Effects Of Chronic Caloric Restriction Research Articles

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 < 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 < 0.05) for CR than for AL, with rpS6 positively associated with protein content (r = 0.611, r2 = 0.372, P < 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 < 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

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.

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]

Chronic caloric restriction induces forestomach hypertrophy with enhanced ghrelin levels during aging

Caloric restriction (CR) is the only preventive intervention that has robust pro-longevity effects in experimental models. Various circulating hormones that regulate the state of negative energy balance may drive the multi-system beneficial effects of the CR phenomenon. Ghrelin, one such stomach-derived circulating peptide hormone stimulates food intake, promotes GH release and inhibits pro-inflammatory cytokines. We have recently demonstrated that ghrelin also reverses age-related thymic involution. Here, we report that chronic CR in aging mice results in reduction in body weight, and spleen size but remarkably, leads to a significant increase in the size and weight of stomach. The increased size of stomach was largely due to increased size of fundus (forestomach) and also smaller but statistically significant enlargement of antrum. The analysis of serial stomach sections revealed that chronic CR leads to a striking hypertrophy of lamina propria, stratum basale, stratum corneum and the stratified squamous epithelium of forestomach of the aged animals. We also report for the first time that chronic CR during aging significantly increases circulating ghrelin levels as well as total ghrelin production in the stomach and reverses age-related loss of ghrelin receptor expression in pituitary. Our data suggests that long-term CR-induced increased ghrelin production from hypertrophic stomach in mice may be an adaptive survival strategy in response to sustained negative energy balance that triggers heightened state of food seeking. Taken together, these data provide new insights into the underlying mechanism behind the salutary effects of chronic caloric restriction during aging process.

Chronic food restriction in young rats results in depression- and anxiety-like behaviors with decreased expression of serotonin reuptake transporter

Evidence of semi-starvation is commonly found in patients with eating disorders. This study was conducted to examine the adverse effects of chronic caloric restriction in young rats, since there have been increasing incidence of eating disorders especially among young populations. Food restriction group was supplied daily with 50% of chow consumed by its ad libitum fed control group from postnatal day 28. After 5 weeks of food restriction, brain contents of serotonin (5-hydroxy-tryptamine; 5-HT) and its metabolite 5-hydroxyindol acetic acid were analyzed by high-performance liquid chromatography and mRNA expression of 5-HT reuptake transporter (5-HTT) by in situ hybridization. Plasma corticosterone levels were determined by radioimmunoassay. Behavioral assessments were performed with Porsolt swim test for depressive behavior and with elevated plus maze test for anxiety. Five weeks of food restriction markedly increased plasma level of corticosterone, and significantly decreased 5-HT turnover rates in the hippocampus and the hypothalamus. 5-HTT mRNA expression decreased in the raphe nucleus of food restricted rats compared with free fed controls. Immobility time during the swim test increased in the food restricted group, compared to the control group. Food restricted rats spent more time in the closed arms, less time in the open arms, of elevated plus maze compared with control rats. These results suggest that chronic caloric restriction in young rats may lead to the development of depressive and/or anxiety disorders, likely, in relation with dysfunction of brain 5-HT system.

Fasting/re‐feeding before initiation enhances the growth of aberrant crypt foci induced by azoxymethane in rat colon and rectum

In contrast to the protective effect of chronic caloric restriction on tumor development, we have shown that fasting sustained tumor initiation in rat liver by a non-initiating dose of diethylnitrosamine. Here we investigated whether fasting had a similar favorable effect on initiation in the colorectal mucosa in 80 male F344 rats. Animals fasted for 4 days were given a single s.c. dose of azoxymethane (AOM) (20 mg/kg) on the first day of re-feeding, and rates of kinetic proliferative parameters, and development of the pre-neoplastic lesions such as aberrant crypt foci (ACF), were evaluated. Starvation before AOM treatment enhanced the growth of ACF, as shown by the significantly higher crypt multiplicity of fasted/re-fed rats as compared with fully fed rats (3.97 ± 0.50 vs. 2.64 ± 0.20, p ≤ 0.025). This difference was associated with perturbations in cell death and cell proliferation. Fasting induced apoptosis and depressed cell division, while re-feeding had opposite effects, resulting in a higher percentage of S-phase cells at the time of AOM injection and 2 days thereafter. Starvation-induced apoptosis may represent the mitogenic stimulus to an increase in the number of cells susceptible to AOM damage, and may favor its fixation, leading to enhanced growth of ACF. Our data therefore suggest that fasting/re-feeding enhances colon cancer. Int. J. Cancer 77:286–294, 1998.© 1998 Wiley-Liss, Inc.

Fasting/re-feeding before initiation enhances the growth of aberrant crypt foci induced by azoxymethane in rat colon and rectum.

In contrast to the protective effect of chronic caloric restriction on tumor development, we have shown that fasting sustained tumor initiation in rat liver by a noninitiating dose of diethylnitrosamine. Here we investigated whether fasting had a similar favorable effect on initiation in the colorectal mucosa in 80 male F344 rats. Animals fasted for 4 days were given a single s.c. dose of azoxymethane (AOM) (20 mg/kg) on the first day of re-feeding, and rates of kinetic proliferative parameters, and development of the pre-neoplastic lesions such as aberrant crypt foci (ACF), were evaluated. Starvation before AOM treatment enhanced the growth of ACF, as shown by the significantly higher crypt multiplicity of fasted/re-fed rats as compared with fully fed rats (3.97 +/- 0.50 vs. 2.64 +/- 0.20, p < or = 0.025). This difference was associated with perturbations in cell death and cell proliferation. Fasting induced apoptosis and depressed cell division, while re-feeding had opposite effects, resulting in a higher percentage of S-phase cells at the time of AOM injection and 2 days thereafter. Starvation-induced apoptosis may represent the mitogenic stimulus to an increase in the number of cells susceptible to AOM damage, and may favor its fixation, leading to enhanced growth of ACF. Our data therefore suggest that fasting/re-feeding enhances colon cancer.

Effects of aging and caloric restriction on extracellular matrix biosynthesis in a model of injury repair in rats.

This study was designed to investigate the effects of aging and caloric restriction on collagen deposition in a model of wound healing. Polyvinyl alcohol sponges were implanted in Fischer 344 rats aged 6, 10, or 24 months. Sponges were removed after 7, 10, or 14 days and analyzed for hydroxyproline content and for enzymatic crosslinks. Aging was associated with a decrease in collagen accumulation as well as a delay in reaching peak rates of collagen accumulation. Levels of enzymatic crosslinks were increased in granulation tissue from old animals relative to young animals. Caloric restriction (60% of ad libitum intake) maintained only during the 2-week experimental period did not affect collagen accumulation, but did result in decreased levels of the difunctional crosslink dihydroxylysinonor-leucine (DHLNL) in sponges implanted for 10 days, but not 14 days. Caloric restriction initiated 4 months before sponges were implanted resulted in decreased collagen accumulation and decreases in DHLNL at all implantation times. These studies suggest that: (1) aging affects total collagen production as well as rate of collagen production in response to injury; (2) aging is not associated with impaired crosslinking in granulation tissue; (3) chronic caloric restriction is associated with decreased collagen production and decreased levels of DHLNL; (4) the adverse effects of chronic caloric restriction on wound healing may have a greater impact on wound healing in old animals.

Effect of chronic caloric restriction: Physiological and behavioral response to alternate day feeding in old female B6C3F1 mice

Physiological and behavioral performance was monitored in old (24 month) female B6C3F1 hybrid mice that were fed either a caloric restricted (CR) diet (60% of ad libitum) or fed ad libitum (AL). The CR group was fed (CR1) and then fasted (CR2) on alternate days starting at 12–14 weeks of age. The main objective of the study was to determine if the effects of intermittent feeding were different from those for daily CR feeding. The average daily body weight, body temperature, oxygen consumption per gram of lean body mass (LBM), and respiratory quotient were higher on the feed day (CR1) than on the fast day (CR2), whereas the average daily body weight, temperature, activity, and oxygen metabolism per gram of LBM were lower on CR1 than in AL. The daily range in temperature, oxygen metabolism per gram of LBM, and RQ was greater on CR1 and CR2 than in AL; the exception was RQ on CR1. No food was eaten on CR2, and water consumption was reduced by 60% compared to CR1. Average food consumption per meal, and average time eating and drinking per meal was greater in CR mice than in AL mice. The results of this study indicated that mice responded to alternate day CR by changing average daily metabolic output per gram of LBM in direct response to food consumption, whereas when CR mice were fed daily, no change was seen in average metabolism per gram of LBM. The fact that a significant reduction in energy output and temperature occurred in CR mice when metabolic pathways shifted from carbohydrate to fat metabolism may indicate that decreased glucose utilization may have triggered these CR-induced effects.

Effect of Chronic Caloric Restriction on the Circadian Regulation of Physiological and Behavioral Variables in Old Male B6C3F1Mice

The circadian rhythms of food and water consumption, the number of feeding and drinking episodes, oxygen consumption, carbon dioxide production, respiratory quotient, gross motor activity, and body temperature were measured in male B6C3F1 mice that were fed ad libitum (AL) or fed a caloric-restricted diet (CR). The CR regimen (60% of the normal AL consumption) was fed to mice during the daytime (5 hr after lights on). CR animals exhibited fewer feeding episodes but consumed more food per feeding bout and spent more total time feeding than AL mice. It appears that CR caused mice to change from their normal "nibbling behavior" to meal feeding. Compared to AL animals, the mean body temperature was reduced in CR animals, while the amplitude of the body temperature rhythm was increased. Spans of reduced activity, metabolism, and body temperature (torpor) occurred in CR mice for several hours immediately before feeding, during times of high fatty acid metabolism (low RQ). The acute availability of exogenous substrates (energy supplies) seemed to modulate metabolism shifting metabolic pathways to promote energy efficiency. CR was also associated with lower DNA damage, higher DNA repair, and decreased proto-oncogene expression. Most of the circadian rhythms studied seemed to be synchronized primarily to the feeding rather than the photoperiod cycle. Night-time CR feeding was found to be better than daytime feeding because the circadian rhythms for AL and AR animals were highly synchronized when this regimen was used.

Effect of Chronic Caloric Restriction on the Circadian Regulation of Physiological and Behavioral Variables in Old Male B6C3F1 Mice

The circadian rhythms of food and water consumption, the number of feeding and drinking episodes, oxygen consumption, carbon dioxide production, respiratory quotient, gross motor activity, and body temperature were measured in male B6C3F, mice that were fed ad libitum (AL) or fed a caloric-restricted diet (CR). The CR regimen (60% of the normal AL consumption) was fed to mice during the daytime (5 hr after lights on). CR animals exhibited fewer feeding episodes but consumed more food per feeding bout and spent more total time feeding than AL mice. It appears that CR caused mice to change from their normal “nibbling behavior” to meal feeding. Compared to AL animals, the mean body temperature was reduced in CR animals, while the amplitude of the body temperature rhythm was increased. Spans of reduced activity, metabolism, and body temperature (torpor) occurred in CR mice for several hours immediately before feeding, during times of high fatty acid metabolism (low RQ). The acute availability of exogenous substrates (energy supplies) seemed to modulate metabolism shifting metabolic pathways to promote energy efficiency. CR was also associated with lower DNA damage, higher DNA repair, and decreased proto-oncogene expression. Most of the circadian rhythms studied seemed to be synchronized primarily to the feeding rather than the photoperiod cycle. Night-time CR feeding was found to be better than daytime feeding because the circadian rhythms for AL and CR animals were highly synchronized when this regimen was used.

Effect of Chronic Caloric Restriction on the Synchronization of Various Physiological Measures in Old Female Fischer 344 Rats

A variety of physiological and behavioral parameters which relate to metabolism were continuously monitored in 18 month old female Fischer 344 rats which were maintained on either ad libitum or reduced calorie diets. Caloric restriction (CR) stimulated average motor activity per day, the duration of each feeding episode, food consumed per episode, and water consumed per gram lean body mass (LBM). However, CR limited total food consumption, feeding time, number of feeding episodes per day, total eating and drinking time, and the daily ratio of food consumed to water consumed, CR also decreased average body temperature per day, O2 consumption, CO2 production, and respiratory quotient. A variety of parameters concerning water consumption were not affected. CR rats ate their food immediately when food was presented during the light span, while ad libitum fed animals ate numerous small meals throughout the entire dark span. An anticipatory response to restricted feeding was also noted. Total motor activity, metabolism, and body temperature increased just prior to scheduled feeding and reached maximum values shortly after feeding, suggesting that these parameters were highly synchronized to feeding. Females and males were found to respond to caloric restriction in a similar fashion. Dramatic changes in respiratory quotient and body temperature suggest rapid shifts between metabolic pathways (glycolysis to gluconeogenesis) to obtain optimal efficiency. Lower body temperature and metabolism may provide protection against DNA damage, thereby increasing the survival potential of restricted rats. These responses may provide insight into the mechanisms by which caloric restriction acts to extend life span.

Effect of chronic caloric restriction on hepatic enzymes of intermediary metabolism in the male Fischer 344 rat

It is well established that caloric restriction extends life span and significantly retards the rate of occurrence of most age-associated degenerative disease processes. A paucity of data exists relative to the mechanisms by which caloric restriction accomplishes these events. We have examined the effect of caloric restriction in rats on several hepatic enzymes of intermediary metabolism. The activities of glycolytic and supporting enzymes including lactate dehydrogenase, pyruvate kinase, sorbitol dehydrogenase, and alcohol dehydrogenase were all decreased in response to caloric restriction. Fructose 1-phosphate aldolase and creatine phpshokinase were not altered. Likewise, enzymes associated with lipid metabolism (malic enzyme and glycerokinase) were reduced (fatty acid synthetase was reduced, but not to a statistically significant degree). Activities of enzymes supporting gluconeogenesis (glutamate oxaloacetate transaminase, tyrosine aminotransferase, glutamate pyruvate transaminase, glutamate dehydrogenase, amino acid oxidase, malate dehydrogenase, and glucose 6-phosphatase) were either unchanged or increased significantly by caloric restriction. Glucagon levels were decreased. Comparisons between young ad libitum fed and older calorically restricted rats revealed similar but not identical metabolic activity. These results suggest that caloric restriction produces an effect on intermediary metabolism, favoring the role of glucagon and glucose synthesis; but limiting the role of insulin and glucose catabolism in the liver. The former observation provides for the efficient support of peripheral tissues and the latter a level of energy production necessary only for self maintenance. Limited lipid metabolism suggests decreased potential for fatty acid epoxide formation and free radical damage to cellular macromolecules. Additionally, caloric restriction may delay the progressive age associated changes in the activities of some of the enzymes investigated.

Effect of chronic caloric restriction on physiological variables related to energy metabolism in the male Fischer 344 rat

In the present study, a number of physiological and behavioral measures that are related to metabolism were continuously monitored in 19-month-old male Fischer 344 rats that were fed ad libitum or fed a caloric restricted diet. Caloric restricted rats ate fewer meals but consumed more food during each meal and spent more time eating per meal than did rats fed ad libitum. Therefore, the timing and duration of meals as well as the total number of calories consumed may be associated with life extension. Average body temperature per day was significantly lower in restricted rats but body temperature range per day and motor activity were higher in restricted rats. Dramatic changes in respiratory quotient, indicating rapid changes in metabolic pathway and lower temperature, occurred in caloric restricted rats when carbohydrate reserves were depleted. Lower body temperature and metabolism during this time interval may result in less DNA damage, thereby increasing the survival potential of restricted rats. Nighttime feeding was found to synchronize physiological performance between ad libitum and caloric restricted rats better than daytime feeding, thereby allowing investigators to distinguish the effects of caloric restriction from those related solely to the time-of-day of feeding.