Chronic Caloric Restriction Research Articles

Perturbation of Hypothalamic MicroRNA Expression Patterns in Male Rats After Metabolic Distress: Impact of Obesity and Conditions of Negative Energy Balance

The hypothalamus plays a crucial role in body weight homeostasis through an intricate network of neuronal circuits that are under the precise regulation of peripheral hormones and central transmitters. Although deregulated function of such circuits might be a major contributing factor in obesity, the molecular mechanisms responsible for the hypothalamic control of energy balance remain partially unknown. MicroRNAs (miRNAs) have been recognized as key regulators of different biological processes, including insulin sensitivity and glucose metabolism. However, the roles of miRNA pathways in the control of metabolism have been mostly addressed in peripheral tissues, whereas the potential deregulation of miRNA expression in the hypothalamus in conditions of metabolic distress remains as yet unexplored. In this work, we used high-throughput screening to define to what extent the hypothalamic profiles of miRNA expression are perturbed in two extreme conditions of nutritional stress in male rats, namely chronic caloric restriction and high-fat diet-induced obesity. Our analyses allowed the identification of sets of miRNAs, including let-7a, mir-9*, mir-30e, mir-132, mir-145, mir-200a, and mir-218, whose expression patterns in the hypothalamus were jointly altered by caloric restriction and/or a high-fat diet. The predicted targets of these miRNAs include several elements of key inflammatory and metabolic pathways, including insulin and leptin. Our study is the first to disclose the impact of nutritional challenges on the hypothalamic miRNA expression profiles. These data will help to characterize the molecular miRNA signature of the hypothalamus in extreme metabolic conditions and pave the way for targeted mechanistic analyses of the involvement of deregulated central miRNAs pathways in the pathogenesis of obesity and related disorders.

The Amen Anti-Addiction Intervention: Theoretical Foundations of an Alternative Protocol for the Prevention and Treatment of Addiction

The Amen Anti-Addiction Intervention (AAI) is an austere intervention intended to eradicate addictive impulses and behaviors. The theoretical and empirical foundations of this intervention are explicated herein. The AAI entails daily cyclic fasting, chronic caloric restriction, supplementation with specific neutraceutical agents, rigorous exercise, and minimalist meditation. The physiological focus of the AAI is twofold: the augmentation of neurogenesis in a manner analogous to the effect of psychotropic drugs and the activation of areas in the brain involved in the regulation of emotional arousal and reward. The psychological focus of the AAI is similarly twofold: the attenuation of anxiety and/or impulsivity and the cultivation of mental mastery and/or self-control. It shall be argued that the AAI, by virtue of its explicit integration of biological, psychological, and sociological elements-each of which evidently influence addiction-will plausibly be an improvement over existing interventions aimed at the treatment of addiction

The Amen Anti-Addiction Intervention: Theoretical Foundations of a Novel Neurotrophic Protocol for the Prevention and Treatment of Addiction

The Amen Anti-Addiction Intervention (AAI) is an austere regimen intended to eradicate addictive impulses and behaviors. The theoretical and empirical foundations of this intervention are explicated herein. The AAI entails daily cyclic fasting, chronic caloric restriction, supplementation with specific neutraceutical agents, rigorous exercise, and minimalist meditation. The physiological focus of the AAI is twofold: the augmentation of neurogenesis in a manner analogous to the effect of psychotropic drugs and the activation of areas in the brain involved in the regulation of emotional arousal and reward. The psychological focus of the AAI is similarly twofold: the attenuation of anxiety and/or impulsivity and the cultivation of mental mastery and/or self-control. It shall be argued that the AAI, by virtue of its explicit integration of biological, psychological, and sociological elements—each of which evidently influences addiction—will plausibly be an improvement over existing interventions aimed at the treatment of addiction.

Cytokines and Diabetes Research

In recent years, the role of the inflammatory system in the pathogenesis of diabetes has been increasingly investigated. Cytokines, a group of proteins that are expressed by several cell types, act as immune mediators and regulators. Depending on the period of pregnancy, a predominant inflammatory profile is defined by increased production of cytokines. Insulin resistance has been associated with abnormal secretion of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and Interleukin-6 (IL-6) and decreased production of anti-inflammatory mediators such as IL-4 and IL-10. Despite some controversies regarding specific cytokine levels, type 2 diabetes mellitus (T2DM) is currently regarded as a chronic inflammatory disease, while type 1 diabetes (T1D) is considered to be a T-helper-(Th)-1 autoimmune disease. Extensive research in animals and in humans over the last decade has revealed important functions of cytokines in diabetes; adiponectin (APN) and leptin can decrease hepatic gluconeogenesis, resistin (REN) can increase hepatic gluconeogenesis and glycogenolysis, IL-6 can decrease glycogen synthesis, and TNF-α can decrease glucose uptake in liver. Both of them can block hepatic insulin signalling by interfection of insulin receptor signalling and insulin signal transduction. Thus, cytokines are involved in nearly every facet of immunity, inflammation, and development of diabetes. In this special issue, we have invited some papers hoping to shed light on some aspects of this very interesting field. We have collected 7 papers by scientists from 5 countries. In the submitted research papers, Y. Li et al. summarize recent findings regarding the relationship between adipocytokines and hepatic insulin resistance. Excessive adipose tissue may be detrimental partially through secretion of the following cytokines: TNF-α, IL-6, and resistin. In contrast, the presence of adipose tissues is vital in the prevention of hepatic insulin resistance via secretion of the following cytokines: leptin and adiponectin. While J. Su and colleagues review the relationship between the endoplasmic reticulum (ER) and autophagy, inflammation, and apoptosis in DM to better understand the molecular mechanisms of diabetes, the authors suggest that the ER is therefore an attractive potential therapeutic target, and maintaining or improving ER function appropriately may prevent diabetes. Z. Meng et al. concluded that ethanol causes glucose intolerance by increasing hepatic expression of 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) and glucocorticoid receptor (GR), which leads to increased expression of gluconeogenic and glycogenolytic enzymes. In the following papers, J. Liu et al. have shown that uncoupling proteins (UCPs) may affect the development of DM through decreasing mitochondrial membrane potential, increasing energy expenditure especially through glucose and lipid metabolisms, downregulating ROS generation, and gene polymorphisms. In a very interesting research paper, J. Vcelakova et al. have shown, that in T1D patients, important immune response-related pathways were involved. These important immune response-related processes largely included the induction of Th17 and Th22 responses, as well as cytoskeletal rearrangements, MHCII presentation, and the upregulation of CD4, TGF-beta, and STAT3. These findings potentially suggest that these processes could be utilised as predictive markers for the development of T1D or as molecular targets for the repression of specific immunocompetent cell populations for the treatment of diabetes. On the other hand, H. Meng and colleagues demonstrate that amyloid precursor protein 17 peptide (APP17 peptide) has a comprehensive therapeutic effect on diabetic encephalopathy, particularly through improving glycol metabolism. Finally, M. Cui et al. have shown that AMPK activation, which was represented by the level of p-AMPK, did not correlate with the improvement of metabolic conditions in diabetes mice, implying that AMPK activation may not participate in mediating the beneficial effects of chronic caloric restriction (CR) or exercise. However, the autophagy activity might be related to the improved metabolic conditions; thus autophagy may play a role in mediating the effects of chronic CR.

Patterns of intraspecific variability in the response to caloric restriction

Caloric restriction (CR) is cited as the most robust means of increasing lifespan across a range of taxa, yet there is a high degree of variability in the response to CR, both within and between species. To examine the intraspecific evolutionary conservation of lifespan extension by CR, we tested the effects of chronic caloric restriction (CCR) at multiple food levels and of intermittent fasting (IF) in twelve isolates from the Brachionus plicatilis species complex of monogonont rotifers. While CCR generally increased or did not change lifespan and total fecundity, IF caused increased, unchanged, or decreased lifespan, depending upon the isolate, and decreased total fecundity in all but one isolate. Lifespan under ad libitum (AL) feeding varied among isolates and predicted the lifespan response to CR: longer-lived isolates under AL were less likely to have a significant increase in lifespan under CCR and were more likely to have a significantly shortened lifespan under IF. Lifespan under AL conditions and the response to CR were not correlated with hydroperiodicity of native habitat or with time in culture. Lack of trade-off between lifespan and fecundity under CCR, and differences in lifespan and fecundity under CCR and IF, even when average food intake was similar, suggest that longevity changes are not always directly determined by energy intake and that CCR and IF regimens extend lifespan through diverse genetic mechanisms.

Age and nutritional state influence the effects of cholecystokinin on energy balance

Cholecystokinin (CCK) is anorexic, irrespective whether it is applied intraperitoneally (IP) or intracerebroventricularly (ICV) in male Wistar rats. The metabolic effects depend on the route of administration: by the IP route it elicits hypothermia (presumably by type-1 receptors, CCK1R-s), while ICV administration is followed by fever-like hypermetabolism and hyperthermia via activation of CCK2R-s, which latter response seems to be most important in the postprandial (compensatory) hypermetabolism. The efficacy of the IP injected CCK varies with age: it causes strong anorexia in young adult 4 and 6-months old and again in old rats (aged 18–24months), but the middle-aged (12-month old) ones seem to be resistant to this effect. Such pattern of effects may contribute to the explanation of age-related obesity observed in middle-aged animals as well as to the aging anorexia and loss of body weight in old ones. Diet-induced obesity accelerates the appearance of CCK-resistance as well as the return of high sensitivity to CCK in further aging, while chronic calorie-restriction prevents the development of resistance, as if the speed of the age-related regulatory changes was altered by the nutritional state. The effects of ICV applied CCK also change with age: the characteristic anorexic and hypermetabolic/hyperthermic effects can be observed in young adult rats, but the effects gradually and monotonically decline with age and disappear by the old age of 24months. These disparate age-related patterns of CCK efficacy upon peripheral or central administration routes may indicate that although both peripheral and central CCKR-s exert anorexic effects, they may have dissimilar roles in the regulation of overall energy balance.

Expression of Serum Retinol Binding Protein and Transthyretin within Mouse Gastric Ghrelin Cells

Ghrelin is an orexigenic peptide hormone produced mainly by a distinct group of dispersed endocrine cells located within the gastric oxyntic mucosa. Besides secreted gene products derived from the preproghrelin gene, which include acyl-ghrelin, desacyl-ghrelin and obestatin, ghrelin cells also synthesize the secreted protein nesfatin-1. The main goal of the current study was to identify other proteins secreted from ghrelin cells. An initial gene chip screen using mRNAs derived from highly enriched pools of mouse gastric ghrelin cells demonstrated high levels of serum retinol-binding protein (RBP4) and transthyretin (TTR), both of which are known to circulate in the bloodstream bound to each other. This high expression was confirmed by quantitative RT-PCR using as template mRNA derived from the enriched gastric ghrelin cell pools and from two ghrelin-producing cell lines (SG-1 and PG-1). RBP4 protein also was shown to be secreted into the culture medium of ghrelin cell lines. Neither acute nor chronic caloric restriction had a significant effect on RBP4 mRNA levels within stomachs of C57BL/6J mice, although both manipulations significantly decreased stomach TTR mRNA levels. In vitro studies using PG-1 cells showed no effect on RBP4 release of octanoic acid, epinephrine or norepinephrine, all of which are known to act directly on ghrelin cells to stimulate ghrelin secretion. These data provide new insights into ghrelin cell physiology, and given the known functions of RBP4 and TTR, support an emerging role for the ghrelin cell in blood glucose handling and metabolism.

Differential Pre-mRNA Splicing Regulates Nnat Isoforms in the Hypothalamus after Gastric Bypass Surgery in Mice

BackgroundNeuronatin (NNAT) is an endoplasmic reticulum proteolipid implicated in intracellular signalling. Nnat is highly-expressed in the hypothalamus, where it is acutely regulated by nutrients and leptin. Nnat pre-mRNA is differentially spliced to create Nnat-α and -β isoforms. Genetic variation of NNAT is associated with severe obesity. Currently, little is known about the long-term regulation of Nnat.MethodsExpression of Nnat isoforms were examined in the hypothalamus of mice in response to acute fast/feed, chronic caloric restriction, diet-induced obesity and modified gastric bypass surgery. Nnat expression was assessed in the central nervous system and gastrointestinal tissues. RTqPCR was used to determine isoform-specific expression of Nnat mRNA.ResultsHypothalamic expression of both Nnat isoforms was comparably decreased by overnight and 24-h fasting. Nnat expression was unaltered in diet-induced obesity, or subsequent switch to a calorie restricted diet. Nnat isoforms showed differential expression in the hypothalamus but not brainstem after bypass surgery. Hypothalamic Nnat-β expression was significantly reduced after bypass compared with sham surgery (P = 0.003), and was positively correlated with post-operative weight-loss (R2 = 0.38, P = 0.01). In contrast, Nnat-α expression was not suppressed after bypass surgery (P = 0.19), and expression did not correlate with reduction in weight after surgery (R2 = 0.06, P = 0.34). Hypothalamic expression of Nnat-β correlated weakly with circulating leptin, but neither isoform correlated with fasting gut hormone levels post- surgery. Nnat expression was detected in brainstem, brown-adipose tissue, stomach and small intestine.Conclusions Nnat expression in hypothalamus is regulated by short-term nutrient availability, but unaltered by diet-induced obesity or calorie restriction. While Nnat isoforms in the hypothalamus are co-ordinately regulated by acute nutrient supply, after modified gastric bypass surgery Nnat isoforms show differential expression. These results raise the possibility that in the radically altered nutrient and hormonal milieu created by bypass surgery, resultant differential splicing of Nnat pre-mRNA may contribute to weight-loss.

The protective effect of intermittent calorie restriction on mammary tumorigenesis is not compromised by consumption of a high fat diet during refeeding

Previously we reported that intermittent calorie restriction (ICR) provided greater prevention of mammary tumors (MTs) than chronic calorie restriction (CCR). Here the impact of increased fat intake during refeeding in an ICR protocol was evaluated. MMTV-TGF-α female mice were assigned to one of three groups: ad libitum (AL) fed (n=45) with free access to a moderately high fat diet (22% fat calories); ICR (n=45) 50% calorie restricted for 3-week intervals followed by 3 weeks of 100% of AL intake; and CCR (n=45) fed 75% of AL mice, matching each 6-week cycle of ICR mice. ICR mice were further designated as ICR-Restricted or ICR-Refed for data obtained during these intervals. All mice consumed the same absolute amount of dietary fat. Mice were followed to assess MT incidence, body weight and serum IGF-1, IGFBP3, leptin and adiponectin levels until 79 (end of final 3-week restriction) or 82 (end of final 3-weeks refeeding) weeks of age. Age of MT detection was significantly extended for CCR (74weeks) and ICR (82weeks) mice, compared to 57.5weeks for AL mice. MT incidence for AL, ICR and CCR mice was 66.7, 4.4, and 52.3%, respectively. Mammary and fat pad weights were reduced significantly following 50% calorie restriction in ICR-Restricted mice compared to AL, CCR and ICR-Refed mice. IGF-1 and leptin levels also tended to be reduced in ICR-Restricted mice over the course of the study while adiponectin was not compared to AL, CCR, and ICR-Refed mice. The adiponectin:leptin ratio was consistently higher following 50% restriction in ICR-Restricted mice. There was no relationship of IGF-1, leptin, or adiponectin with the presence of MTs in any groups. Thus the manner in which calories are restricted impacts the protective effect of calorie restriction independently of high fat intake.

The 2010 ESPEN Sir David Cuthbertson Lecture: New and old proteins: Clinical implications

The past century had witnessed vast advances in biomedical research, particularly in the fields of genomics and proteomics, yet the translation of these discoveries into clinical practice has been hindered by gaps in mechanistic understanding of variability governing disease susceptibility and pathogenesis. Among the greatest challenges are the dynamic nature of the proteome and the imperfect methodologies currently available to study it. Here, we review key recently developed proteomic techniques that have allowed for dynamic characterization of protein quality, as well as quantity, and discuss their potential applications in understanding aging and metabolic disorders including diabetes. These methodologies revealed that senescence is characterized, in part, by decreased rates of de novo protein synthesis and potentially also degradation, in addition to concomitantly increased levels of oxidative stress, ultimately resulting in excessive accumulation of damaged and dysfunctional proteins. Insulin may be a key mediator in these pathologies, as hyperinsulinemia has been shown to hinder protein degradation while transient insulin deficiency may accelerate oxidative damage. We also discuss two interventions that have been proposed to delay, and possibly reverse, senescence by augmenting protein degradation: chronic caloric restriction and aerobic exercise.

Effects of caloric restriction on oxidative stress parameters

Moderate caloric restriction prolongs lifespan. Changes in oxidative stress and hormesis may be involved in this process. The aim of this study is to examine the effects of different levels of chronic caloric restriction (CR) and acute fasting on stress response and oxidative stress parameters in rat liver and plasma. Forty-two rats were divided into groups: control group, calorie-restricted groups with intake of 80-90%, 60-70%, 40-50%, 20-30% of daily caloric needs and acute fasting group. To determine alanine aminotransferase (ALT), aspartate aminotransferase (AST) and superoxide dismutase (SOD) activity, concentration of corticosterone, nitrites and nitrates (NOx), malondialdehyde (MDA) and glutathione (GSH), liver samples and blood were collected. Increase in plasma corticosterone concentration and AST and ALT activity was found in severe CR. Ingestion 40-50% daily caloric needs or less increased liver MDA and NOx concentration and decreased SOD activity. Ingestion 60-70% daily caloric needs increased Mn-SOD activity, GSH and NOx. In acute fasting group and group taking 20-30% daily caloric needs, GSH was significantly lower than in control group. Severe CR and acute fasting increase oxidative damage and decrease antioxidative capacity of hepatocytes. Moderate CR increases antioxidative capacity of hepatocytes due to increase in Mn-SOD activity and GSH concentration, which might have a role in anti-aging and hormetic mechanism of CR.

Chronic Caloric Restriction and Exercise Improve Metabolic Conditions of Dietary-Induced Obese Mice in Autophagy Correlated Manner without Involving AMPK

Aim. To investigate the role of AMPK activation and autophagy in mediating the beneficial effects of exercise and caloric restriction in obesity. Methods. Dietary-induced obesity mice were made and divided into 5 groups; one additional group of normal mice serves as control. Mice in each group received different combinations of interventions including low fat diet, caloric restriction, and exercise. Then their metabolic conditions were assessed by measuring serum glucose and insulin, serum lipids, and liver function. AMPK phosphorylation and autophagy activity were detected by western blotting. Results. Obese mice models were successfully induced by high fat diet. Caloric restriction consistently improved the metabolic conditions of the obese mice, and the effects are more prominent than the mice that received only exercise. Also, caloric restriction, exercise, and low fat diet showed a synergistic effect in the improvement of metabolic conditions. Western blotting results showed that this improvement was not related with the activation of AMPK in liver, skeletal muscle, or heart but correlates well with the autophagy activity. Conclusion. Caloric restriction has more prominent beneficial effects than exercise in dietary-induced obese mice. These effects are correlated with the autophagy activity and may be independent of AMPK activation.

Effect of Growth Hormone and Calorie Restriction on the Expression of Antioxidant Enzymes in the Liver and Kidney of Growth Hormone Receptor Knockout Mice.

Caloric restriction (CR) can delay aging and prolong life span and these actions may be related to reduced oxidative damage. Mice with disrupted growth hormone (GH) receptor/binding protein knockout (GHRKO) live significantly longer than their normal siblings. Therefore, it is of interest to examine the effects of chronic CR on hepatic and renal antioxidant enzymes as well as lipid peroxidation (LP) as an oxidative stress marker in GHRKO mice. Female GHRKO and normal mice were either fed ad libitum (AL) or subjected to 30% CR starting at 2 months of age and examined at the age of 9 months. In the liver, catalase (CAT) activity was significantly increased in GHRKO-AL as compared to normal control -AL animals. CR reduced CAT activity in both GHRKO and normal phenotypes. Cu/Zn superoxide dismutase (SOD1) activity was also higher in GHRKO-AL as compared to normal-AL mice. However, CR reduced SOD1 activity in GHRKO mutants. Glutathione peroxidase (GPx) activity was significantly decreased in GHRKO-AL mice and further reduced in GHRKO-CR group of animals. CR significantly increased LP in GHRKOs while its activity was not altered in GHRKO-AL group of mice. In the kidney, CAT activity was lower in GHRKO-AL as compared to normal-AL, however CR did not induce any significant effect in both phenotypes. Similarly, SOD1 levels were significantly lower in GHRKO than in normal mice. GPx expression was higher in GHRKO-AL as compared to control-AL. CR reduced GPx activity in GHRKO mice but increased it in controls as compared to their AL counterparts. There was no difference in LP expression between GHRKO-AL and normal-AL mice. However, CR significantly increased its levels in both phenotypes. Although these findings do not support the hypothesis that CR would increase the capacity of ROS defense mechanisms in GHRKO mice by increasing antioxidant enzymes levels, they do agree with some of the reported effects of CR on their expression. We suspect that GH resistance and CR may affect aging by different mechanisms and if CR delays aging in GHRKO animals it is not due to changes in the activity of antioxidant enzymes.

Effects of chronic caloric restriction on kidney and heart redox status and antioxidant enzyme activities in Wistar rats

Caloric restriction (CR) has been associated with health benefits and these effects have been attributed, in part, to modulation of oxidative status by CR; however, data are still controversial. Here, we investigate the effects of seventeen weeks of chronic CR on parameters of oxidative damage/modification of proteins and on antioxidant enzyme activities in cardiac and kidney tissues. Our results demonstrate that CR induced an increase in protein carbonylation in the heart without changing the content of sulfhydryl groups or the activities of superoxide dismutase and catalase (CAT). Moreover, CR caused an increase in CAT activity in kidney, without changing other parameters. Protein carbonylation has been associated with oxidative damage and functional impairment; however, we cannot exclude the possibility that, under our conditions, this alteration indicates a different functional meaning in the heart tissue. In addition, we reinforce the idea that CR can increase CAT activity in the kidney. [BMB Reports 2012; 45(11): 671-676]

Life-Span Extension by Caloric Restriction Is Determined by Type and Level of Food Reduction and by Reproductive Mode in Brachionus manjavacas (Rotifera)

We measured life span and fecundity of three reproductive modes in a clone of the monogonont rotifer Brachionus manjavacas subjected to chronic caloric restriction (CCR) over a range of food concentrations or to intermittent fasting (IF). IF increased life span 50%-70% for all three modes, whereas CCR increased life span of asexual females derived from sexually or asexually produced eggs, but not that of sexual females. The main effect of CR on both asexual modes was to delay death at young ages, rather than to prevent death at middle ages or to greatly extend maximum life span; in contrast CR in sexual females greatly increased the life span of a few long-lived individuals. Lifetime fecundity did not decrease with CCR, suggesting a lack of resource allocation trade-off between somatic maintenance and reproduction. Multiple outcomes for a clonal lineage indicate that different responses are established through epigenetic programming, whereas differences in life-span allocations suggest that multiple genetic mechanisms mediate life-span extension.

Fatty Acid Composition of the Brain, Retina, Liver and Adipose Tissue of the Grey Mouse Lemur (Microcebus murinus, Primate)

The particular interest in supplementing human foods with n-3 fatty acids has arisen from the findings that this series of polyunsaturated fatty acids (PUFA) have an impact on neuronal functions. Indeed vertebrates, including humans, preferentially use docosahexaenoic acid (DHA, 22:6n-3) over other long-chain n-3 PUFA for the genesis of their neuronal and retinal membranes. The grey mouse lemur is a nocturnal prosimian primate originating from Madagascar. The increased use of this omnivorous primate in nutritional studies (chronic caloric restriction, n-3 fatty acids supplementation), justifies the interest of determining their fatty acids body composition. In the present study, we report the fatty acid composition in lipid classes from the main target tissues (brain, retina, liver and adipose tissue) of six adult mouse lemurs raised under laboratory nutritional conditions. Among the main findings, n-6-docosapentaenoic acid (n-6-DPA; 22:5n-6) is very low in the brain cortex and retina, whereas there is a very high accumulation of docosahexaenoic acid (DHA, 22:6n-3) in the neural tissues compared to liver and plasma. In particular, DHA accounts for about one half of the total fatty acids in the retina ethanolamine glycerophospholipids. This high concentration clearly indicates that DHA is efficiently transferred from blood lipids to the outer segment of the mouse lemur retina. We conclude that the mouse lemur n-3 PUFA metabolism efficiently drives DHA to neural tissues, through the blood-brain barrier and the blood-retina barrier.

Chronic caloric restriction partially protects against age-related alteration in serum metabolome

Calorie restriction (CR) remains the most robust metabolic intervention to extend lifespan and improve healthspan in several species. Using global and targeted mass spectrometry-based metabolomics approaches, here we show that chronic CR prevents age-related changes in specific metabolic signatures. Global metabolomic analysis using ultra-performance liquid chromatography-tandem mass spectrometry detected more than 7,000 metabolites in sera from ad-libitum-fed young, aged, and aged C57BL/6 mice maintained on 40 % CR. Multivariate statistical analysis of mass spectrometry data revealed a clear separation among the young, aged, and aged-CR mice demonstrating the potential of this approach for producing reliable metabolic profiles that discriminate based on age and diet. We have identified 168 discriminating features with high statistical significance (p ≤ 0.001) and validated and quantified three of these metabolites using targeted metabolite analysis. Calorie restriction prevented the age-related alteration in specific metabolites, namely lysophosphatidylcholines (16:1 and 18:4), sphingomyelin (d18:1/12:0), tetracosahexaenoic acid, and 7α-dihydroxy-4-cholesten-3-one, in the serum. Pathway analysis revealed that CR impacted the age-related changes in metabolic byproducts of lipid metabolism, fatty acid metabolism, and bile acid biosynthesis. Our data suggest that metabolomics approach has the potential to elucidate the metabolic mechanism of CR's potential anti-aging effects in larger-scale investigations.

SIRT3 Protein Deacetylates Isocitrate Dehydrogenase 2 (IDH2) and Regulates Mitochondrial Redox Status

Mitochondria play a central role in oxidative energy metabolism and age-related diseases such as cancer. Accumulation of spurious oxidative damage can cause cellular dysfunction. Antioxidant pathways that rely on NADPH are needed for the reduction of glutathione and maintenance of proper redox status. The mitochondrial matrix protein isocitrate dehydrogenase 2 (IDH2) is a major source of NADPH. Previously, we demonstrated that the NAD(+)-dependent deacetylase SIRT3 was essential for the prevention of age-related hearing loss in mice fed a calorically restricted diet. Here we provide direct biochemical and biological evidence establishing an exquisite regulatory relationship between IDH2 and SIRT3 under acute and chronic caloric restriction. The regulated site of acetylation was mapped to Lys-413, an evolutionarily invariant residue. Site-specific, genetic incorporation of N(ε)-acetyllysine into position 413 of IDH2 revealed that acetylated IDH2 displays a dramatic 44-fold loss in activity. Deacetylation by SIRT3 fully restored maximum IDH2 activity. The ability of SIRT3 to protect cells from oxidative stress was dependent on IDH2, and the deacetylated mimic, IDH2(K413R) variant was able to protect Sirt3(-/-) mouse embryonic fibroblasts from oxidative stress through increased reduced glutathione levels. Together these results uncover a previously unknown mechanism by which SIRT3 regulates IDH2 under dietary restriction. Recent findings demonstrate that IDH2 activities are a major factor in cancer, and as such, these results implicate SIRT3 as a potential regulator of IDH2-dependent functions in cancer cell metabolism.

Effects of Chronic Calorie Restriction or Dietary Resveratrol Supplementation on Insulin Sensitivity Markers in a Primate, Microcebus murinus

The prevalence of diabetes and hyperinsulinemia increases with age, inducing metabolic failure and limiting lifespan. Calorie restriction (CR) without malnutrition delays the aging process, but its long-term application to humans seems difficult. Resveratrol (RSV), a dietary polyphenol, appears to be a promising CR mimetic that can be easily administered in humans. In this work, we hypothesized that both CR and RSV impact insulin sensitivity in a non-human primate compared to standard-fed control (CTL) animals. Four- to five-year-old male grey mouse lemurs (Microcebus murinus) were assigned to three dietary groups: a CTL group, a CR group receiving 30% fewer calories than the CTL and a RSV group receiving the CTL diet supplemented with RSV (200 mg·day−1·kg−1). Insulin sensitivity and glycemia were assessed using an oral glucose tolerance test (OGTT) and the homeostasis model assessment of insulin resistance (HOMA-IR index) evaluation after 21 or 33 months of chronic treatment. Resting metabolic rate was also measured to assess the potential relationships between this energy expenditure parameter and insulin sensitivity markers. No differences were found after a 21-month period of treatment, except for lower glucose levels 30 min after glucose loading in CR animals. After 33 months, CR and RSV decreased glycemia after the oral glucose loading without decreasing fasting blood insulin. A general effect of treatment was observed on the HOMA-IR index, with an 81% reduction in CR animals and 53% in RSV animals after 33 months of treatment compared to CTL. Chronic CR and dietary supplementation with RSV affected insulin sensitivity by improving the glucose tolerance of animals without disturbing their baseline insulin secretion. These results suggest that both CR and RSV have beneficial effects on metabolic alterations, although these effects are different in amplitude between the two anti-aging treatments and potentially rely on different metabolic changes.

Chronic caloric restriction attenuates a loss of sulfatide content in PGC-1α mouse cortex: a potential lipidomic role of PGC-1α in neurodegeneration

Peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), a key regulator of energy metabolism and lipid homeostasis in multiple highly oxidative tissues, has been implicated in the metabolic derangements of diabetes and obesity. However, relatively less is known regarding its role in neurological functions. Using shotgun lipidomics, we investigated the lipidome of mouse cerebral cortex with generalized deficiency of PGC-1α (PGC-1α(-/-)) versus wild-type (WT) mice under standard diet and chronically calorically restricted conditions. Specific deficiency in sulfatide, a myelin-specific lipid class critically involved in maintaining neurological function, was uncovered in the cortex of PGC-1α(-/-) mice compared with WT mice at all ages examined. Chronic caloric restriction (CR) for 22 months essentially restored the sulfatide reduction in PGC-1α(-/-) mice compared with WT, but sulfatide reduction was not restored in PGC-1α(-/-) with CR for a short term (i.e., 3 months). Mechanistic studies uncovered and differentiated the biochemical mechanisms underpinning the two conditions of altered sulfatide homeostasis. The former is modulated through PGC-1α-MAL pathway, whereas the latter is under the control of LXR/RXR-apoE metabolism pathway. These results suggest a novel mechanistic role of PGC-1α in sulfatide homeostasis, provide new insights into the importance of PGC-1α in neurological functions, and indicate a potential therapeutic approach for treatment of deficient PGC-1α-induced alterations in sulfatide homeostasis.