Calorie restriction roundup

Once again the booming literature on calorie restriction (CR) has bested me, and I’ve fallen hopelessly behind. Therefore, without comment, I’ll just run through the last month’s abstracts, with a smattering of brief commentary here and there. Each paper deserves its own entry, but we’re just going to have to make do with this. Quoted passages are all abstract excerpts.

The Nrf2 pathway: Mechanisms Underlying Caloric Restriction and Lifespan Regulation: Implications for Vascular Aging, Ungvari et al.:

We propose that caloric restriction increases bioavailability of NO, decreases vascular reactive oxygen species generation, activates the Nrf2/antioxidant response element pathway, inducing reactive oxygen species detoxification systems, exerts antiinflammatory effects, and, thereby, suppresses initiation/progression of vascular disease that accompany aging.

More on Nrf2 and aging here and here.

Protein vs. sugar in insulin signaling: Opposing Effects of Dietary Protein and Sugar Regulate a Transcriptional Target of Drosophila Insulin-like Peptide Signaling, Buch et al.

Through microarray analysis of flies in which the insulin-producing cells (IPCs) were ablated, we identified a target gene, target of brain insulin (tobi), that encodes an evolutionarily conserved -glucosidase. Flies with lowered tobi levels are viable, whereas tobi overexpression causes severe growth defects and a decrease in body glycogen. Interestingly, tobi expression is increased by dietary protein and decreased by dietary sugar.

Inactivity and inflammation: Calorie restriction modulates inactivity-induced changes in the inflammatory markers CRP and PTX3, Busutti et al.:

Calorie restriction prevents the inflammatory response induced by 14 days of bed rest. We suggest an inverse regulation of CRP and PTX3 in response to changes in energy balance.

*** This was a human study.

“Nutritional emphysema”: Effect of Severe Calorie Restriction on the Lung in Two Strains of Mice, Bishai and Mitzner:

Although the baseline mechanics and alveolar size were quantitatively different in the two strains, both strains showed similar qualitative changes during the starvation and refeeding periods. Thus, in two strains of mice with genetically determined differences in alveolar size neither the mechanics nor the histology show any evidence of emphysema-like changes with this severe caloric insult.

SIRT1 stabilization: Regulation of SIRT1 protein levels by nutrient availability, Kanfi et al.:

We show here that levels of SIRT1 increased in response to nutrient deprivation in cultured cells, and in multiple tissues of mice after fasting. The increase in SIRT1 levels was due to stabilization of SIRT1 protein, and not an increase in SIRT1 mRNA. In addition, p53 negatively regulated SIRT1 levels under normal growth conditions and is also required for the elevation of SIRT1 under limited nutrient conditions.

Protein modification in the heart: Aging and dietary restriction effects on ubiquitination, sumoylation, and the proteasome in the heart, Li et al.:

Cumulatively, our data indicate that DR has many beneficial effects towards the UPP [ubiquitin-proteasome pathway] in the heart, and suggests that a preservation of the UPP may be a potential mechanism by which DR mediates beneficial effects on the cardiovascular system.

Males vs. females, round 1: The brain: Conserved and Differential Effects of Dietary Energy Intake on the Hippocampal Transcriptomes of Females and Males, Martin et al.:

Genes involved in energy metabolism, oxidative stress responses and cell death were affected by the HFG diet in both males and females. The gender-specific molecular genetic responses of hippocampal cells to variations in dietary energy intake identified in this study may mediate differential behavioral responses of males and females to differences in energy availability.

Males vs, females, round 2: The gonad: Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary, Sharov et al.:

CR-mediated reversal of age-associated gene expression changes, reported in somatic organs previously, was limited to a small number of genes in gonads. Instead, in both ovary and testis, CR caused small and mostly gonad-specific effects: suppression of ovulation in ovary and activation of testis-specific genes in testis.

Whew. OK, have a great weekend, everyone.

 

Low-dose resveratrol as a calorie restriction mimetic

Resveratrol, a natural product derived from grape skins and other plant sources, is widely (but not universally) believed to be an activator of the longevity assurance genes known as sirtuins. Despite some debate about its mechanism of action, the compound has received a great deal of attention as a possible pharmaceutical remedy for diseases of aging such as late-onset diabetes.

Most famously, resveratrol has been reported to increase the median lifespan of mice fed a high-fat diet, but that study has been subject to numerous criticisms. The diet in question was so unhealthy it would have made Morgan Spurlock blush, raising questions about its fairness as a model even for the most deranged Western diet. Furthermore, the quantity of resveratrol administered to the mice in the study corresponded to something like 1000 bottles of red wine per day. A skeptical reader could fairly claim that such a study, in which ridiculously high doses of a compound have an effect on an obscenely unhealthy animal, teaches us exactly nothing about what manageable doses of the same compound might accomplish in reasonably healthy people (which is, arguably, the point).

So: do manageable doses of resveratrol have health benefits — specifically, with respect to diseases of aging or aging itself? The first evidence in the affirmative has recently been published by Barger et al., who demonstrate that mice eating a normal ad libitum calorie-controlled* diet supplemented with resveratrol (at a much lower dose than in previous studies) undergo many of the same transcriptional changes as animals undergoing caloric restriction (CR):

A Low Dose of Dietary Resveratrol Partially Mimics Caloric Restriction and Retards Aging Parameters in Mice

Resveratrol in high doses has been shown to extend lifespan in some studies in invertebrates and to prevent early mortality in mice fed a high-fat diet. We fed mice from middle age (14-months) to old age (30-months) either a control diet, a low dose of resveratrol (4.9 mg/kg per day), or a calorie restricted (CR) diet and examined genome-wide transcriptional profiles. We report a striking transcriptional overlap of CR and resveratrol in heart, skeletal muscle and brain. Both dietary interventions inhibit gene expression profiles associated with cardiac and skeletal muscle aging, and prevent age-related cardiac dysfunction. Dietary resveratrol also mimics the effects of CR in insulin mediated glucose uptake in muscle. Gene expression profiling suggests that both CR and resveratrol may retard some aspects of aging through alterations in chromatin structure and transcription. Resveratrol, at doses that can be readily achieved in humans, fulfills the definition of a dietary compound that mimics some aspects of CR.

In addition to altered gene expression, the resveratrol-treated mice also exhibit delays in aging parameters (cardiovascular, endocrinological, metabolic) comparable to those caused by CR. The physiological and gene expression changes are observed in multiple tissues; taken together, they strongly support the hypothesis that resveratrol acts as a CR mimetic. Based on patterns of SIRT1 activity, however, the authors conclude that a subset of these changes (specifically, the delay in age-related cardiac decline) are not due to activation of SIRT1 by resveratrol.

The next question: Given that resveratrol and CR stimulate similar transcriptional changes, and that resveratrol yields some of the same physiological benefits as long-term CR, does low-dose resveratrol also have a favorable effect on median or maximum longevity? Based on these findings, I know how I’d bet, but for the ultimate answer, we’ll have to wait for the next paper.

* See Jamie Barger’s comment below.

 

Sirtuins come under fire

Over the past few years, sirtuins have generated great excitement — both in the basic study of biogerontology and (more recently) in the private sector. In just over a decade, the field has moved from its founding observations in yeast to wide-ranging results in mammals. Among the adherents of a widely held theory, it is believed that sirtuins act to extend lifespan via similar mechanisms to calorie restriction (CR), and that small-molecule activators of sirtuins (such as resveratrol) are CR mimetics — therefore, the sirtuins are the first molecular target to guide drug design in a bona fide anti-aging pharmacopoeia.

As theories reach maturity (and middle age), they are naturally subject to challenge, and the sirtuin story is no exception. The role of sirtuins in CR has been challenged, sometimes by the very founders of the field. The mechanism(s) of action of resveratrol are also under close scrutiny. Even some of the most famous studies of sirtuins — specifically, regarding effects on median lifespan and exercise tolerance — used animals eating such horrifyingly fatty diets or ingesting such gigantic doses of resveratrol that their relevance to humans must be questioned.

It’s therefore high time that we turned a skeptical eye to the sirtuin story. Ken Garber, reporting for Nature Biotechnology, has assembled a very accessible short review of the subject that lines up the arguments regarding sirtuins’ role in aging, the relationship between sirtuin activity and CR, and the value of “known” sirtuin activators as preclinical leads (link):

… But there is another sirtuin narrative that has received much less attention. To begin with, there is no published evidence that resveratrol or sirtuin activators can extend lifespan in normal mammals. Calorie restriction does extend lifespan in many organisms (though not all), but its effects in mammals may have little to do with the sirtuins: other pathways may be more important. And resveratrol may not be a general sirtuin activator in the first place—the compound’s beneficial effects may arise from completely different mechanisms. Finally, credible research in yeast suggests that sirtuins may actually function to limit chronological lifespan, not increase it.

The piece summarizes, thoroughly and fairly, the arguments for and against the competing narratives regarding sirtuins’ importance; in the process, it gives a nice historical overview of the evolution of the sirtuin field since its foundations in yeast.

By pointing out the importance of narrative, Garber reminds us that sometimes we tend to preferentially remember facts that improve the consistency of a story, and conversely, to preferentially forget completely valid observations that add rough edges and sharp corners to a favored view. This field is rife with examples. Here, we are reminded of some of the prima facie weaknesses of some of founding studies, including ones that led to such fundamental beliefs as the idea that resveratrol activates sirtuins. We’re also pointed toward the work of dissenting scholars who find that sirtuin mutations and resveratrol have minimal, if any, effect on lifespan — raising the possibility that any such effects observed in other studies are sensitively dependent on the choice of culture conditions and the genetic backgrounds of the animals used.

On the balance, the piece doesn’t argue that sirtuins aren’t involved in aging or that they’re not worth further study — but after reading it, I found myself realizing that some of the parts of the big machine don’t fit together as smoothly as I thought they had. Especially when a theory is widely accepted — and widely used as an inspiration for future studies — it’s crucial to be regularly reminded of what we know for sure, why we think we know it, and (most importantly) of the magnitude of what we don’t yet know.

 

A glutamine a day keeps senescence away

Cellular senescence is regarded as a tumor suppressor mechanism: damaged cells permanently leave the cell cycle (preventing tumor initiation), and also secrete factors that trigger both tissue repair and inflammation in the vicinity. This is probably good at first but bad later on: persistent senescent cells also secrete growth factors and metalloproteases that degrade the tissue microenvironment and encourage nearby preneoplastic cells to progress into full-blown tumors. Thus, senescence has been implicated in late-life cancer and age-related decline in tissue function.

The “damage” in question is usually genotoxic in nature: telomere shortening, indicating that a cell has undergone many rounds of potentially mutagenic cell division, or high levels of DNA damage such as that resulting from ionizing radiation or exposure to chemical clastogens. Oncogene expression probably also induces senescence via DNA damage, by triggering over-firing of replication origins and generating broken ends and weird chromatin structures that are interpreted as damage.

Now it appears that falling cellular ATP levels may also result in cellular senescence. Unterluggauer et al. report that inhibition of glutaminolysis (preventing cells from generating ATP from glutamine, an unglamorous and occasionally overlooked pathway that is nonetheless an important energy source in many cellular lineages) results in increased senescence in human vascular endothelial cells (HUVECs):

Premature senescence of human endothelial cells induced by inhibition of glutaminase

Cellular senescence is now recognized as an important mechanism of tumor suppression, and the accumulation of senescent cells may contribute to the aging of various human tissues. Alterations of the cellular energy metabolism are considered key events in tumorigenesis and are also known to play an important role for aging processes in lower eukaryotic model systems. In this study, we addressed senescence-associated changes in the energy metabolism of human endothelial cells, using the HUVEC model of in vitro senescence. We observed a drastic reduction in cellular ATP levels in senescent endothelial cells. Although consumption of glucose and production of lactate significantly increased in senescent cells, no correlation was found between both metabolite conversion rates, neither in young endothelial cells nor in the senescent cells, which indicates that glycolysis is not the main energy source in HUVEC. On the other hand, glutamine consumption was increased in senescent HUVEC and inhibition of glutaminolysis by DON, a specific inhibitor of glutaminase, led to a significant reduction in the proliferative capacity of both early passage and late passage cells. Moreover, inhibition of glutaminase activity induced a senescent-like phenotype in young HUVEC within two passages. Together, the data indicate that glutaminolysis is an important energy source in endothelial cells and that alterations in this pathway play a role in endothelial cell senescence.

The authors provide good evidence that endothelial cells rely heavily on glutaminolysis, and that removal of this energy source both drastically reduces cellular ATP levels and results in a “senescent-like” growth arrest. They then show fairly convincingly that this arrest is very similar to the arrest induced by telomere shortening, DNA damage or oncogene expression (i.e., cellular senescence) — in particular, by demonstrating that the arrested HUVECs express a panoply of senescence-associated gene expression and cytological markers. No word, as far as I could tell, on the reversibility of the arrest upon resumption of glutaminolysis (irreversibility is a hallmark of senescence); I mention this because growth arrest is a fairly obviously sensible response to an energy deficit, but it’s not clear why it ought to be permanent.

The reason I’m interested in this paper is that it might point toward a unifying principle underlying two major subjects within the field of biogerontology — cellular senescence and sirtuins — which both receive a great deal of individual attention but so far have not been demonstrated to have much to do with one another. Sirtuins such as SIRT1 are regulated by cellular energy state (in particular, by the NAD+/NADH ratio); if it turns out that perturbations in the cellular energy budget are an important means of senescence induction, it might be interesting to take a closer look and see whether sirtuin signaling might influence the establishment of cellular senescence.

 

Is there anything SIRT1 can’t do?

In honor of the pending acquisition of Sirtris by GlaxoSmithKline — and the advent of truly big pharma getting into the biology of aging — I wanted to pay tribute to SIRT1, the principal target of the sirtuin activators under development.

SIRT1 plays a variety of roles in regulatory biology and lifespan determination, and the list is growing: it inhibits p53, blocks inflammatory signaling, extends the healthspan of mice, and improves exercise tolerance It slices, it dices, and that’s not all: SIRT1 also

• regulates energy metabolism and mediates lifespan extension in response to calorie restriction (CR);
• directs neurogenesis;
• suppresses tumorigenesis in the gut (and, in tumors that do form, may negatively regulate the oncogenic form of β-catenin);
• antagonizes cellular senescence (though, just as with inhibiting p53, this might not be a crazy good idea in in all tissues);
• gives Lenny Guarente something to write reviews about; and
• has some really interesting relatives, including SIRT6, which was recently shown to maintain telomeric chromatin in a healthy and functional state.

Watch for more functional news, as well as novel connections between SIRT1 functions and human disease, as industry starts generating more (and more specific) activators of this multi-talented protein and its relatives.

 

Sirtris to be acquired by GlaxoSmithKline

Following closely on news that products in their pipeline can decrease blood glucose and may have tumor suppressor potential, Sirtris Pharmaceuticals (SIRT) has been snapped up by GlaxoSmithKline (GSK).

Sirtris has focused on the commercial development of clinically useful sirtuin activators, which are predicted to be useful as anti-diabetic drugs. Data from academic labs have suggested they could be of even wider use, e.g., in increasing exercise tolerance or treating inflammatory disease. Underneath it all, of course, is the knowledge that the the sirtuins were initially identified as longevity assurance genes; the subtext of all discussions of sirtuin activators is that they may mediate their beneficial effects by slowing aspects of the aging process itself.

The acquisition of an small company at a large premium (the offer was more than 80% higher than Sirtris’ market cap) by a pharmaceutical giant is one of the first demonstrations that the drug industry is taking seriously the idea that there’s money to be made in treating aging per se rather than all of the associated conditions separately (link):

“Through the acquisition of Sirtris, GlaxoSmithKline will significantly enhance its metabolic, neurology, immunology and inflammation research efforts by establishing a presence in the field of sirtuins, a recently discovered class of enzymes that are believed to be involved in the aging process,” the companies said in a joint release.

Then again, even in the best case, those who take sirtuin activators will get age-related diseases eventually anyway, so the question of whether to treat aging or age-related disease isn’t really an either/or choice.

I am currently wondering whether recent findings that indiscriminate activation of SIRT1 might lead to cancer (e.g., when DBC1 is deleted) will temper the enthusiasm for these compounds.

 

DBC1 stimulates p53 by inhibiting SIRT1

SIRT1, the most widely studied of the protein family known as sirtuins, is a histone deacetylase that has been implicated in regulation of aging in mammals. Activators of SIRT1, such as resveratrol, have been demonstrated to extend the lifespan as well as boost mitochondrial function in mice.

More recently, SIRT1 has been demonstrated to regulate p53 function: deacetylation by SIRT1 makes p53 less active, thereby decreasing apoptosis in response to specific types of DNA-damaging stress (e.g., ionizing radiation). This would be advantageous in some circumstances and deleterious in others, depending on the relative value placed on cellular survival vs. elimination of potentially neoplastic cells. Thus, this observation raises questions regarding how SIRT1 is itself regulated.

In back-to-back papers in Nature earlier this year, two labs report the discovery that the DBC1 (”deleted in breast cancer”) protein specifically inhibits SIRT1, in turn increasing p53 activity and thereby stimulating p53-mediated apoptosis in response to genotoxic stress. Of the two papers, Zhao et al. have the more elaborate abstract, reproduced below; Kim et al. reach similar conclusions:

Negative regulation of the deacetylase SIRT1 by DBC1

SIRT1 is an NAD-dependent deacetylase critically involved in stress responses, cellular metabolism and, possibly, ageing. The tumour suppressor p53 represents the first non-histone substrate functionally regulated by acetylation and deacetylation; we and others previously found that SIRT1 promotes cell survival by deacetylating p53. These results were further supported by the fact that p53 hyperacetylation and increased radiation-induced apoptosis were observed in Sirt1-deficient mice. Nevertheless, SIRT1-mediated deacetylase function is also implicated in p53-independent pathways under different cellular contexts, and its effects on transcriptional factors such as members of the FOXO family and PGC-1 directly modulate metabolic responses. These studies validate the importance of the deacetylase activity of SIRT1, but how SIRT1 activity is regulated in vivo is not well understood. Here we show that DBC1 (deleted in breast cancer 1) acts as a native inhibitor of SIRT1 in human cells. DBC1-mediated repression of SIRT1 leads to increasing levels of p53 acetylation and upregulation of p53-mediated function. In contrast, depletion of endogenous DBC1 by RNA interference (RNAi) stimulates SIRT1-mediated deacetylation of p53 and inhibits p53-dependent apoptosis. Notably, these effects can be reversed in cells by concomitant knockdown of endogenous SIRT1. Our study demonstrates that DBC1 promotes p53-mediated apoptosis through specific inhibition of SIRT1.

The fact that the DBC1 protein was originally identified as one that is frequently deleted in breast tumors suggests that there are indeed tissues in which unchecked SIRT1 deacetylation of p53 would be a bad thing (i.e., in which it makes sense to kill off damaged cells, even at a cost to regenerative capacity — another example of the evolutionary tradeoffs between regenerative capacity and tumor suppression).

One obvious question is whether DBC1 is also commonly deleted in other epithelial tumors; if so, is there a pattern in the tissue types that develop such tumors? e.g., perhaps DBC1 is particularly important in epithelial populations that, like the breast, lie dormant for much of the lifespan but possess the latent ability to proliferate rapidly in response to hormones — in cells like these, it makes sense to have a relatively “hair-trigger” apoptotic response to potentially carcinogenic insults. In less proliferative tissues, however, the system might be tuned quite differently, with DBC1 levels set relatively low in order to preserve self-renewal capacity even after a manageable level of genotoxic damage.

Next step, of course: What regulates DBC1?

 

Guarente on mitochondria, sirtuins and CR

Lenny Guarente, grandpappy of the sirtuin field, has a nice review of the connection between mitochondria, calorie restriction, and sirtuin protein function in a recent issue of Cell.

 

A bridge between sirtuins and Werner’s

Because every longevity-control gene eventually is eventually shown to interact with every other longevity-control gene, it is perhaps not surprising that SIRT1 deacetylates WRN, the protein whose gene is mutated in the devastating human progeria Werner’s Syndrome. Both the helicase and exonuclease activities of the WRN protein are more active in the deacetylated state; thus, the longevity-assurance gene (SIRT1) is responsible for boosting the activity of the major player in the cellular response to DNA damage (WRN), which is the way we’d expect it to work.

Two other recent reports describing progress on WRN reveal that the protein plays a significant role in DNA metabolism under normal growth and after DNA damage: WRN is required both for replication fork progression after genotoxic stress as well as suppressing the spontaneous formation of telomeric DNA circles. These latter structures (which remind me of the extrachromosomal ribosomal DNA circles from the early years of the sirtuin field) are associated with both telomere shortening and cellular senescence.

 

SIRT1 inhibits NF-κB and blocks inflammation

Resveratrol decreases tobacco-induced inflammation in the lungs of smokers, suggesting that SIRT1 is involved in regulating the inflammatory response to cigarette smoke. Further evidence for a role for sirtuins in this process comes from Rajendrasozhan et al., in a study that confirms and extends the results of previous work.

The authors show once again that SIRT1 levels are low in the lungs of smokers, and that these levels diminish further in response to cigarette smoke. Smoke-exposed lung cells and macrophages exhibited increased inflammatory signaling (assayed primarily by looking at IL-8), as do cells in which SIRT1 has been artificially knocked out. Consistent with this, the RelA subunit of the master inflammation regulator NF-κB is hyperacetylated — recall that SIRT1 is a deacetylase — and consequently more active. Conversely, overexpression of SIRT1 decreases inflammatory cytokine production, echoing earlier results that described SIRT1 activation by resveratrol treatment.

Thus, a key longevity-assurance gene is also involved in restricting inflammation, which is a risk factor for some of smoking’s worst complications — chronic obstructive pulmonary disease (COPD) — as well as tumorigenesis. Tobacco is a major public health issue, so it’s not surprising that SIRT1 is getting attention in this context, but there is reason to believe that the phenomenon is general to organs other than the lung — e.g., see this review from Salminen et al., which describes how signaling from SIRT1 and FoxO transcription factors (mammalian homologs of the worm longevity gene DAF-16) can inhibit NF-κB signaling in a variety of systems. The authors close the circle by discussing the connection between longevity assurance and the mitigation of one specific age-related phenotype, inflammaging.

COPD is a leading cause of death worldwide, and it arises not only in smokers but in members of their households, as well as people exposed to environmental pollutants. A host of other inflammatory diseases, such as arthritis, beset the elderly and decrease quality of life. Increasing evidence that SIRT1 activity could mitigate the health harms of runaway inflammation point to even more potential uses for the new class of sirtuin-activating drugs that are currently under consideration as therapies against diabetes and metabolic syndrome.