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.

 

Of mice and men: Deleterious psychological effects of CR may be limited to rodents

Calorie restriction (CR) extends lifespan and boosts physical health in most organisms studied, but is it any fun? Here I’m referring not to any diminishment of gustatory pleasures (leading to the oft-repeated jest, “You might not live longer, but it would sure seem longer”), but rather to the prospect that CR might have anhedonic psychological effects.

Earlier we’ve discussed evidence that this is indeed the case: Very serious food deprivation (more like starvation or anorexia than well-managed CR) in rats can lead to depression, possibly by exerting an effect on the expression of synaptic vesicle proteins and other molecules involved in neuronal signal transmission.

CR of rodent parents can even have negative effects on offspring: specifically, gestational protein restriction in mother rats leads increased DNA damage and other progeroid symptoms in their young. To these deleterious effects we must now add anxiety: rats whose mothers were gestationally restricted show less interest in exploration and more desire to hide in familar places. This is consistent with the idea that some of CR’s behavioral effects reflect adaptive responses to a hostile world in which the risk-to-reward ratio has grown suddenly higher: if you’re a forager and there’s nothing to forage, then you might as well stay in your burrow.

But to what extent do these rodent experiments reflect the situation in humans? Up until very recently, we’ve had only anecdotes — which, even ignoring inevitable biases in reporting, are inconclusive to say the least. (In my own anecdotal experience, the self-reporting of CR subjects ranges from thoughtful and positive sharing to behavior that is, well, neither of those.) Fortunately, however, a major test of the effects of CR in humans — the CALERIE study — is currently underway, and already yielding interesting data. Williamson et al. report that CR in humans is not associated with either harmful mood changes or any symptom of eating disorders. Indeed, the authors find, CR in humans is associated primarily with neutral or positive psychological and behavioral effects:

Is caloric restriction associated with development of eating-disorder symptoms? Results from the CALERIE trial.

Objective: This study tested a secondary hypothesis of the CALERIE trial (Heilbronn et al., 2006) that a 12-month period of intentional dietary restriction would be associated with an increase in eating disorder symptoms. Design: To test this hypothesis, 48 overweight adults were randomly assigned to four treatment arms in a 12-month study: (1) 25% calorie restriction, (2) 12.5% calorie restriction and 12.5% increased energy expenditure by structured exercise, (3) low-calorie diet, and (4) healthy diet (no-calorie restriction). Main Outcome Measures: Primary outcome measures for the study were changes in: eating disorder symptoms, mood, dietary restraint, body weight, and energy balance. Results: All three dietary restriction arms were associated with increased dietary restraint and negative energy balance, but not with increased ED symptoms or other harmful psychological effects. Participants in the three calorie restriction arms lost significant amounts of body weight. The psychological and behavioral effects were maintained during a 6-month follow-up period. Conclusion: These results did not support the hypothesis that caloric restriction causes increased eating disorder symptoms in overweight adults. In general, caloric restriction had either benign or beneficial psychological and behavioral effects.

Why the difference? Having neither the opportunity nor inclination to construct a panel of rodent-human hybrids, I’m stuck with speculation: It’s entirely possible that rodents and humans are different enough from one another that they have different psychological responses to similar sorts of shortage issues. Perhaps, not having a burrow in which to hide when the going got tough, our own ancestors had to think and act more energetically and enthusiastically in order to survive the lean times. Perhaps consuming 60% of the calories found in our modern diets is closer to the “natural” (evolutionarily optimized) state of affairs for humans — i.e., ad libitum humans might be basically overfed zoo animals, living hazy full-tummied lives far away from the types of stimuli that really light us up, mentally speaking.

Another possibility is less optimistic: the CALERIE subjects may have been screened so stringently that they are no longer a legitimate sample of the general population. Potential subjects were screened both physically and psychologically; fewer than 10% of the initial applicants were ultimately accepted. While only a few percent (8/599) were rejected explicitly on psychological grounds, I’m guessing that several of the larger classes of automatically excluded candidates (”weight instability,” “medications”, “smoker”, “other” and “withdrew during screening” add up to more than 80% of the rejections) included some individuals who might have been eliminated in psychological testing if they’d made it that far. Based on the proportion of “surviving” candidates, one might argue that CALERIE subjects were chosen in part because they were physically and psychically unusual, at least among overweight individuals. On those grounds, one could reasonably wonder whether any of the study’s findings about CR will prove true for those of us in the remaining 90% of the general population.

 

Your pot belly is trying to kill you

I have two words for you: rat lipsuction.

One of the common features of aging throughout the Class Mammalia is the accumulation of body fat in specific deposits — specifically, the growth of visceral (or abdominal fat). We do it, monkeys do it, dogs do it, and rodents do it. Visceral fat (VF) has been implicated in a variety of age-related disorders, including metabolic syndrome and chronic inflammation, both of which have in turn been linked to frailty.

If VF produces a factor or factors that limits lifespan (either by promoting the aforementioned conditions or by another mechanism), then removing it should make those factors go away and concomitantly lengthen lifespan. (I suppose the alternative would be a parabiosis experiment in which a rat with lots of VF shared a blood supply with a rat with little VF, though (a) the results would be difficult to interpret due to myriad confounding factors; (b) I’m not sure what one would do if the fat rat died while the thin rat was still alive; and (c) the entire exercise would be a horrifying abomination.) Muzumdar et al. have collected just that sort of data: They removed the VF from rats consuming an ad libitum chow diet, and showed that the rats aged almost as successfully as calorie-restricted (CR) rats (which, by the way, never accumulate VF):

Visceral adipose tissue modulates mammalian longevity

Caloric restriction (CR) can delay many age-related diseases and extend lifespan, while an increase in adiposity is associated with enhanced disease risk and accelerated aging. Among the various fat depots, the accrual of visceral fat (VF) is a common feature of aging, and has been shown to be the most detrimental on metabolic syndrome of aging in humans. We have previously demonstrated that surgical removal of VF in rats improves insulin action; thus, we set out to determine if VF removal affects longevity. We prospectively studied lifespan in three groups of rats: ad libitum-fed (AL-fed), CR (Fed 60% of AL) and a group of AL-fed rats with selective removal of VF at 5 months of age (VF-removed rats). We demonstrate that compared to AL-fed rats, VF-removed rats had a significant increase in mean (p < 0.001) and maximum lifespan (p < 0.04) and significant reduction in the incidence of severe renal disease (p < 0.01). CR rats demonstrated the greatest mean and maximum lifespan (p < 0.001) and the lowest rate of death as compared to AL-fed rats (0.13). Taken together, these observations provide the most direct evidence to date that a reduction in fat mass, specifically VF, may be one of the possible underlying mechanisms of the anti-aging effect of CR.

The authors argue (a bit too strongly, in my opinion) that their experiment demonstrates that prevention of VF accumulation is a major mechanism of the lifespan extension due to CR. A skeptic could easily argue that it works the other way: VF represents a really large storehouse of energy, and its removal could force the rat to deplete other fat reserves and enter a state that mimics CR. The parabiosis experiment that I parenthetically described above (or some less repellent and [not entirely incidentally] more scientifically valuable version thereof, e.g., one in which candidate factors secreted by VF were introduced back into lipectomized rats) would go a long way toward bolstering the authors’ interpretation.

Regardless, it’s a good reminder not to skip yoga and jogging this weekend.

 

A candle that burns twice as bright and twice as long: the PEPCK-Cmus transgenic mouse

Increased expression of a metabolic enzyme, phosphoenolpyruvate carboxykinase (PEPCK, an enzyme that most of us learned about in freshman biology and then promptly forgot, reasoning that the descriptive name and the ability to look it up if necessary would suffice if it ever came up again) results in mice that are muscular, have lower body fat than a runway model, and able to run 25 times farther than a wildtype control.

Even more interesting, according to proud parents Hanson and Hakimi, the females of the PEPCK-C^mus strain mate and have normal-sized litters at 35 months, an age when the blood of wildtype mice has cooled substantially (and, indeed, the mice themselves are starting to check out). The implication is that aging is slowed, and longevity extended, as a result of the transgene.

It’s become reflexive to ask whether a long-lived mutant is living longer because it’s calorie-restricted for some reason, incidental to the main phenotype conferred by the mutation, but this is not the case here: In order to preserve their enviable bods, PEPCK-C^mus mice eat 60% more than controls — so they’re not extending their lifespan by dieting. If anything, they’re anti-dieting: their increased metabolic efficiency means they’re harvesting more calories per gram of carb or fat than normal animals. No word yet on what happens if you do try to calorie-restrict them; I can imagine it going either way but am holding out hope for tiny explosions.

The PEPCK-C^mus seem to have it all: great bodies, long lives, extended reproductive and sexual lifespans, and no need to limit their appetites. The down side? Apparently, they are complete assholes: the mutants are aggressive and hyperactive, traits heretofore unheard-of among muscular, fit humans (and, indeed, in the field of biogerontology).

Rigorous lifespan and aging studies in these animals are ongoing, but are not yet complete, so the authors are reserving final judgment on the question of whether PEPCK-C^mus transgenics are bona fide longevity mutants. Hopefully we’ll have an answer within a couple of years. In the meantime, I hope they’re busily cross-breeding the transgene into short-lived DNA repair mutants — recently shown to induce longevity-assurance pathways in a last-ditch effort to stave off progeria — both to accelerate the progress of research and to see whether the metabolic benefits of PEPCK-C^mus might be used to treat premature aging syndromes.

(Hat tip to Longevity Meme.)

 

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.

 

Give it to us raw and wriggling

Star Trek fans know that Klingons prefer to eat their worms while they are still alive. It therefore seems fitting that biogerontologist’s favorite worm, C. elegans, in turn prefer their own food alive. Lenaerts et al. report that C. elegans has a nutritional requirement for some component of metabolically live microbes. This compound (or set of compounds) remains to be identified.

These findings may make it necessary to re-interpret a number of calorie restriction (CR) studies performed in axenic (bacteria-free) media — which deprives the worm of this required factor. Thus worms in axenic media may be undergoing not only CR but nutritional deficiency. If the required compound is diffusible, it might also go some way toward explaining why olfactory cues can influence health and lifespan. (The authors demonstrate that the component in question is probably not even soluble, but then again, many odorants aren’t.)

(More analysis and discussion of this article can be found at PharmacoNutrition Reviewed).

 

In mouse, not every antioxidant gene extends lifespan

We recently discussed evidence that the Drosophila gene Nrf2 is involved in antioxidant defense, cancer prevention and longevity assurance — at least in the fly. A new study by Pearson et al., however, suggests the system may be more complicated in mammals. In mice, it appears that the Nrf2 homolog is required for cancer prevention by calorie restriction (CR), but dispensable for the lifespan phenotype of CR.

Open questions: Outside of a CR paradigm, is mNrf2 involved in regulation of lifespan in ad libitum-fed animals? If not, has some other gene picked up Nrf2’s longevity-related functions, or is the role in lifespan determination an idiosyncrasy of the fly?

 

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.