take resveratrol supplements.  I have been doing so for about four years now.

A 2010 publication confirms the central perceptions of the previously-cited one: SIRT1 and p53, effect on cancer, senescence and beyond: “NAD(+)-dependent Class III histone deacetylase SIRT1 is a multiple function protein critically involved in stress responses, cellular metabolism and aging through deacetylating a variety of substrates including p53, forkhead-box transcription factors, PGC-1alpha, NF-kappaB, Ku70 and histones. The first discovered non-histone target of SIRT1, p53, is suggested to play a central role in SIRT1-mediated functions in tumorigenesis and senescence. SIRT1 was originally considered to be a potential tumor promoter since it negatively regulates the tumor suppressor p53 and other tumor suppressors. There is new evidence that SIRT1 acts as a tumor suppressor based on its role in negatively regulating beta-catenin and survivin. This review provides an overview of current knowledge of SIRT1-p53 signaling and controversies regarding the functions of SIRT1 in tumorigenesis.”

There have been a number of additional recent and interesting publications related to SIRT1, its role in longevity pathways, and its molecular activation and inactivation.  I expect to cover these in another blog entry.