Prolonged Caloric Restriction Research Articles

Hippocampal neuroimmune response in mice undergoing serial daily torpor induced by calorie restriction.

Hibernating animals demonstrate a remarkable ability to withstand extreme physiological brain changes without triggering adverse neuroinflammatory responses. While hibernators may offer valuable insights into the neuroprotective mechanisms inherent to hibernation, studies using such species are constrained by the limited availability of molecular tools. Laboratory mice may serve as an alternative, entering states of hypometabolism and hypothermia similar to the torpor observed in hibernation when faced with energy shortage. Notably, prolonged calorie restriction (CR) induces serial daily torpor patterns in mice, comparable to species that utilize daily hibernation. Here, we examined the neuroinflammatory response in the hippocampus of male C57BL/6 mice undergoing serial daily torpor induced by a 30% CR for 4 weeks. During daily torpor episodes, CR mice exhibited transient increases in TNF-α mRNA expression, which normalized upon arousal. Concurrently, the CA1 region of the hippocampus showed persistent morphological changes in microglia, characterized by reduced cell branching, decreased cell complexity and altered shape. Importantly, these morphological changes were not accompanied by evident signs of astrogliosis or oxidative stress, typically associated with detrimental neuroinflammation. Collectively, the adaptive nature of the brain's inflammatory response to CR-induced torpor in mice parallels observations in hibernators, highlighting its value for studying the mechanisms of brain resilience during torpor. Such insights could pave the way for novel therapeutic interventions in stroke and neurodegenerative disorders in humans.

The Beneficial and Adverse Effects of Autophagic Response to Caloric Restriction and Fasting.

Each cell is equipped with a conserved housekeeping mechanism, known as autophagy, to recycle exhausted materials and dispose of injured organelles via lysosomal degradation. Autophagy is an early-stage cellular response to stress stimuli in both physiological and pathological situations. It is thought that the promotion of autophagy flux prevents host cells from subsequent injuries by removing damaged organelles and misfolded proteins. As a correlate, the modulation of autophagy is suggested as a therapeutic approach in diverse pathological conditions. Accumulated evidence suggests that intermittent fasting or calorie restriction can lead to the induction of adaptive autophagy and increase longevity of eukaryotic cells. However, prolonged calorie restriction with excessive autophagy response is harmful and can stimulate a type II autophagic cell death. Despite the existence of a close relationship between calorie deprivation and autophagic response in different cell types, the precise molecular mechanisms associated with this phenomenon remain unclear. Here, we aimed to highlight the possible effects of prolonged and short-term calorie restriction on autophagic response and cell homeostasis.

Prolonged caloric restriction ameliorates age-related atrophy in slow and fast muscle fibers of rat soleus muscle.

Aging causes loss of skeletal muscle mass and function, which is called sarcopenia. While sarcopenia impairs the quality of life of older adults and is a major factor in long-term hospitalization, its detailed pathogenic mechanism and preventive measures remain to be identified. Caloric restriction (CR) suppresses age-related physiological and pathological changes in many species and prolongs the average and healthy life expectancy. It has recently been reported that CR suppresses the onset of sarcopenia; however, few studies have analyzed the effects of long-term CR on age-related skeletal muscle atrophy. Thus, we investigated the aging and CR effects on soleus (SOL) muscles of 9-, 24-, and 29-month-old ad libitum-fed rats (9AL, 24AL, and 29AL, respectively) and of 29-month-old CR (29CR) rats. The total muscle cross sectional area (mCSA) of the entire SOL muscle significantly decreased in the 29AL rats, but not in the 24AL rats, compared with the 9AL rats. SOL muscle of the 29AL rats exhibited marked muscle fiber atrophy and increases in the number of muscle fibers with a central nucleus, in fibrosis, and in adipocyte infiltration. Additionally, although the decrease in the single muscle fiber cross-sectional area (fCSA) and the muscle fibers' number occurred in both slow-type and fast-type muscle fibers, the degree of atrophy was more remarkable in the fast-type fibers. However, CR suppressed the muscle fiber atrophy observed in the 29AL rats' SOL muscle by preserving the mCSA and the number of muscle fibers that declined with aging, and by decreasing the number of muscle fibers with a central nucleus, fibrosis and denervated muscle fibers. Overall, these results revealed that advanced aging separately reduces the number and fCSA of each muscle fiber type, but long-term CR can ameliorate this age-related sarcopenic muscle atrophy.

Role of Growth Hormone in Ghrelin’s Metabolic Actions

Objective: Ghrelin regulates eating, body weight, and blood glucose. Upon binding to its receptor (growth hormone secretagogue receptor; GHSR), administered ghrelin increases food intake, body weight, and blood glucose. In contrast, blocking ghrelin lowers body weight and food intake. Also, mice that lack ghrelin or GHSR develop life-threatening hypoglycemia when submitted to a prolonged caloric restriction protocol providing only 40% of usual daily calories. Although GHSR was first identified in the pituitary, ghrelin was first defined by its ability to stimulate GH secretion via GHSRs, GH replacement prevents hypoglycemia in ghrelin-KO mice undergoing prolonged caloric restriction, and GH is known to modulate body composition, relatively little attention has been devoted to the role of GH-secreting pituitary somatotrophs (“GH cells”) in ghrelin action. The objective here was to determine the requirement for GHSR-expressing GH cells in mediating ghrelin’s metabolic actions. Methods: Mice with GH cell-selective GHSR deletion were generated by crossing novel GH-IRES-Cre mice to novel floxed-GHSR mice. GH cell-selective GHSR knockout mice and three control littermate groups were studied. Plasma GH, food intake, and blood glucose were measured after ip or sc ghrelin administration. Blood glucose and plasma GH were measured over the course of a 15-d calorie restriction protocol providing only 40% of usual daily calories. Results: In mice with GH cell-selective GHSR deletion, ghrelin-induced GH secretion and food intake were attenuated (by 84.1% at 15 min and by 35.3% at 45 min, respectively) as compared to controls; ghrelin-induced blood glucose elevation was unchanged. Mice with GH cell-selective GHSR deletion exhibited an attenuated GH rise (by 76.8%) over the 15-d calorie restriction period, yet they nonetheless resisted life-threatening hypoglycemia which is observed in similarly-treated ghrelin-KO mice, GHSR-null mice, and mice with hepatocyte-selective GH receptor deletion. Conclusions: These results suggest that GH cell-expressed GHSRs are required for ghrelin’s acute orexigenic and GH secretory actions but are dispensable for ghrelin’s glucoregulatory actions, at least in the settings assessed here. Although GH cell-expressed GHSRs are required for the progressive GH elevations associated with prolonged calorie restriction, they are not required for ghrelin’s overall protective effects to block prolonged calorie restriction-associated hypoglycemia.

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 < 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.

β-Hydroxybutyrate Suppresses Lipid Accumulation in Aged Liver through GPR109A-mediated Signaling.

Dietary interventions such as prolonged calorie restriction (CR) and intermittent fasting provide health benefits including a reduction in the inflammatory burden and regulation of energy metabolism. During CR, β-hydroxybutyrate (BHB) level is elevated in the serum. BHB is a ligand of GPR109A, which inhibits lipolysis and exerts anti-inflammatory effects on cells. During aging, comorbidities related to dyslipidemia are significantly associated with fatty liver. However, the underlying mechanisms of BHB in hepatic ER stress and dyslipidemia are unclear and remain to be elucidated. Here, we used aged rats that were administered with BHB and compared the modulatory effects of BHB through the GPR109A/AMPK pathway on the hepatic endoplasmic reticulum (ER) stress and lipid accumulation to CR rats. BHB caused suppression of hepatic ER stress and lipid accumulation through GPR109A/AMPK pathway in the aged rats. Aged rats of both treatment groups showed reduced cAMP level and PKA phosphorylation. Furthermore, AMPK-Ser173 phosphorylation via PKA was decreased, whereas AMPK-Thr172 phosphorylation was increased by BHB and CR. Further supporting evidence was provided in HepG2 cells that BHB inhibited ER stress and lipid accumulation induced by palmitate. These results suggest that BHB activates GPR109A and regulates the activation of AMPK. These findings were further confirmed by GPR109A-siRNA transfection in vitro. In addition, BHB treatment elevated the protein levels of AMPK leading to significant inhibition of hepatic steatosis, whereas AMPK-siRNA treatment abolished these effects. Taken together, these findings suggest that BHB could be a effective molecule that mimics CR in ameliorating age-related hepatic lipid accumulation via GPR109A signaling pathway.

Time-Limited Eating in Pediatric Patients with Obesity: A Case Series.

BackgroundTime Limited Eating (TLE) is an effective strategy for management of obesity in adults, but there is a paucity of data that have examined its use in the clinical management of children with obesity. A TLE approach involves interspersing normal daily caloric intake with periods of prolonged calorie restriction several times per week. TLE may actually be more feasible, non-stigmatizing, flexible and effective in children, especially for adolescents, compared to alternatives like daily caloric or macronutrient restriction. This is because TLE removes the need for intensive counting of daily calorie intake or macronutrient content and focuses on a straightforward task of consuming food during a pre-specified time period. Also it avoids periods of extended caloric restriction which may interfere with growth and/or risk evoking development of eating behaviors. This case series describes four patients who trialed a TLE approach in a clinical weight management clinic and describes BMI reduction at 4 months.Case PresentationTo date, 4 patients, ages 5–15, with varying underlying pathologies (i.e. Bardet Biedl Syndrome (BBS), previously healthy, craniopharyngioma and epilepsy) have tried a TLE type approach (16-hour fast/8-hour feed for 3–5 days per week) for 4 months and have demonstrated an average decrease in their BMI z-score compared to baseline of −0.24 SD. Patients and their families reported high degrees of satisfaction with this dietary approach.ConclusionsFamilies were very satisfied with the TLE intervention and reported it was feasible, flexible and sustainable to implement in a real life setting and associated with decreased zBMI. Further investigation is required to determine if this approach is effective in both the short and long term as a weight management technique.

Change in self-efficacy, eating behaviors and food cravings during two years of calorie restriction in humans without obesity

Calorie restriction (CR) enhances longevity in humans who are normal weight, overweight and obese. While dietary regimens can change self-efficacy, eating behaviors, and food cravings in individuals with obesity, the responses of these measures to prolonged CR in individuals who are exclusively not obese is unknown. The aim of this analysis was to test the effects of a two-year CR intervention on self-efficacy and eating attitudes and behaviors in humans without obesity by analyzing data from the Comprehensive Assessment of Long-term Effects of Reducing Intake of Energy Phase 2 (CALERIE 2) study. Participants (n = 218, BMI range = 21.3–29.0 kg/m2) were randomized to a 25% CR group or an ad libitum (AL) group. Eating attitudes and behaviors and self-efficacy were assessed using validated questionnaires at baseline, month 12, and month 24. Dietary restraint and self-efficacy increased in the CR compared to the AL group (ES ≥ 0.32). Increased self-efficacy was negatively related to weight change (ρ < −0.24). In the CR group, males showed a reduction in cravings for carbohydrates and fats at month 24, whereas females did not. The CR group showed elevations in state hunger, which were transient, and disinhibited eating (ES ≥ 0.37). In individuals without obesity, dietary restraint and self-efficacy could be important in promoting long-term CR for individuals looking to use CR as a tool to improve longevity.

Peripheral interleukin-6 levels and working memory in non-obese adults: A post-hoc analysis from the CALERIE study.

This analysis aimed to investigate the association among interleukin 6 (IL-6) levels, caloric intake, and working memory and to explore the potential mediators of these associations using the public dataset from the Comprehensive Assessment of Long-Term Effects of Reducing Intake of Energy (CALERIE) clinical trial. The CALERIE study was designed to evaluate the effects of 2 y of prolonged caloric restriction in humans. Individuals were randomized to caloric restriction (CR; n = 145) or an ad libitum diet (AL; n = 75) for 2 y. The outcome measures used herein were spatial working memory tests (i.e., total number of errors and strategy). Generalized estimating equations were used to assess the effects of treatment, time, and potential moderators (e.g., sleep and physical activities). At baseline, there was an effect of hours of sleep, alcohol intake, and physical activities (i.e., mean total metabolic equivalent of task hours per day [MET-hours/day]) on IL-6 levels. The association between IL-6 and energy intake was moderated by MET-hours/day. The longitudinal analysis indicated that there was an effect of time, but not of treatment, on IL-6 levels, with decreasing values in both the CR and AL groups. Changes in IL-6 levels were associated with changes in working memory performance, but there were no between-group (i.e., CR vs. AL) differences. We observed an association between changes in IL-6 levels and improvement in spatial working memory tests. IL-6 was associated with higher caloric consumption, poorer sleep quality, and lower levels of physical activity.

THE USE OF NUTRIENT RESTRICTION IN COMBINATION WITH CHEMOTHERAPY IN LEWIS’ LUNG CARCINOMA

The aim of the study was to evaluate the effect of nutrient restriction in combination with chemotherapy with methotrexate on the development of Lewis lung carcinoma (LLC) in mice. Material and methods. LLC cells were inoculated into the thigh of adult C57BL/6j mice. Mice fasted twice, for 2 days each, with a day interval. Methotrexate (MTX) was used alone and in combination with nutrient restriction. The drug was administered intraperitoneally at the maximally tolerated dose. Treatment efficacy was evaluated by the tumor growth inhibition (TGI), the level of inhibition of lung metastases (IM) and the kinetic tumor growth index (GI). Results. TGI was 34 %, 27 % and 46 % in mice with nutrient restriction alone, MTX alone and with combination of nutrient restriction and MTX, respectively. IM index was 90% and 70% in mice treated with MTX alone and in combination with nutrient restriction, respectively. There was no difference in IM index between mice subjected to restricted feeding and mice in the control group. GI was 0.7 in mice with nutrient restriction and combination of nutrient restriction and MTX. GI was 0.81 in mice treated with MTX monotherapy. The toxic effect of MTX was manifested by weight loss in mice. Weigh loss by 13% and 17% was observed in mice subjected to nutrient restriction alone and in combination with MTX, respectively. Conclusion. MTX inhibited cancer cell growth, however, the therapeutic effect of MTX was leveled by toxic effect on mice. Prolonged caloric restriction inhibited primary tumor growth. However, after returning to a normal diet, tumor growth resumed quickly. The use of MTX in combination with caloric restriction increased its anti-tumor effect on the primary tumor, decreased its toxic effect, but reduced its anti-metastatic activity by 20 %.

Caloric restriction lowers endocannabinoid tonus and improves cardiac function in type 2 diabetes

Background/ObjectivesEndocannabinoids (ECs) are associated with obesity and ectopic fat accumulation, both of which play a role in the development of cardiovascular disease (CVD) in type 2 diabetes (T2D). The effect of prolonged caloric restriction on ECs in relation to fat distribution and cardiac function is still unknown. Therefore, our aim was to investigate this relationship in obese T2D patients with coronary artery disease (CAD).Subjects/MethodsIn a prospective intervention study, obese T2D patients with CAD (n = 27) followed a 16 week very low calorie diet (VLCD; 450–1000 kcal/day). Cardiac function and fat accumulation were assessed with MRI and spectroscopy. Plasma levels of lipid species, including ECs, were measured using liquid chromatography-mass spectrometry.ResultsVLCD decreased plasma levels of virtually all measured lipid species of the class of N-acylethanolamines including the EC anandamide (AEA; −15%, p = 0.016), without decreasing monoacylglycerols including the EC 2-arachidonoylglycerol (2-AG). Baseline plasma AEA levels strongly correlated with the volume of subcutaneous white adipose tissue (SAT; R2 = 0.44, p < 0.001). VLCD decreased the volume of SAT (−53%, p < 0.001), visceral white adipose tissue (VAT) (−52%, p < 0.001), epicardial white adipose tissue (−15%, p < 0.001) and paracardial white adipose tissue (−28%, p < 0.001). VLCD also decreased hepatic (−86%, p < 0.001) and myocardial (−33%, p < 0.001) fat content. These effects were accompanied by an increased left ventricular ejection fraction (54.8 ± 8.7–56.2 ± 7.9%, p = 0.016).ConclusionsCaloric restriction in T2D patients with CAD decreases AEA levels, but not 2-AG levels, which is paralleled by decreased lipid accumulation in adipose tissue, liver and heart, and improved cardiovascular function. Interestingly, baseline AEA levels strongly correlated with SAT volume. We anticipate that dietary interventions are worthwhile strategies in advanced T2D, and that reduction in AEA may contribute to the improved cardiometabolic phenotype induced by weight loss.

Dietary restriction reduces hippocampal neurogenesis and granule cell neuron density without affecting the density of mossy fibers

The hippocampal formation undergoes significant morphological and functional changes after prolonged caloric and dietary restriction (DR). In this study we tested whether prolonged DR results in deleterious alterations in hippocampal neurogenesis, density of granule cell neurons and mossy fibers, all of which support plasticity in the dentate gyrus. Young adult animals either experienced free access to food (control condition), or every-other-day feeding regimen (DR condition) for 3months. The number of Ki-67 cells and 28-day old 5-bromo-2′-deoxyuridine (BrdU) cells were quantified in the dorsal and ventral dentate gyrus to determine the effect of DR on cellular proliferation and survival of neural progenitor cells in the anatomically defined regions of the dentate gyrus. The density of granule cell neurons and synaptoporin were also quantified to determine the effect of DR on granule cell neurons and mossy fiber projections in the dentate gyrus. Our results show that DR increases cellular proliferation and concurrently reduces survival of newly born neurons in the ventral dentate gyrus without effecting the number of cells in the dorsal dentate gyrus. DR reduced density of granule cell neurons in the dorsal dentate gyrus. These alterations in the number of granule cell neurons did not affect mossy fiber density in DR animals, which was visualized as no differences in synaptoporin expression. Our findings demonstrate that granule cell neurons in the dentate gyrus are vulnerable to chronic DR and that the reorganization of granule cells in the dentate gyrus subregions is not producing concomitant alterations in dentate gyrus neuronal circuitry with this type of DR.

Prolonged Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Intake and Associated microRNA Expression.

Short-term (5–10 days) calorie restriction (CR) downregulates muscle protein synthesis, with consumption of a high protein-based diet attenuating this decline. Benefit of increase protein intake is believed to be due to maintenance of amino acid-mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1), however, there is limited evidence to support this contention. The purpose of this investigation was to determine the effects of prolonged CR and high protein diets on skeletal muscle mTORC1 signaling and expression of associated microRNA (miR). Twelve-week old male Sprague Dawley rats consumed ad libitum (AL) or calorie restricted (CR; 40%) adequate (10%, AIN-93M) or high (32%) protein milk-based diets for 16 weeks. Body composition was determined using dual energy X-ray absorptiometry and muscle protein content was calculated from muscle homogenate protein concentrations expressed relative to fat-free mass to estimate protein content. Western blot and RT-qPCR were used to determine mTORC1 signaling and mRNA and miR expression in fasted mixed gastrocnemius. Independent of dietary protein intake, muscle protein content was 38% lower (P < 0.05) in CR compared to AL. Phosphorylation and total Akt, mTOR, rpS6, and p70S6K were lower (P < 0.05) in CR vs. AL, and total rpS6 was associated with muscle protein content (r = 0.64, r2 = 0.36). Skeletal muscle miR expression was not altered by either energy or protein intake. This study provides evidence that chronic CR attenuates muscle protein content by downregulating mTORC1 signaling. This response is independent of skeletal muscle miR and dietary protein.

Chronic Caloric Restriction Alters Nutrient Sensing in Skeletal Muscle in High‐Fat Diet Fed Rats

Caloric restriction (CR) is regarded as one of the most effective strategies for weight loss, decreasing chronic disease risk markers. However, the reduction on body weight induces both lean and fat mass loss, and the molecular mechanism underlying excessive loss of skeletal muscle mass remains largely unknown. We hypothesize that CR alters nutrient sensing pathways in muscle, causing phenotypical loss of muscle mass. To that end, the aim of our study was to analyze the changes in body composition after prolonged CR and the effect on nutrient sensing pathways. In our study, 5 to 6 week old male Sprague‐Dawley rats were fed a high fat diet (45% Kcal from fat) Ad libitum for 3 months. Rats were then divided into HF group Ad libitum (HF), maintenance group fed a control diet (ML, 100% energy requirement at baseline adjusted with body weight), and the caloric restriction group (CR, 25% energy reduction of baseline requirement with age adjustment). Food intake, body weight, and body composition were regularly monitored and recorded. Six months after the dietary intervention rats were sacrificed, nutrient sensing intermediates in serum were measured, and skeletal muscle samples were analyzed for gene expression. The HF group showed an increase of 30.5% in body weight, of which 83.0% is fat and 7.0% is lean mass, respectively. At the same time, ML maintained body weight gain (8.4%). A negative balance was observed in the CR group, with a marked 16.9% decrease in body weight, and a significant fat and lean mass reduction of 61.0% and 12.4%, respectively at the end of the experiment. Gene expression analysis of skeletal muscle revealed a 2.2‐fold increase in the expression of Activating Transcription Factor 4 (Atf4) in CR group compared to HF, as well as a 3.1‐fold upregulation of proteosomal degradation factor Muscle‐Specific Ring Finger Protein 1 (Murf1) and a 2.3‐fold increase in master myogenic regulator Follistatin (Fst). Nutrient sensing during the caloric restriction is further characterized by investigation of regulatory mechanisms of these pathways. Overall, chronic caloric restriction resulted in the activation of nutrient sensing and protein degradation pathways, which correlates to the reduced fat mass and concomitant lean mass loss in response to the reduced energy.

Chronic Calorie Restriction Downregulates Skeletal Muscle mTORC1 Signaling Independent of Dietary Protein Level and Associated microRNA Expression in Male Rats

Short‐term (5–10 days) calorie restriction downregulates muscle protein synthesis, with consumption of a high protein‐based diet attenuating this decline. Benefit with increased protein intake is likely due to maintenance of amino acid‐mediated anabolic signaling through the mechanistic target of rapamycin complex 1 (mTORC1) pathway. However, the effects of chronic caloric restriction and high protein diets on mTORC1 signaling and the expression of microRNA (miR) that regulates this pathway remain unclear. This study determined skeletal muscle mTORC1 and miR responses to a 16 week ad libitum (AL; ~25 g/d) or calorie restricted (CR; 40%; ~15 g/d) standard (10%; ~96 g/kg) or high (32%; ~327 g/kg) protein milk‐based diet in 12‐wk old male Sprague Dawley rats. Crude and clear mixed gastrocnemius homogenate protein concentrations (BSA Protein Assay) were used to calculate protein content, an index of hypertrophy. Immunoblotting was performed to quantify total protein and phosphorylation status of mTORC1 signaling proteins. Expression of miR‐99a‐5p, miR‐100‐5p, miR‐128a‐3p, miR‐133a‐3p, miR‐199a‐3p, and miR‐221‐3p were determined using RT‐qPCR. Data were expressed as fold change (mean ± SEM), compared to AL 10% as the study control. Protein content was higher (P &lt; 0.05) for AL (21.9 ± 0.9 mg) compared to CR (17.3 ± 0.9 mg) rats, regardless of dietary protein intake. Total AKT, mTOR, rpS6 and p70S6K were 1.85 ± 0.04, 1.71 ± 0.08, 2.57 ± 0.05, and 2.23 ± 0.05 fold lower (P &lt; 0.05) for CR than for AL, with rpS6 positively associated with protein content (r = 0.611, r2 = 0.372, P &lt; 0.05). Phosphorylation status of AKTSer473, mTORSer2448, rpS6Ser235/236 and p70S6KThr389 were 1.72 ± 0.11, 1.41 ± 0.08, 3.47 ± 0.05, and 1.95 ± 0.05 fold lower (P &lt; 0.05) for CR than for AL. No effects were observed for dietary protein level on mTORC1 signaling protein, and neither energy nor protein intake altered skeletal muscle miR expression. These data show that prolonged caloric restriction downregulates mTORC1 intracellular signaling, independent of skeletal muscle miR known to regulate mTORC1 protein expression. Consuming a high protein does not appear to attenuate the negative effects of chronic caloric restriction on the intracellular regulation of skeletal muscle mass.Support or Funding InformationT32 NIDDK training grant # 5T32DK062032‐23, USDA, USAMRMC, and the Dairy Research Institute

Metabolic Damage: do Negative Metabolic Adaptations During Underfeeding Persist After Refeeding in Non-Obese Populations?

Several researches have proposed that a prolonged period of caloric restriction (CR) may have a permanent, adverse effect on basal metabolism, fostering the development of obesity. This reported metabolic slowing has been associated with a reduction in resting metabolic rate (RMR) beyond what is predicted by the change in body composition, promoting the idea that metabolism can be permanently damaged. This systematic review investigates if prolonged CR exhibits a permanent, negative effect on basal metabolism.Here we review the literature reporting weight loss and weight regain of individuals who were initially within a healthy weight range, such as the long-term Minnesota starvation experiment, in addition to research on chronically undernourished individuals, such as patients with anorexia nervosa, before and after recovery. Quantification of basal metabolism before and after prolonged CR revealed that body composition is the most critical factor in determining absolute RMR in neutral energy balance. Changes in energy balance induce a rapid yet reversible increase or decrease in RMR. Previous reports may have come to erroneous conclusions in favor of the metabolic damage hypothesis because they did not examine the full recovery period in the Minnesota experiment or neglected the influence of energy balance on RMR. Our findings indicate that the theory of permanent, diet-induced metabolic slowing in non-obese individuals is not supported by the current literature.

Minireview: CNS Mechanisms of Leptin Action.

Leptin is an adipocytokine that circulates in proportion to body fat to signal the repletion of long-term energy stores. Leptin acts via its receptor, LepRb, on specialized neuronal populations in the brain (mainly in the hypothalamus and brainstem) to alter motivation and satiety, as well as to permit energy expenditure and appropriate glucose homeostasis. Decreased leptin, as with prolonged caloric restriction, promotes a powerful orexigenic signal, decreases energy use via a number of neuroendocrine and autonomic axes, and disrupts glucose homeostasis. Here, we review what is known about cellular leptin action and focus on the roles for specific populations of LepRb-expressing neurons for leptin action.

Caloric restriction in lean and obese strains of laboratory rat: effects on body composition, metabolism, growth and overall health.

What is the central question of this study? How do lean and obese rats respond physiologically to caloric restriction? What is the main finding and its importance? Obese rats show marked benefits compared with lean animals. Reduced body fat is associated with improved longevity with caloric restriction (CR) in rodents. Little is known regarding effects of CR in genetically lean versus obese strains. Long-Evans (LE) and Brown Norway (BN) rats make an ideal comparison for a CR study because the percentage body fat of young adult LE rats is double that of BN rats. Male LE and BN rats were either fed ad libitum (AL) or were calorically restricted to 80 or 90% of their AL weight. The percentages of fat, lean and fluid mass were measured non-invasively at 2- to 4-week intervals. Metabolic rate and respiratory quotient were measured after 3, 6, 9 and 12months of CR. Overall health was scored monthly. The percentage of fat of the LE strain decreased with CR, whereas the percentage of fat of the BN strain remained above the AL group for several months. The percentage of lean mass increased above the AL for both strains subjected to CR. The percentage of fluid was unaffected by CR. The average metabolic rate over 22h of the BN rats subjected to CR was reduced, whereas that of LE rats was increased slightly above the AL group. The respiratory quotient of BN rats was decreased with CR. Overall health of the CR LE group was significantly improved compared with that of the AL group, whereas health of the CR BN rats was impaired compared with the AL group. Overall, the lean BN and obese LE strains differ markedly in fat utilization and metabolic response to prolonged CR. There appears to be little benefit of CR in the lean strain.

Effect of Two-Year Caloric Restriction on Bone Metabolism and Bone Mineral Density in Non-Obese Younger Adults: A Randomized Clinical Trial

Although caloric restriction (CR) could delay biologic aging in humans, it is unclear if this would occur at the cost of significant bone loss. We evaluated the effect of prolonged CR on bone metabolism and bone mineral density (BMD) in healthy younger adults. Two-hundred eighteen non-obese (body mass index [BMI] 25.1 ± 1.7 kg/m(2) ), younger (age 37.9 ± 7.2 years) adults were randomly assigned to 25% CR (CR group, n = 143) or ad libitum (AL group, n = 75) for 2 years. Main outcomes were BMD and markers of bone turnover. Other outcomes included body composition, bone-active hormones, nutrient intake, and physical activity. Body weight (-7.5 ± 0.4 versus 0.1 ± 0.5 kg), fat mass (-5.3 ± 0.3 versus 0.4 ± 0.4 kg), and fat-free mass (-2.2 ± 0.2 versus -0.2 ± 0.2 kg) decreased in the CR group compared with AL (all between group p < 0.001). Compared with AL, the CR group had greater changes in BMD at 24 months: lumbar spine (-0.013 ± 0.003 versus 0.007 ± 0.004 g/cm(2) ; p < 0.001), total hip (-0.017 ± 0.002 versus 0.001 ± 0.003 g/cm(2) ; p < 0.001), and femoral neck (-0.015 ± 0.003 versus -0.005 ± 0.004 g/cm(2) ; p = 0.03). Changes in bone markers were greater at 12 months for C-telopeptide (0.098 ± 0.012 versus 0.025 ± 0.015 μg/L; p < 0.001), tartrate-resistant acid phosphatase (0.4 ± 0.1 versus 0.2 ± 0.1 U/L; p = 0.004), and bone-specific alkaline phosphatase (BSAP) (-1.4 ± 0.4 versus -0.3 ± 0.5 U/L; p = 0.047) but not procollagen type 1 N-propeptide; at 24 months, only BSAP differed between groups (-1.5 ± 0.4 versus 0.9 ± 0.6 U/L; p = 0.001). The CR group had larger increases in 25-hydroxyvitamin D, cortisol, and adiponectin and decreases in leptin and insulin compared with AL. However, parathyroid hormone and IGF-1 levels did not differ between groups. The CR group also had lower levels of physical activity. Multiple regression analyses revealed that body composition, hormones, nutrients, and physical activity changes explained ∼31% of the variance in BMD and bone marker changes in the CR group. Therefore, bone loss at clinically important sites of osteoporotic fractures represents a potential limitation of prolonged CR for extending life span. Further long-term studies are needed to determine if CR-induced bone loss in healthy adults contributes to fracture risk and if bone loss can be prevented with exercise.

Caloric restriction increases ratio of estrogen to androgen receptors expression in murine ovaries--potential therapeutic implications.

Both estrogens and androgens are involved in the development and normal functioning of the ovaries. It is also known that ovarian function is regulated by diet. The goal of this study was to estimate the expression of sex hormone receptors in ovaries of mice that were on a 9-month caloric restriction (alternate-day feeding) as compared to normal control animals fed ad libitum. We found that prolonged caloric restriction in mouse ovaries led to increased expression of estrogen receptors (ERs) but did not affect expression of the androgen receptor (AR). This increase in ER:AR ration as result of caloric restriction may lead to higher sensitivity to estrogens and upon return to normal diet may increase ovulation. Thus our observation shed more light on a role of beneficial effect of calorie restriction on female reproduction.