Assessing Markers of Cellular Senescence in the CALERIE Study of Calorie Restriction

The practice of calorie restriction, eating up to 40% fewer calories while structuring the diet to continue to obtain sufficient micronutrients, is well demonstrated to slow aging and extend life in short-lived species. It is thought that the primary mechanism for this effect is upregulation of the cellular maintenance process of autophagy, given that sabotaging autophagy prevents extension of life span resulting from calorie restriction. Calorie restriction produces such sweeping changes in cell and tissue function that there remains plenty of room to argue for other mechanisms to be important, however. For example, we might consider the loss of visceral fat mass as likely a sizable contribution, given that visceral fat promotes chronic inflammation, and surgically removing visceral fat from mice produces significant benefits.

Calorie restriction is thought unlikely to produce sizable gains in human life span, on the grounds that this would have become well known in ancient times if it was the case. Calorie restriction has been formally assessed in humans in recent years. In the short-term, health benefits look quite similar to those produced in mice. Over the longer term, data is lacking. The longest and largest formal study today was the second phase of the CALERIE trial, in which 128 participants underwent two years of an average 12% calorie restriction compared to the 71 control participants. This study produced a great deal of data that continues to be mined for insights into human aging and effects of calorie restriction in a long-lived species such as our own, to contrast with the sizable effects on health and longevity in short-lived species such as mice.

In particular, and the topic for today, cellular senescence and its role in degenerative aging has garnered far greater interest in the research community in the years since the CALERIE study took place. Thus in today's open access paper, scientists examine CALERIE study data to find evidence for calorie restriction to reduce the burden of cellular senescence that is characteristic of aging. It is known that calorie restriction reduces the burden of senescent cells in mice. The CALERIE data is not as convincing, however. This is probably because the participants were largely not old enough to have a sizable number of senescent cells present in their tissues. It is also the case that other researchers have found it hard to correlate levels of circulating proteins known to be generated by senescent cells with senescent cell burden, for reasons yet to be fully explored.

Calorie restriction reduces biomarkers of cellular senescence in humans

Compelling evidence from a wide range of animal studies suggests that calorie restriction (CR) with adequate nutrient intake is a promising strategy to extend lifespan and delay the onset of several age-related chronic diseases. In humans, the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) has been the most rigorous study to investigate the effects of CR. Phase 2 of CALERIE was a 2-year, multicenter, randomized controlled trial in healthy non-obese young-to-middle-aged individuals to examine the safety and effects of moderate CR compared to an ad libitum (AL) diet on predictors of longevity, disease risk factors, and quality of life. Although the average CR attained over the 2 years was ~12% rather than the prescribed 25%, the intervention was deemed safe and effective in promoting cardiometabolic risk reduction. The long-term implications of the intervention on healthspan and longevity remain to be established, but the biospecimens collected during the CALERIE study represent a unique resource to explore the influence of CR on the biology of aging in humans.

In this study we found that 2 years of moderate CR with adequate nutrient intake compared to AL significantly decreased the circulating levels of several senescence-associated biomarkers in healthy, young to middle-aged individuals without obesity. A greater number of biomarkers were modulated at 12 months than at 24 months, but PAI1, PARC, TARC, and TNFR1 were lower in CR participants at both timepoints. Using a machine learning approach, we observed that the changes in several biomarkers were important predictors of the change in CALERIE metabolic outcomes, including HOMA-IR, insulin sensitivity index, and resting metabolic rate residual. Our results advance the mechanistic understanding of CR and suggest a potential link between cellular senescence and metabolic health in humans.

Previous findings from the CALERIE study evidenced a reduction in systemic markers of inflammation, such as C-reactive protein, in participants randomized to the CR intervention. Low grade "sterile" systemic inflammation is considered a risk factor for several age-related chronic diseases and, although its pathogenesis is multifactorial, senescent cells through their senescence-associated secretory phenotype (SASP) are a plausible source of proinflammatory molecules. Unfortunately, none of the SASP components studied to date is unique to senescent cells and, certainly, the secretome of other cell types may be affected by CR. At this time, however, we cannot disentangle whether the reduced levels in circulating senescence-related biomarkers observed in response to CR reflect reduced senescent cell accumulation, increased clearance, or inhibition of their SASP. It is also not clear what organs are the main targets of the intervention. Results from our in silico analysis would suggest that CR may target senescent cells in adipose tissue, but the small sample size limits the generalizability of these results.

To corroborate our data demonstrating a reduction in circulating senescence biomarkers in response to CR, we examined tissue-level changes in a recently defined gene set of 125 secreted factors, transmembrane proteins, and intracellular proteins centered on cellular senescence and the SASP, named SenMayo. Through gene set enrichment analysis we observed a significant reduction in SenMayo in response to CR, which is reflected by an enrichment at baseline compared to 12 months. We note that we did not detect a significant change by RNA sequencing in expression of CDKN1A (P21) or CDKN2A (P16), two prototypical markers of cellular senescence. This may be related to their variable and low levels of expression, particularly in younger and healthier adults.