Comprehensive Assessment Of Long-term Effects Research Articles

Effect of long-term caloric restriction on telomere length in healthy adults: CALERIE™ 2 trial analysis.

Caloric restriction (CR) modifies lifespan and aging biology in animal models. The Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE™) 2 trial tested translation of these findings to humans. CALERIE™ randomized healthy, nonobese men and premenopausal women (age 21-50y; BMI 22.0-27.9 kg/m2 ), to 25% CR or ad-libitum (AL) control (2:1) for 2 years. Prior analyses of CALERIE™ participants' blood chemistries, immunology, and epigenetic data suggest the 2-year CR intervention slowed biological aging. Here, we extend these analyses to test effects of CR on telomere length (TL) attrition. TL was quantified in blood samples collected at baseline, 12-, and 24-months by quantitative PCR (absolute TL; aTL) and a published DNA-methylation algorithm (DNAmTL). Intent-to-treat analysis found no significant differences in TL attrition across the first year, although there were trends toward increased attrition in the CR group for both aTL and DNAmTL measurements. When accounting for adherence heterogeneity with an Effect-of-Treatment-on-the-Treated analysis, greater CR dose was associated with increased DNAmTL attrition during the baseline to 12-month weight-loss period. By contrast, both CR group status and increased CR were associated with reduced aTL attrition over the month 12 to month 24 weight maintenance period. No differences were observed when considering TL change across the study duration from baseline to 24-months, leaving it unclear whether CR-related effects reflect long-term detriments to telomere fidelity, a hormesis-like adaptation to decreased energy availability, or measurement error and insufficient statistical power. Unraveling these trends will be a focus of future CALERIE™ analyses and trials.

EFFECT OF LONG-TERM CALORIC RESTRICTION ON THE PACE OF BIOLOGICAL AGING IN HEALTHY ADULTS FROM THE CALERIE TRIAL

Abstract The geroscience hypothesis proposes that therapy to slow or reverse molecular changes that occur with aging can delay or prevent multiple chronic diseases and extend healthy lifespan. Caloric restriction (CR), defined as lessening caloric intake without depriving essential nutrients, can change molecular processes associated with aging, including DNA methylation (DNAm), and increases healthy lifespan in multiple species. Here we report results of a post-hoc analysis of the influence of CR on DNAm measures of aging in blood samples from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trial, a randomized controlled trial in which n=220 non-obese adults were randomized to 25% CR or ad libitum control diet for 2years. We found that CALERIE intervention slowed the pace of aging, as measured by the DunedinPACE DNAm clock, but did not lead to significant changes in PhenoAge- or GrimAge-clock-measured biological age. Treatment effects were small; changes in DunedinPACE suggest a ~3% slowing of the pace of aging. Nevertheless, this small effect may be substantive; in an independent study of older adults, 3% slower DunedinPACE is associated with a 15% lower risk of death. The finding that CR modified DunedinPACE in a randomized controlled trial supports the geroscience hypothesis, building on evidence from small and uncontrolled studies and contrasting with reports that biological aging may not be modifiable. Ultimately, a conclusive test of the geroscience hypothesis will require trials with long-term follow-up to establish effects of intervention on primary healthy-aging endpoints, including incidence of chronic disease and mortality.

Calorie restriction reduces biomarkers of cellular senescence in humans.

Calorie restriction (CR) with adequate nutrient intake is a potential geroprotective intervention. To advance this concept in humans, we tested the hypothesis that moderate CR in healthy young-to-middle-aged individuals would reduce circulating biomarkers of cellular senescence, a fundamental mechanism of aging and aging-related conditions. Using plasma specimens from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE™) phase 2 study, we found that CR significantly reduced the concentrations of several senescence biomarkers at 12 and 24 months compared to an adlibitum diet. Using machine learning, changes in biomarker concentrations emerged as important predictors of the change in HOMA-IR and insulin sensitivity index at 12 and 24 months, and the change in resting metabolic rate residual at 12 months. Finally, using adipose tissue RNA-sequencing data from a subset of participants, we observed a significant reduction in a senescence-focused gene set in response to CR at both 12 and 24 months compared to baseline. Our results advance the understanding of the effects of CR in humans and further support a link between cellular senescence and metabolic health.

Reduction of SPARC protects mice against NLRP3 inflammasome activation and obesity.

The comprehensive assessment of long-term effects of reducing intake of energy (CALERIE-II; NCT00427193) clinical trial established that caloric restriction (CR) in humans lowers inflammation. The identity and mechanism of endogenous CR-mimetics that can be deployed to control obesity-associated inflammation and diseases are not well understood. Our studies have found that 2 years of 14% sustained CR in humans inhibits the expression of the matricellular protein, secreted protein acidic and rich in cysteine (SPARC), in adipose tissue. In mice, adipose tissue remodeling caused by weight loss through CR and low-protein diet feeding decreased, while high-fat diet-induced (HFD-induced) obesity increased SPARC expression in adipose tissue. Inducible SPARC downregulation in adult mice mimicked CR's effects on lowering adiposity by regulating energy expenditure. Deletion of SPARC in adipocytes was sufficient to protect mice against HFD-induced adiposity, chronic inflammation, and metabolic dysfunction. Mechanistically, SPARC activates the NLRP3 inflammasome at the priming step and downregulation of SPARC lowers macrophage inflammation in adipose tissue, while excess SPARC activated macrophages via JNK signaling. Collectively, reduction of adipocyte-derived SPARC confers CR-like metabolic and antiinflammatory benefits in obesity by serving as an immunometabolic checkpoint of inflammation.

Effect of long-term caloric restriction on DNA methylation measures of biological aging in healthy adults from the CALERIE trial

The geroscience hypothesis proposes that therapy to slow or reverse molecular changes that occur with aging can delay or prevent multiple chronic diseases and extend healthy lifespan1–3. Caloric restriction (CR), defined as lessening caloric intake without depriving essential nutrients4, results in changes in molecular processes that have been associated with aging, including DNA methylation (DNAm)5–7, and is established to increase healthy lifespan in multiple species8,9. Here we report the results of a post hoc analysis of the influence of CR on DNAm measures of aging in blood samples from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trial, a randomized controlled trial in which n = 220 adults without obesity were randomized to 25% CR or ad libitum control diet for 2 yr (ref. 10). We found that CALERIE intervention slowed the pace of aging, as measured by the DunedinPACE DNAm algorithm, but did not lead to significant changes in biological age estimates measured by various DNAm clocks including PhenoAge and GrimAge. Treatment effect sizes were small. Nevertheless, modest slowing of the pace of aging can have profound effects on population health11–13. The finding that CR modified DunedinPACE in a randomized controlled trial supports the geroscience hypothesis, building on evidence from small and uncontrolled studies14–16 and contrasting with reports that biological aging may not be modifiable17. Ultimately, a conclusive test of the geroscience hypothesis will require trials with long-term follow-up to establish effects of intervention on primary healthy-aging endpoints, including incidence of chronic disease and mortality18–20.

No Effect of Calorie Restriction or Dietary Patterns on Spatial Working Memory During a 2-Year Intervention: A Secondary Analysis of the CALERIE Trial

BackgroundThe effect of calorie restriction (CR) on cognitive function is not well understood, and the impact of the dietary patterns consumed during CR has not been investigated. ObjectivesWe analyzed the combined association of CR and dietary quality with spatial working memory (SWM) in healthy adults without obesity. MethodsThe Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trial was a 2-y, multisite clinical trial. This trial was registered at clinicaltrials.gov as NCT00427193. Participants were randomized to a 25% reduction in EI (n = 143) or an ad libitum Control (n = 76). The Dietary Inflammatory Index (DII) and the Healthy Eating Index (HEI) captured dietary quality, with a lower DII and higher HEI score indicating a healthier diet. The Cambridge Neuropsychological Test Automated Battery battery was used to assess SWM. Longitudinal associations between each dietary pattern index and SWM for CR and Control were assessed by multivariable negative binomial models that included baseline, 12-mo, and 24-mo visits. ResultsParticipants were aged 38.1 ± 7.2 y with a BMI of 25.1 ± 1.7 kg/m2. A total of 70% of the participants were female. Baseline mean DII and HEI scores were −0.15 (range: −3.77, 4.21) and 59.1 (24.1, 91.0) and did not differ between groups. Improvements in DII and HEI were significantly different between CR and Control over 2 y (both P-interaction = 0.001). In longitudinal analyses, there was no association of either index with SWM. Furthermore, though within-group improvements in SWM were observed at 12 mo, there was no statistically significant difference between CR (SWM errors: 9.0; 95% CI: 1.9, 41.6) and Control (11.7; 95% CI: 2.6, 53.5; P > 0.99), holding DII constant. Comparable results were observed at 24 mo and for the HEI. ConclusionsDietary quality during CR was not associated with measures of SWM over 2 y in healthy adults without obesity. These results suggest that, in healthy populations, dietary patterns and CR may have a limited impact on working memory. Further research is required to understand the concurrent effect of these nutritional strategies.

DOES CALORIC RESTRICTION SLOW THE PROCESS OF BIOLOGICAL AGING? EVIDENCE FROM THE CALERIE TRIAL

Abstract Calorie restriction (CR) slows aging and increases healthy lifespan in model organisms. We tested if CR slowed biological aging in humans using DNA methylation analysis of blood samples from N=197 participants in the Comprehensive Assessment of Long-term Effects ofReducing Intake of Energy (CALERIE™) randomized controlled trial. We quantified CR effects on biological aging by comparing change scores for six epigenetic-clock and Pace-of-Aging measures between n=128 CR-group and n=69 ad-libitum-control-group participants at 12- and24-month follow-ups. CR effects were strongest for DunedinPACE Pace of Aging (12-month Cohen’s d=0.3; 24-month Cohen’s d=0.2, p<0.01 for both), followed by DunedinPoAm and the GrimAge epigenetic clock, although effects for these measures were not statistically differentfrom zero (p>0.08). CR effects for other epigenetic clocks were in the opposite direction (all p>0.15). CALERIE intervention slowed Pace of Aging but showed minimal effect on epigenetic clocks hypothesized to reflect longer term accumulation of aging burden.

Epigenome-wide Association Study Analysis of Calorie Restriction in Humans, CALERIETM Trial Analysis.

Calorie restriction (CR) increases healthy life span and is accompanied by slowing or reversal of aging-associated DNA methylation (DNAm) changes in animal models. In the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIETM) human trial, we evaluated associations of CR and changes in whole-blood DNAm. CALERIETM randomized 220 healthy, nonobese adults in a 2:1 allocation to 2 years of CR or ad libitum (AL) diet. The average CR in the treatment group through 24 months of follow-up was 12%. Whole blood (baseline, 12, and 24 months) DNAm profiles were measured. Epigenome-wide association study (EWAS) analysis tested CR-induced changes from baseline to 12 and 24 months in the n = 197 participants with available DNAm data. CR treatment was not associated with epigenome-wide significant (false discovery rate [FDR] < 0.05) DNAm changes at the individual-CpG-site level. Secondary analysis of sets of CpG sites identified in published EWAS revealed that CR induced DNAm changes opposite to those associated with higher body mass index and cigarette smoking (p < .003 at 12- and 24-month follow-ups). In contrast, CR altered DNAm at chronological-age-associated CpG sites in the direction of older age (p < .003 at 12- and 24-month follow-ups). Although individual CpG site DNAm changes in response to CR were not identified, analyses of sets CpGs identified in prior EWAS revealed CR-induced changes to blood DNAm. Altered CpG sets were enriched for insulin production, glucose tolerance, inflammation, and DNA-binding and DNA-regulation pathways, several of which are known to be modified by CR. DNAm changes may contribute to CR effects on aging.

Associations between the timing of eating and weight-loss in calorically restricted healthy adults: Findings from the CALERIE study.

Calorie restriction (CR) and time-restricted eating (TRE) are distinctly different dietary management strategies with overlapping health outcomes. After two years of CR, healthy participants in the Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE) study showed significant weight-loss relative to the ad libitum intake control group and achieved 12% CR on average. Preclinical rodent studies have shown that sustaining a consistent eating interval of 8-12h between the first and last calories of each day-without reducing daily calorie intake-can impart health benefits that partly overlap with those imparted by CR. Preclinical CR protocols often inadvertently restrict eating interval, and conversely, clinical studies of TRE often inadvertently result in modest CR. Other factors related to daily timing of food intake, such as breakfast skipping, and early food intake also impact health outcomes. These observations have raised the possibility that CR protocols can be further optimized by adopting relevant aspects of eating patterns to boost weight loss and health outcomes. With a goal to inform CR protocols that aim to optimize eating patterns, the objective of this secondary analysis was to test aspects of daily timing of food intake associated with greater weight loss in the CALERIE study participants. We found no difference in the daily time window of energy intake between the CR and control arms. In the CALERIE trial, weight change was used as a proxy for adherence to CR, and hence we used linear models to test the relationships among CR, weight loss, and temporal aspects of daily eating pattern. We found that CR alone could explain 41% of the variance in weight loss. We tested the contribution of eating interval, time to 50% daily calorie intake, and day-to-day shifts in the time of the first (breakfast) or last meal consumed. We found that eating interval and variation in the timing of the first and last meals significantly influenced weight loss after controlling for CR. Our models suggest that shorter eating intervals are associated with greater CR (1% of the variance explained) and facilitate additional weight loss. Our models suggest that less day to day variation in first mealtime is directly associated with weight loss (6% of the variance explained). More regular first meal timing is also associated with greater CR (2% of the variance explained). Likewise, regular timing of the last daily meal is directly associated with weight loss (1% of the variance explained) and greater CR (1% of the variance explained). The time to 50% of daily calorie intake or consuming half the caloric intake earlier in the day is associated with additional CR (2% of the variance explained). In summary, these secondary analyses on CALERIE data suggest that - in order to maximize CR and weight loss - future CR protocols should encourage participants to adopt consistent timing of their first and last meals, a shorter eating window, and earlier consumption of food.

Nutritional quality of calorie restricted diets in the CALERIE™ 1 trial.

The aim was to determine the nutritional adequacy of calorie restricted (CR) diets during CR interventions up to 12months. The Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE™) phase 1 trial consisted of 3 single-site studies to test the feasibility and effectiveness of CR in adults without obesity. After baseline assessments, participants who were randomized to a CR intervention received education and training from registered dietitians on how to follow a healthful CR diet. Food diaries were completed at baseline and during the CR interventions (~6, 9, and 12months) when participants were self-selecting CR diets. Diaries were analyzed for energy, macronutrients, fiber, 11 vitamins, and 9 minerals. Nutritional adequacy was defined by sex- and age-specific Estimated Average Requirement (EAR) or Adequate Intake (AI) criteria for each nutrient. Diet quality was evaluated using the PANDiet diet quality index. Eighty-eight CR participants (67% women, age 40±9 y, BMI 27.7±1.5kg/m2) were included in the analysis. Dietary intake of fiber and most vitamins and minerals increased during CR. More than 90% of participants achieved 100% of EAR or AI during CR for 2 of 4 macronutrients (carbohydrate and protein), 6 of 11 vitamins (A, B1, B2, B3, B6, B12), and 6 of 9 minerals assessed (copper, iron, phosphorus, selenium, sodium, zinc). Nutrients for which <90% of participants achieved adequacy included fiber, omega-3 fatty acids, vitamins B5, B9, C, E, and K, and the minerals calcium, magnesium, and potassium. The PANDiet diet quality index improved from 72.9±6.0% at baseline to 75.7±5.2% during CR (p<0.0001). Long-term, calorie-restricted diets were nutritionally equal or superior to baseline ad libitum diets among adults without obesity. Our results support modest calorie restriction as a safe strategy to promote healthy aging without compromising nutritional adequacy or diet quality.

Modestly Increased Incidence of Ketosis in Caloric Restriction Does not Significantly Alter the Effects of Caloric Restriction.

Caloric restriction (CR) has been shown to slow the aging processes in a number of preclinical studies and reduces expression of aging-associated biomarkers in human trials. We hypothesized that CR would lead to increased incidence of ketosis and that ketosis in CR individuals would alter the aging-protective effects of CR or biomarkers thereof. We analyzed data from the "Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE, Phase 2)" Public Use Database available at calerie.duke.edu. In this study, non-obese adults between the ages of 21 and 50 were randomized to 25% CR or control (ad lib) diet groups and extensively monitored for two years. Given our focus on the effect of caloric restriction on ketosis, individuals with detectible ketones during the baseline visit (pre-randomization) and those with missing data for ketone testing were excluded from the analysis, leaving 71 control and 117 CR participants. We analyzed the incidence of ketosis as well as ketosis free survival in control and CR participants and assessed the effect of ketosis on a number of clinical lab values, functional assessments, and participant survey data related to aging biology. We report that CR was associated with modestly increased incidence of ketosis (4.4% in CR vs 1.9% in control), though CR-associated changes in T3, VO2, SUMPT-WT (weight normalized composite strength score - peak torque), physical functioning, and general health did not appear to be altered by the presence or absence of ketosis. Additional observations of interest include: 1) striking patterns of biomarker expression changes (MCP-1, TNFα, TGF-β1, GH) in both the control and CR participants between the baseline visit and the 24-month post-randomization visit and 2) pro-growth/anti-inflammatory baseline (pre-randomization) biomarker expression profile in CR individuals that later test ketone positive relative to other CR individuals. CR modestly increases the incidence of ketosis in healthy adults, yet the increase in ketosis in CR patients did not significantly affect the aging-protective effects of CR. However, given the relatively small number of participants who were ketone positive, further investigation in larger study cohorts is still required for definitive conclusions.

Anti-aging mechanism of calorie restriction in humans

Animal studies have demonstrated that calorie restriction lowers the incidence of age-related diseases and leads to longer lifespans.1 However, a question that has not been answered is whether long-term calorie restriction has similar beneficial effects in humans. A recent study by Spadaro et al. attempted to address this question by leveraging data from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) clinical trial.2

Tissue losses and metabolic adaptations both contribute to the reduction in resting metabolic rate following weight loss

ObjectiveTo characterize the contributions of the loss of energy-expending tissues and metabolic adaptations to the reduction in resting metabolic rate (RMR) following weight loss.MethodsA secondary analysis was conducted on data from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy study. Changes in RMR, body composition, and metabolic hormones were examined over 12 months of calorie restriction in 109 individuals. The contribution of tissue losses to the decline in RMR was determined by weighing changes in the size of energy-expending tissues and organs (skeletal muscle, adipose tissue, bone, brain, inner organs, residual mass) assessed by dual-energy X-ray absorptiometry with their tissue-specific metabolic rates. Metabolic adaptations were quantified as the remaining reduction in RMR.ResultsRMR was reduced by 101 ± 12 kcal/d as participants lost 7.3 ± 0.2 kg (both p < 0.001). On average, 60% of the total reduction in RMR were explained by energy-expending tissues losses, while 40% were attributed to metabolic adaptations. The loss of skeletal muscle mass (1.0 ± 0.7 kg) was not significantly related to RMR changes (r = 0.14, p = 0.16), whereas adipose tissue losses (7.2 ± 3.0 kg) were positively associated with the reduction in RMR (r = 0.42, p < 0.001) and metabolic adaptations (r = 0.31, p < 0.001). Metabolic adaptations were correlated with declines in leptin (r = 0.27, p < 0.01), triiodothyronine (r = 0.19, p < 0.05), and insulin (r = 0.25, p < 0.05).ConclusionsDuring weight loss, tissue loss and metabolic adaptations both contribute to the reduction in RMR, albeit variably. Contrary to popularly belief, it is not skeletal muscle, but rather adipose tissue losses that seem to drive RMR reductions following weight loss. Future research should target personalized strategies addressing the predominant cause of RMR reduction for weight maintenance.

Calorie restriction improves lipid-related emerging cardiometabolic risk factors in healthy adults without obesity: Distinct influences of BMI and sex from CALERIE™ a multicentre, phase 2, randomised controlled trial.

SummaryBackgroundFor many cardiovascular risk factors there is no lower limit to which further reduction will result in decreased disease risk; this includes values within ranges considered normal for healthy adults. This seems to be true for new emerging metabolic risk factors identified by innovative technological advances. Further, there seems to be ever evolving evidence of differential responses to lifestyle interventions by sex and body compositions in the normal range. In this secondary analysis, we had the opportunity to test these principles for newly identified molecular biomarkers of cardiometabolic risk in a young (21–50 years), normal weight healthy population undergoing calorie restriction for two years.MethodsThe Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE™) was a 24-month, multicenter, randomized controlled trial (May 2007-November 2012) in healthy, adults without obesity to evaluate the potential for calorie restriction (CR) to promote anti-aging adaptations, including those associated with disease risk. 218 participants (age 37.9 ± 7.2 years and body mass index (BMI) 25.1 ± 1.7 kg/m2, mean±SD) were randomized 2:1 to 24 months of CR (prescribed as 25% reduction from baseline calorie intake) versus ad libitum (AL). Fasting plasma from baseline, 12, and 24 months was used for assessments of lipoproteins, metabolites, and inflammatory markers using nuclear magnetic resonance spectroscopy.FindingsAveraging 11.9% CR, the CR group had reductions at 12 and 24 months in the cardiovascular disease risk markers, apolipoprotein B and GlycA, and risks for insulin resistance and type 2 diabetes—Lipoprotein Insulin Resistance Index and Diabetes Risk Index (all PCRvsAL≤0.0009). Insulin resistance and diabetes risk improvements resulted from CR-induced alterations in lipoproteins, specifically reductions in triglyceride-rich lipoprotein particles and low-density lipoprotein particles, a shift to larger high-density lipoprotein particles (more effective cholesterol transporters), and reductions in branched chain amino acids (BCAAs) (all PCRvsAL≤0.004). These CR responses were more pronounced in overweight than normal weight participants and greater in men than women.InterpretationIn normal to slightly overweight adults without overt risk factors or disease, 12 months of ∼12% CR improved newly identified risk markers for atherosclerotic cardiovascular disease, insulin resistance and type 2 diabetes. These markers suggest that CR improves risks by reducing inflammation and BCAAs and shifting lipoproteins from atherogenic to cholesterol transporting. Additionally, these improvements are greater for men and for those with greater BMIs indicating sex and BMI-influences merit attention in future investigations of lifestyle-mediated improvements in disease risk factors.FundingThe CALERIE™ trial design and implementation were supported by a National Institutes of Health (NIH) U-grant provided to four institutions, the three intervention sites and a coordinating center (U01 AG022132, U01 AG020478, U01 AG020487 U01 AG020480). For this secondary analysis including sample acquisition and processing, data analysis and interpretation, additional funding was provided by the NIH to authors as follows: R01 AG054840 (MO, VBK); R33 AG070455 (KMH, DCP, MB, SBR, CKM, LMR, SKD, CFP, CJR, WEK); P30 DK072476 (CKM, LMR); and U54 GM104940 (CKM, LMR).

Effect of 2-year caloric restriction on organ and tissue size in nonobese 21- to 50-year-old adults in a randomized clinical trial: the CALERIE study

Sustained calorie restriction (CR) promises to extend the lifespan. The effect of CR on changes in body mass across tissues and organs is unclear. We used whole-body MRI to evaluate the effect of 2 y of CR on changes in body composition. In an ancillary study of the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trial, 43 healthy adults [25-50 y; BMI (kg/m2): 22-28] randomly assigned to 25% CR (n = 28) or ad libitum (AL) eating (n = 15) underwent whole-body MRI at baseline and month 24 to measure adipose tissue in subcutaneous, visceral, and intermuscular depots (SAT, VAT, and IMAT, respectively); skeletal muscle; and organs including brain, liver, spleen, and kidneys but not heart. The CR group lost more adipose tissue and lean tissue than controls (P < 0.05). In the CR group, at baseline, total tissue volume comprised 32.1%, 1.9%, and 1.0% of SAT, VAT, and IMAT, respectively. The loss of total tissue volume over 24 mo comprised 68.4%, 7.4%, and 2.2% of SAT, VAT, and IMAT, respectively, demonstrating preferential loss of fat vs. lean tissue. Although there is more muscle loss in CR than AL (P < 0.05), the loss of muscle over 24 mo in the CR group comprised only 17.2% of the loss of total tissue volume. Changes in organ volumes were not different between CR and AL. The degree of CR (% decrease in energy intake vs. baseline) significantly (P < 0.05) affected changes in VAT, IMAT, muscle, and liver volume (standardized regression coefficient ± standard error of estimates: 0.43 ± 0.15 L, 0.40 ± 0.19 L, 0.55 ± 0.17 L, and 0.45 ± 0.18 L, respectively). Twenty-four months of CR (intended, 25%; actual, 13.7%) in young individuals without obesity had effects on body composition, including a preferential loss of adipose tissue, especially VAT, over the loss of muscle and organ tissue. This trial was registered at www.clinicaltrials.gov as NCT02695511.

303-OR: Calorie Restriction Equalizes Glycemic Control between Short and Adequate Sleepers

Many adults receive inadequate sleep, which elevates obesity and type 2 diabetes risk and may hinder benefits of calorie restriction (CR). We hypothesized that short sleepers would experience less weight loss and diminished glycemic benefits compared to adequate sleepers during CR. We performed secondary analyses in participants randomized to 25% CR in the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy- Phase 2 (CALERIE 2) trial. Body weight, dual-energy x-ray absorptiometry-measured body composition, fasting glucose, insulin, and HOMA-IR were assessed at baseline, 12, and 24 months. Self-reported sleep duration identified participants with stable sleep as short (≤7 hrs, n=45) or adequate sleepers (&amp;gt;7 hrs, n=65). Sex, BMI, weight, and race-adjusted linear mixed models identified differences over time and between sleepers. Short and adequate sleepers had similar body weight and composition values at all timepoints and experienced equivalent CR-induced reductions over time. Baseline HOMA-IR (1.2 vs. 1.0; P=0.047) was higher in short sleepers, with trends noted for glucose (81.7 vs. 83.5 mg/dL; P=0.093) and insulin (5.7 vs. 4.9 µU/mL; P=0.077). Both phenotypes demonstrated enhanced glycemic control relative to baseline at 12 and 24 months. While there were no 12-month differences in absolute glycemic values between sleepers, initial differences re-emerged at 24 months, with higher HOMA-IR (0.9 vs. 0.7; P=0.01) and insulin concentrations (4.3 vs. 3.4 µU/mL; P&amp;lt;0.01) in short vs. adequate sleepers. Short sleepers had worse baseline glycemic control than adequate sleepers. This difference dissipated during active weight loss but returned during weight maintenance. While short sleepers responded to CR in the present analysis, subsequent work must examine the likely synergy of extending sleep in short sleepers during CR to sustain improvements in glycemic control long-term. Importantly, these data also point to CR as a potential metabolic countermeasure to short sleep. Disclosure K. K. Hoddy: None. P. Singh: None. J. L. Dorling: None. R. A. Beyl: None. J. P. Kirwan: None. K. Huffman: None. S. B. Racette: None. S. Das: None. C. K. Martin: Advisory Panel; Self; EHE Health, Board Member; Self; NaturallySlim, Other Relationship; Self; ABGIL, Academy of Nutrition and Dietetics, Research Support; Self; American Society for Nutrition, Leona M. and Harry B. Helmsley Charitable Trust, Lilly, National Institutes of Health, Patient-Centered Outcomes Research Institute, U. S. Department of Agriculture, WW. Funding National Institute on Aging (U01AG022132, U01AG020478, U01AG020487, U01AG020480, R01AG060499, U24AG047121, U24AG047121); Agricultural Research Service (1950-51000-071-01S); American Heart Association (20POST35210907); National Institute of Diabetes and Digestive and Kidney Diseases (5P30DK07247615); National Institute of General Medical Sciences (U54GM104940)

Calorie Restriction Improves Lipid-Related Emerging Cardiometabolic Risk Factors in Healthy Non-Obese Adults: Distinct Influences of BMI and Sex From CALERIE™ - A Multicentre, Phase 2, Randomised Controlled Trial

Background: For many cardiovascular risk factors there is no lower limit to which further reduction will result in decreased disease risk; this includes values extending ranges of normal healthy adults. This seems to be true for new emerging metabolic risk factors identified by innovative technological advances. Further, there seems to be ever evolving evidence of differential responses to lifestyle interventions by sex and body compositions in the normal range. In this secondary analysis, we had the opportunity to test these principles for newly identified molecular biomarkers of cardiometabolic risk in a young, normal weight healthy population undergoing calorie restriction for two years. Methods: The Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE™) was a 24-month, randomized controlled trial in healthy, non-obese adults to evaluate the potential for calorie restriction (CR) to promote anti-aging adaptations, including those associated with disease risk. 218 participants (age 37.9±7.19 years and BMI 25.1±1.73 kg/m2, mean±SD) were randomized 2:1 to 24 months of CR (prescribed as 25% reduction from baseline calorie intake) versus ad libitum (AL). Fasting plasma from baseline, 12, and 24 months was used for assessments of lipoproteins, metabolites, and inflammatory markers using nuclear magnetic resonance spectroscopy.­­­­­­­­­ Results: Averaging 11.9% CR, the CR group had reductions at 12 and 24 months in the cardiovascular disease risk markers, apolipoprotein B and GlycA, and risks for insulin resistance and type 2 diabetes—Lipoprotein Insulin Resistance Index and Diabetes Risk Index (all PCRvsAL ≤0.0009). Insulin resistance and diabetes risk improvements resulted from CR-induced alterations in lipoproteins, specifically reductions in triglyceride-rich lipoprotein particles and LDL particles, a shift to larger HDL particles (more effective cholesterol transporters), and reductions in branched chain amino acids (BCAAs) (all PCRvsAL ≤0.004). These CR responses were more pronounced in overweight than normal weight participants and greater in men than women. Conclusions: In normal to slightly overweight adults without overt risk factors or disease, 12 months of ~12% CR improved risks of atherosclerotic cardiovascular disease, insulin resistance and type 2 diabetes. These CR-mediated benefits accrued via reduced inflammation and branched chain amino acids, and a shift from atherogenic to cholesterol transporting lipoproteins. Additionally, CR benefits were influenced by sex and BMI, factors meriting close attention in future investigations of lifestyle-mediated improvements in disease risk factors. Clinical Trial Registration Details: NCT00427193. Funding Information: CALERIETM was conducted as a National Institutes of Health (NIH) U-grant (U01 AG022132, U01 AG020478, U01 AG020487 U01 AG020480 ). As defined by this mechanism, the study design and conduct was a collaborative effort between internal NIH and external study investigators. Additional project funding was provided by NIH R01 AG054840 (MO, VBK); R33 AG070455 (KMH, DCP, MB, SBR, CKM, LMR, SKD, CFP, CJR, WEK); P30 DK072476 (CKM, LMR); and U54 GM104940 (CKM, LMR) and without any additional scientific contribution or other role in this report. Declaration of Interests: MAC, IS, and JDO are employees of Labcorp. All other authors have no conflicts interests. Ethics Approval Statement: The study protocol (NCT00427193) was approved by the institutional review boards at all participating clinical centers (Washington University School of Medicine, St Louis, MO; Pennington Biomedical Research Center, Baton Rouge, LA; Tufts University, Boston, MA), and the coordinating center (Duke University, Durham, NC). All study participants provided written informed consent and study oversight was provided by a Data and Safety Monitoring Board.

Abstract 13507: The Role of Weight Loss in Mediating the Effects of a 2-year Caloric Restriction Regimen on Cardiometabolic Risk Markers in Individuals Without Obesity

Introduction: Caloric restriction (CR) improves cardiometabolic risk, even among individuals without obesity. However, it is unclear whether these aging-related benefits are mediated by weight loss. Mediation analyses inform mechanisms underlying relationships between an exposure and outcome. Using mediation analyses, our aim was to test if 2-year weight loss mediates the beneficial effects of CR on cardiometabolic risk markers in individuals without obesity. Methods: Participants without obesity were randomized 2:1 to CR or ad libitum (AL) as part of the 2-year trial, Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE). The CR group aimed to enact 25% CR for 2 years, while AL maintained habitual energy intake. Baseline and year 2 assessments included weight and cardiometabolic risk markers. Using the approaches of Valeri and VanderWeele, mediation was quantified as the natural indirect effect (NIE), defined as the impact of an exposure on an outcome through a mediator. Here, the NIE was the effect of CR (exposure) on cardiometabolic risk markers (outcome) that was accounted for by weight change (mediator). Results: In total, 117 and 71 participants in the CR and AL groups, respectively, completed the trial. The CR group achieved 11.9 (± 0.7)% CR and 7.6 (± 0.3) kg of weight loss ( P &lt; 0.01 versus AL). Weight loss significantly mediated the CR-induced improvements in total cholesterol (NIE = -10.4 ± 3.5 mg/dL), low-density lipoprotein cholesterol (NIE = -8.5 ± 2.8 mg/dL), high-density lipoprotein cholesterol (NIE = 2.9 ± 1.3 mg/dL), triglycerides (NIE = -0.20 ± 0.05 log mg/dL), the homeostatic model assessment of insulin resistance (NIE = -0.22 ± 0.06), and C-reactive protein (NIE = -0.39 ± 0.15 log ug/mL) ( P ≤ 0.02). Weight loss did not mediate CR-induced reductions in systolic (NIE = -0.9 ± 1.4 mmHg) and diastolic (NIE = -1.0 ± 1.1 mmHg) blood pressure ( P ≥ 0.37). Conclusion: In individuals without obesity, CR-induced improvements in multiple cardiometabolic risk markers are driven by weight loss after 2 years. These findings emphasize that, even in individuals without obesity, weight loss after prolonged CR plays a role in improving cardiometabolic disease risk; however, some CR benefits still occur independent of weight loss.

Effects of caloric restriction on human physiological, psychological, and behavioral outcomes: highlights from CALERIE phase 2

Caloric restriction (CR) is a strategy that attenuates aging in multiple nonhuman species. The Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy (CALERIE) trials are part of a research program aiming to test the effects of CR on aging and longevity biomarkers in humans. Building on CALERIE phase 1, CALERIE phase 2 (CALERIE 2) was the largest study to date to assess sustained CR in healthy humans without obesity. In a 24-month randomized controlled trial comprising 218 participants at baseline, CALERIE 2 showed that moderate CR, 11.9% on average, induced improvements in aging-related biomarkers without adversely affecting psychological or behavioral outcomes. The objectives of this report are to summarize and review the highlights of CALERIE 2 and report previously unpublished results on eating disorder symptoms and cognitive function. This article specifically summarizes the physiological, psychological, aging, behavioral, and safety results of the trial. Also provided are research directions beyond CALERIE 2 that highlight important opportunities to investigate the role of CR in aging, longevity, and health span in humans.

Changes in body weight, adherence, and appetite during 2 years of calorie restriction: the CALERIE 2 randomized clinical trial.

Background/Objectives:The Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy Phase 2 (CALERIE) study showed that individuals who are non-obese were able to undergo significant calorie restriction (CR), yet the time course changes in adherence, weight, and appetite are unknown. This analysis aimed to investigate the time course changes in adherence, body weight, and appetite during the CALERIE study.Subjects/Methods:Overall, 143 participants (body mass index: 21.9–28.0 kg/m2) were randomized to a CR group that aimed to achieve 25% CR for 2 years. Throughout the intervention, body weight was measured, and appetite was assessed through visual analogue scales. Algorithms were utilized with body weight measurements to calculate adherence percentile score. Participants targeted an adherence percentile score of 50, though being between 80 (lowest acceptable adherence) and 10 (highest acceptable adherence) was adequate. Polynomial regression analyses were used to assess time course changes.Results:Polynomials indicated that adherence percentile score increased above 50 after approximately week 20, although adherence remained acceptable (adherence percentile score less than 80) (R2 = 0.89; P < 0.001). Weight loss occurred until approximately week 60 and then plateaued (R2 ≥ 0.92; P < 0.001). Hunger and thirst increased (R2 ≥ 0.30; P < 0.001), but the total increase in scale scores were less than 10 mm throughout the intervention.Conclusions:In individuals who are non-obese, adherence to 25% CR declines after 20 weeks, but two years of CR that stimulates a meaningful reduction in weight, promotes aging-related benefits and negligibly affects appetite is viable.