Protective Effects Of Calorie Restriction Research Articles

Calorie restriction activates a gastric Notch-FOXO1 pathway to expand ghrelin cells.

Calorie restriction increases lifespan. Among the tissue-specific protective effects of calorie restriction, the impact on the gastrointestinal tract remains unclear. We report increased numbers of chromogranin A-positive (+), including orexigenic ghrelin+ cells, in the stomach of calorie-restricted mice. This effect was accompanied by increased Notch target Hes1 and Notch ligand Jag1 and was reversed by blocking Notch with DAPT, a gamma-secretase inhibitor. Primary cultures and genetically modified reporter mice show that increased endocrine cell abundance is due to altered Lgr5+ stem and Neurog3+ endocrine progenitor cell proliferation. Different from the intestine, calorie restriction decreased gastric Lgr5+ stem cells, while increasing a FOXO1/Neurog3+ subpopulation of endocrine progenitors in a Notch-dependent manner. Further, activation of FOXO1 was sufficient to promote endocrine cell differentiation independent of Notch. The Notch inhibitor PF-03084014 or ghrelin receptor antagonist GHRP-6 reversed the phenotypic effects of calorie restriction in mice. Tirzepatide additionally expanded ghrelin+ cells in mice. In summary, calorie restriction promotes Notch-dependent, FOXO1-regulated gastric endocrine cell differentiation.

Calorie restriction anti-hypertrophic effects are associated with improved mitochondrial content, blockage of Ca2+-induced mitochondrial damage, and lower reverse electron transport-mediated oxidative stress

Calorie restriction is a nutritional intervention that reproducibly protects against the maladaptive consequences of cardiovascular diseases. Pathological cardiac hypertrophy leads to cellular growth, dysfunction (with mitochondrial dysregulation), and oxidative stress. The mechanisms behind the cardiovascular protective effects of calorie restriction are still under investigation. In this study, we show that this dietetic intervention prevents cardiac protein elevation, avoids fetal gene reprogramming (atrial natriuretic peptide), and blocks the increase in heart weight per tibia length index (HW/TL) seen in isoproterenol-induced cardiac hypertrophy. Our findings suggest that calorie restriction inhibits cardiac pathological growth while also lowering mitochondrial reverse electron transport-induced hydrogen peroxide formation and improving mitochondrial content. Calorie restriction also attenuated the opening of the Ca2+-induced mitochondrial permeability transition pore. We also found that calorie restriction blocked the negative correlation of antioxidant enzymes (superoxide dimutase and glutatione peroxidase activity) and HW/TL, leading to the maintenance of protein sulphydryls and glutathione levels. Given the nature of isoproterenol-induced cardiac hypertrophy, we investigated whether calorie restriction could alter cardiac beta-adrenergic sensitivity. Using isolated rat hearts in a Langendorff system, we found that calorie restricted hearts have preserved beta-adrenergic signaling. In contrast, hypertrophic hearts (treated for seven days with isoproterenol) were insensitive to beta-adrenergic activation using isoproterenol (50 nM). Despite protecting against cardiac hypertrophy, calorie restriction did not alter the lack of responsiveness to isoproterenol in isolated hearts harvested from isoproterenol-treated rats. These results suggest (through a series of mitochondrial, oxidative stress, and cardiac hemodynamic studies) that calorie restriction possesses beneficial effects against hypertrophic cardiomyopathy.

Protective effects of calorie restriction and 17-β estradiol on cardiac hypertrophy in ovariectomized obese rats.

Obesity and menopause lead to cardiovascular diseases. Calorie restriction (CR) can modulate estrogen deficiency and obesity-related cardiovascular diseases. The protective effects of CR and estradiol on cardiac hypertrophy in ovariectomized obese rats were explored in this study. The adult female Wistar rats were divided into sham and ovariectomized (OVX) groups that received a high-fat diet (60% HFD) or standard diet (SD) or 30% CR for 16 weeks, and then, 1mg/kg E2 (17-β estradiol) was injected intraperitoneally every 4 days for four weeks in OVX-rats. Hemodynamic parameters were evaluated before and after each diet. Heart tissues were collected for biochemical, histological, and molecular analysis. HFD consumption led to weight gain in sham and OVX rats. In contrast, CR and E2 led to body weight loss in these animals. Also, heart weight (HW), heart weight/body weight (HW/BW) ratio, and left ventricular weight (LVW) were enhanced in OVX rats that received SD and HFD. E2 reduced these indexes in both diet conditions but reduction effects of CR were seen only in HFD groups. HFD and SD feeding increased hemodynamic parameters, ANP (atrial natriuretic peptide) mRNA expression, and TGF-β1(transforming growth factor-beta 1) protein level in the OVX animals, while CR and E2 reduced these factors. Cardiomyocyte diameter and hydroxyproline content were increased in the OVX-HFD groups. Nevertheless, CR and E2 decreased these indicators. The results showed that CR and E2 treatment reduced obesity-induced-cardiac hypertrophy in ovariectomized groups (20% and 24% respectively). CR appears to have almost as reducing effects as estrogen therapy on cardiac hypertrophy. The findings suggest that CR can be considered a therapeutic candidate for postmenopausal cardiovascular disease.

Calorie Restriction Provides Kidney Ischemic Tolerance in Senescence-Accelerated OXYS Rats

Kidney diseases belong to a group of pathologies, which are most common among elderly people. With age, even outwardly healthy organisms start to exhibit some age-related changes in the renal tissue, which reduce the filtration function of kidneys and increase the susceptibility to injury. The therapy of acute kidney injury (AKI) is aggravated by the absence of targeted pharmacotherapies thus yielding high mortality of patients with AKI. In this study, we analyzed the protective effects of calorie restriction (CR) against ischemic AKI in senescence-accelerated OXYS rats. We observed that CR afforded OXYS rats with significant nephroprotection. To uncover molecular mechanisms of CR beneficial effects, we assessed the levels of anti- and proapoptotic proteins of the Bcl-2 family, COX IV, GAPDH, and mitochondrial deacetylase SIRT-3, as well as alterations in total protein acetylation and carbonylation, mitochondrial dynamics (OPA1, Fis1, Drp1) and kidney regeneration pathways (PCNA, GDF11). The activation of autophagy and mitophagy was analyzed by LC3 II/LC3 I ratio, beclin-1, PINK-1, and total mitochondrial protein ubiquitination. Among all considered protective pathways, the improvement of mitochondrial functioning may be suggested as one of the possible mechanisms for beneficial effects of CR.

Protective effects of calorie restriction on insulin resistance and islet function in STZ-induced type 2 diabetes rats

BackgroundCaloric restriction (CR) has become increasingly attractive in the treatment of type 2 diabetes mellitus (T2DM) because of the increasingly common high-calorie diet and sedentary lifestyle. This study aimed to evaluate the role of CR in T2DM treatment and further explore its potential molecular mechanisms.MethodsSixty male Sprague–Dawley rats were used in this study. The diabetes model was induced by 8 weeks of high-fat diet (HFD) followed by a single dose of streptozotocin injection (30 mg/kg). Subsequently, the diabetic rats were fed HFD at 28 g/day (diabetic control) or 20 g/day (30% CR regimen) for 20 weeks. Meanwhile, normal rats fed a free standard chow diet served as the vehicle control. Body mass, plasma glucose levels, and lipid profiles were monitored. After diabetes-related functional tests were performed, the rats were sacrificed at 10 and 20 weeks, and glucose uptake in fresh muscle was determined. In addition, western blotting and immunofluorescence were used to detect alterations in AKT/AS160/GLUT4 signaling.ResultsWe found that 30% CR significantly attenuated hyperglycemia and dyslipidemia, leading to alleviation of glucolipotoxicity and thus protection of islet function. Insulin resistance was also markedly ameliorated, as indicated by notably improved insulin tolerance and homeostatic model assessment for insulin resistance (HOMA-IR). However, the improvement in glucose uptake in skeletal muscle was not significant. The upregulation of AKT/AS160/GLUT4 signaling in muscle induced by 30% CR also attenuated gradually over time. Interestingly, the consecutive decrease in AKT/AS160/GLUT4 signaling in white adipose tissue was significantly reversed by 30% CR.ConclusionCR (30%) could protect islet function from hyperglycemia and dyslipidemia, and improve insulin resistance. The mechanism by which these effects occurred is likely related to the upregulation of AKT/AS160/GLUT4 signaling.

Roles of adiponectin and leptin signaling-related microRNAs in the preventive effects of calorie restriction in mammary tumor development.

Calorie restriction (CR) is suggested to prevent the development of mammary tumors (MTs); however, the mechanism remains to be clarified. We aimed to determine the microRNA (miRNA) profile in mice applied to 2 different CR protocols; chronic (CCR) and intermittent (ICR) and follow the MT development. In addition, the roles of miRNAs involved in adiponectin and/or leptin signaling pathways were investigated. Mice were divided into 3groups: ad-libitum (AL), CCR, or ICR, which comprised 3 weeks of AL feeding followed by 1 week of 60% CR in a cyclic manner. Blood and tissue collection were performed at weeks 10, 17/18, 49/50 and 81/82. Long-term CCR provided better protection compared with ICR for MT development with a delay in the MT occurrence. Adiponectin expression in mammary fat pad were significantly higher in CCR group compared with AL. Using GeneChip Array, 250 of 3195 miRNAs were differentially expressed among the dietary groups. Thirteen of 250 miRNAs were related to adiponectin and/or leptin signaling genes. Results were verified by reverse transcription polymerase chain reaction. Specifically, miR-326-3p, miR-500-3p and miR-129-5p, which are related to adiponectin and/or leptin signaling, may play important roles in the preventive effects of CR in MT development and in ageing. Thus, these miRNAs might be putative biomarkers to target for diagnostic and treatment purposes. Novelty: Type of CR and micro RNA interaction is related to ageing. miR-326-3p, miR-500-3p and miR-129-5p expression levels were differentially expressed in MT development and in ageing. The genes associated with adiponectin and/or leptin signaling pathways are regulated by certain miRNAs in the protective effects of CR.

Calorie Restriction Protects against Contrast-Induced Nephropathy via SIRT1/GPX4 Activation.

Calorie restriction (CR) extends lifespan and increases resistance to multiple forms of stress, including renal ischemia-reperfusion (I/R) injury. However, whether CR has protective effects on contrast-induced nephropathy (CIN) remains to be determined. In this study, we evaluated the therapeutic effects of CR on CIN and investigated the potential mechanisms. CIN was induced by the intravenous injection of iodinated contrast medium (CM) iopromide (1.8 g/kg) into Sprague Dawley rats with normal food intake or 40% reduced food intake, 4 weeks prior to iopromide administration. We found that CR was protective of CIN, assessed by renal structure and function. CM increased apoptosis, reactive oxygen species (ROS), and inflammation in the renal outer medulla, which were decreased by CR. The silent information regulator 1 (SIRT1) participated in the protective effect of CR on CIN, by upregulating glutathione peroxidase 4 (GPX4), a regulator of ferroptosis, because this protective effect was reversed by EX527, a specific SIRT1 antagonist. Our study showed that CR protected CIN via SIRT1/GPX4 activation. CR may be used to mitigate CIN.

Targeting hepatocyte carbohydrate transport to mimic fasting and calorie restriction.

The pervasion of three daily meals and snacks is a relatively new introduction to our shared experience and is coincident with an epidemic rise in obesity and cardiometabolic disorders of overnutrition. The past two decades have yielded convincing evidence regarding the adaptive, protective effects of calorie restriction (CR) and intermittent fasting (IF) against cardiometabolic, neurodegenerative, proteostatic, and inflammatory diseases. Yet, durable adherence to intensive lifestyle changes is rarely attainable. New evidence now demonstrates that restricting carbohydrate entry into the hepatocyte by itself mimics several key signaling responses and physiological outcomes of IF and CR. This discovery raises the intriguing proposition that targeting hepatocyte carbohydrate transport to mimic fasting and caloric restriction can abate cardiometabolic and perhaps other fasting-treatable diseases. Here, we review the metabolic and signaling fates of a hepatocyte carbohydrate, identify evidence to target the key mediators within these pathways, and provide rationale and data to highlight carbohydrate transport as a broad, proximal intervention to block the deleterious sequelae of hepatic glucose and fructose metabolism.

Cognitive Impact of Calorie Restriction: A Narrative Review

The impairment of cognitive function can cause substantial emotional and financial burdens. A recent global increasing trend in cognitive impairment and associated disorders has been observed, which will continue to grow as the population ages rapidly. As a nonpharmaceutical approach, calorie restriction (CR) has received extensive research interests due to its health benefits, including maintaining cognitive function. In this narrative review, we first briefly introduce the role of cognitive function in activities of daily living and CR as a part of healthy lifestyle behaviors to protect against cognitive decline. Second, we present results from human studies demonstrating that CR might be beneficial for improving age-related cognitive decline and cognitive impairment in the clinical population such as obesity and type 2 diabetes. Third, the potential mechanisms regarding the protective effects of CR on cognition are discussed. Fourth, specific suggestions are highlighted to be considered in future human studies. Overall, although there are few data available from human studies, CR appears to be beneficial for cognitive protection for both healthy and clinical populations. Further scientific investigations are needed before a firm conclusion can be made.

Calorie restriction attenuates hypertrophy-induced redox imbalance and mitochondrial ATP-sensitive K+ channel repression

Oxidative stress has been implicated in the pathogenesis of cardiac hypertrophy and associated heart failure. Cardiac tissue grows in response to pressure or volume overload, leading to wall thickening or chamber enlargement. If sustained, this condition will lead to a dysfunctional cardiac tissue and oxidative stress. Calorie restriction (CR) is a powerful intervention to improve health and delay aging. Here, we investigated whether calorie restriction in mice prevented isoproterenol-induced cardiac hypertrophy in vivo by avoiding reactive oxygen species (ROS) production and maintaining antioxidant enzymatic activity. Additionally, we investigated the involvement of mitochondrial ATP-sensitive K+ channels (mitoKATP) in cardiac hypertrophy. CR was induced by 40% reduction in daily calorie ingestion. After 3 weeks on CR or ad libitum (Control) feeding, Swiss mice were treated intraperitoneally with isoproterenol (30 mg/kg per day) for 8 days to induce hypertrophy. Isoproterenol-treated mice had elevated heart weight/tibia length ratios and cardiac protein levels. These gross hypertrophic markers were significantly reduced in CR mice. Cardiac tissue from isoproterenol-treated CR mice also produced less H2O2 and had lower protein sulfydryl oxidation. Additionally, calorie restriction blocked hypertrophic-induced antioxidant enzyme (catalase, superoxide dismutase and glutathione peroxidase) activity repression during cardiac hypertrophy. MitoKATP opening was repressed in isolated mitochondria from hypertrophic hearts, in a manner sensitive to calorie restriction. Finally, mitoKATP inhibition significantly blocked the protective effects of calorie restriction. Altogether, our results suggest that CR improves intracellular redox balance during cardiac hypertrophy and prevents this process in a mechanism involving mitoKATP activation.

Abstract 4986: Association of sirtuins and diet in cancer development: Studying the roles of SIRT2/3

Abstract Lifestyle factors, especially diet, affects health and lifespan. Dietary restriction (DR) has been shown as a beneficial regimen to increase longevity and protect from cancer. On the other hand, high fat intake has been found to be associated with greater risk of several cancer types. In this regard, several studies suggest that the beneficial effect of calorie restriction (CR) is mediated by the increased activity of sirtuins, a family of NAD-dependent deacetylases that plays a role in aging and age-related diseases. However, the specific role of individual family members in the inhibitory role of CR in tumorigenesis has not been elucidated. With the exception of SIRT1, there is no evidence for the contribution of other sirtuins in the diet-induced effects on tumorigenesis. In this study, we investigated, for the first time, the role of SIRT2 and SIRT3, which are found predominantly in the cytoplasm and mitochondria respectively, in this context. To do this, Sirt2-/- and Sirt3-/- mice were crossed with p53-/- mice to generate: p53+/-, (Sirt2-/-; p53+/-), (Sirt3-/-; p53+/-), p53-/-, (Sirt2-/-; p53-/-), and (Sirt3-/-; p53-/-) mice. Subsequently, mice with the different genotypes were subjected to 3 dietary regimes; ad libitum control diet, 30% calorie restriction diet (CR), and high-fat diet (HFD). As expected, CR reduced tumor incidence and increased survival of mice wild type for both SIRT2 and SIRT3. In contrast, HFD significantly increased body weight, tumor incidence and decreased overall survival. Interestingly, the protective effect of CR was enhanced in (Sirt3-/-; p53-/-) compared to (Sirt3+/+; p53-/-) mice, as evidenced by delayed tumor incidence (median age 257 days vs 189 days) and increased survival. On the other hand, Sirt2 deletion in both p53+/- and p53-/- backgrounds abolished the protective effect of CR. In addition, loss of Sirt2 decreased tumor incidence age (111 days vs 184 days) and accelerated the death of p53 deficient mice on HFD. Our results highlight a new role of SIRT2 in mediating the diet-induced effect on tumorigenesis. Citation Format: Mohamed A. Ahmed, Carol O'Callaghan, Athanasios Vassilopulos. Association of sirtuins and diet in cancer development: Studying the roles of SIRT2/3 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4986.

Calorie restriction improves brain health in aging rhesus macaques

Calorie restriction (CR) without malnutrition slows the aging and disease process, prolonging median and maximum lifespan in yeast, worms, fish, flies, and rodents. Studies of CR in non-human primates (Macaca mulatta) indicate a protective effect of CR against aging-related diseases and a salutary effect on brain health. Our group has studied the specific effects of CR on the non-human primate brain using neuroimaging, behavioral, and post-mortem histological techniques and this talk will provide an overview of these findings. Animals in this study were part of the longitudinal “Dietary Restriction and Aging Study” at the Wisconsin National Primate Research Center (WNPRC). Animals were either fed a normal diet or maintained on a moderately restricted diet (approximately 30% reduced intake from baseline), with both groups receiving comparable diet supplements. Using volumetric imaging we demonstrated that CR preserves gray matter cortex in limbic and heteromodal association areas, indicating a positive effect of CR against age-related brain atrophy. Diffusion tensor imaging showed preservation of white matter integrity in the corpus callosum and fronto-occipital fasciculus, superior longitudinal fasciculus, external capsule, and brainstem. CR also attenuated age-related iron accumulation in the basal ganglia, red nucleus, and parietal, temporal, and perirhinal cortex. Decreased iron accumulation was in turn associated with faster performance on fine motor function tests, signifying a protective effect against motor slowness that results during aging. CR moderated the effect of important plasma-based inflammatory markers (e.g. IL-6) on gray and white matter changes in several brain areas, including the parietal and temporal gray matter regions that are sensitive to aging. CR improved glucoregulatory profiles and positively influenced gray matter volume in the hippocampus. Histopathology studies reveal that CR monkeys express significantly lower (∼30%) levels of microglial activation in the hippocampus. Energy metabolism in the hippocampus as indexed by PGC1alpha and GSK3B was preserved in CR. Number of MTL amyloid plaques was however equivalent between groups. Overall, these results recapitulate the neuroprotective effects of CR from lower animal models. Taken together, these findings point to an overall beneficial effect of CR on the brain in this non-human primate model of aging.

Calorie Restriction Attenuates Monocrotaline-induced Pulmonary Arterial Hypertension in Rats.

:Calorie restriction (CR) is one of the most effective nonpharmacological interventions protecting against cardiovascular disease, such as hypertension in the systemic circulation. However, whether CR could attenuate pulmonary arterial hypertension (PAH) is largely unknown. The PAH model was developed by subjecting the rats to a single subcutaneous injection of monocrotaline. CR lowered mean pulmonary arterial pressure (mPAP) and reduced vascular remodeling and right ventricular hypertrophy in PAH rats. Meanwhile, CR attenuated endothelial dysfunction as evidenced by increased relaxation in response to acetylcholine. The beneficial effects of CR were associated with restored sirtuin-1 (SIRT1) expression and endothelial nitric oxide synthase (eNOS) phosphorylation and reduced eNOS acetylation in pulmonary arteries of PAH rats. To further clarify the role of SIRT1 in the protective effects of CR, adenoviral vectors for overexpression of SIRT1 were administered intratracheally at 1 day before monocrotaline injection. Overexpression of SIRT1 exhibited similar beneficial effects on mPAP and endothelial function, and increased eNOS phosphorylation and reduced eNOS acetylation in the absence of CR. Moreover, SIRT1 overexpression attenuated the increase in mPAP in hypoxia-induced PAH animals. Overall, the present data demonstrate that CR may serve as an effective treatment of PAH, and targeting the SIRT1/eNOS pathway may improve treatment of PAH.

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.

Dietary fat modifies mitochondrial and plasma membrane apoptotic signaling in skeletal muscle of calorie-restricted mice

Calorie restriction decreases skeletal muscle apoptosis, and this phenomenon has been mechanistically linked to its protective action against sarcopenia of aging. Alterations in lipid composition of membranes have been related with the beneficial effects of calorie restriction. However, no study has been designed to date to elucidate if different dietary fat sources with calorie restriction modify apoptotic signaling in skeletal muscle. We show that a 6-month calorie restriction decreased the activity of the plasma membrane neutral sphingomyelinase, although caspase-8/10 activity was not altered, in young adult mice. Lipid hydroperoxides, Bax levels, and cytochrome c and AIF release/accumulation into the cytosol were also decreased, although caspase-9 activity was unchanged. No alterations in caspase-3 and apoptotic index (DNA fragmentation) were observed, but calorie restriction improved structural features of gastrocnemius fibers by increasing cross-sectional area and decreasing circularity of fibers in cross sections. Changing dietary fat with calorie restriction produced substantial alterations of apoptotic signaling. Fish oil augmented the protective effect of calorie restriction decreasing plasma membrane neutral sphingomyelinase, Bax levels, caspase-8/10, and -9 activities, while increasing levels of the antioxidant coenzyme Q at the plasma membrane, and potentiating the increase of cross-sectional area and the decrease of fiber circularity in cross sections. Many of these changes were not found when we used lard. Our data support that dietary fish oil with calorie restriction produces a cellular anti-apoptotic environment in skeletal muscle with a downregulation of components involved in the initial stages of apoptosis engagement, both at the plasma membrane and the mitochondria.

Sir-2.1 modulates ‘calorie-restriction-mediated’ prevention of neurodegeneration in Caenorhabditis elegans: Implications for Parkinson’s disease

The phenomenon of aging is known to modulate many disease conditions including neurodegenerative ailments like Parkinson’s disease (PD) which is characterized by selective loss of dopaminergic neurons. Recent studies have reported on such effects, as calorie restriction, in modulating aging in living systems. We reason that PD, being an age-associated neurodegenerative disease might be modulated by interventions like calorie restriction. In the present study we employed the transgenic Caenorhabditis elegans model (Pdat-1::GFP) expressing green fluorescence protein (GFP) specifically in eight dopaminergic (DA) neurons. Selective degeneration of dopaminergic neurons was induced by treatment of worms with 6-hydroxy dopamine (6-OHDA), a selective catecholaminergic neurotoxin, followed by studies on effect of calorie restriction on the neurodegeneration. Employing confocal microscopy of the dopaminergic neurons and HPLC analysis of dopamine levels in the nematodes, we found that calorie restriction has a preventive effect on dopaminergic neurodegeneration in the worm model. We further studied the role of sirtuin, sir-2.1, in modulating such an effect. Studies employing RNAi induced gene silencing of nematode sir-2.1, revealed that presence of Sir-2.1 is necessary for achieving the protective effect of calorie restriction on dopaminergic neurodegeneration.Our studies provide evidence that calorie restriction affords, an sir-2.1 mediated, protection against the dopaminergic neurodegeneration, that might have implications for neurodegenerative Parkinson’s disease.

Abstract 809: Dietary L-Leucine diminishes protective effects of calorie restriction in a transplant model of pancreatic cancer

Abstract Branched chain amino acids (BCAA), prevalent in soy proteins, are commonly consumed at high levels in the form of food supplements. Of the BCAAs, leucine is the most potent activator of mTOR, a kinase at the crux of energy and growth factor signaling. mTOR is also frequently involved in dysregulated proliferation and survival signals in various cancers. We have previously shown that calorie restriction (CR) exerts potent anti-pancreatic cancer effects through reduced activation of the IGF-1/mTOR pathway. Thus we hypothesized that leucine supplementation would offset the anticancer effects of CR through restoration of mTOR signaling. To test this, 5×105 Panc02 mouse pancreatic cancer cells were subcutaneously injected into C57BL/6 male mice (n=15/group) that were fed one of three diets for 23 weeks beginning 4-6 weeks of age: AIN-76A control fed ad libitum (CON, 12 kcal%fat diet); CR (70% of weekly CON intake); and CR supplemented with L-Leucine (CR+Leu, 91-120 mg/day/mouse). Assigned diets were continued and tumors were palpated weekly for four more weeks (when 50% of CON tumors reached 1 cm in diameter) then tumors were measured ex vivo with calipers. Prior to tumor injection, body composition and insulin sensitivity were assessed by magnetic resonance imaging and a glucose tolerance test, respectively, at week 21 (n=11/group). Levels of energy balance-responsive hormones were assayed using serum collected at week 22 (CR groups, n=10; CON group, n=9). At week 21, the CR and CR+Leu groups had significantly reduced body weight (19.7 ± 0.3g and 21.3 ± 0.3g), lean mass (14.8 ± 0.4g and 15.6 ± 0.3g) and percent body fat (14.1 ± 1.1% and 16.3 ± 0.7%) relative to the CON group (36.0 ± 0.5g, 21.3 ± 0.6g and 35.0 ± 1.0%, p<0.01), respectively. CR and CR+Leu displayed an enhanced rate of glucose clearance relative to the CON diet (p<0.05). CR reduced circulating levels of IGF-1 (22.5 ± 1.2ng/mL), insulin (0.19 ± 0.08ng/mL) and leptin (0.69 ± 0.17ng/mL) relative to CON (61.3 ± 3.3ng/mL, 0.65 ± 0.13ng/mL and 13.3 ± 2.9ng/mL; p<0.01), as did CR+Leu (29.9 ±1.9ng/mL, 0.19 ± 0.06ng/mL, 1.7 ± 0.25ng/mL; p<0.01), respectively. CR and CR+Leu did not differ with respect to these parameters. CR+Leu tumors (103.8 ± 18.4mm3) were significantly larger than CR (46.4 ± 13.4mm3, p<0.05), yet were smaller than CON (229.7 ± 40.1mm3, p<0.05). CR significantly reduced tumor burden relative to CON (p<0.0001). CR+Leu tumors also had higher levels of p-mTOR than CR, but not CON. We found that supplementation with L-Leucine blunts the anti-cancer effects of a CR diet in association with increases in mTOR signaling but does not alter other factors typically associated with the protective properties of CR (body composition, insulin sensitivity, and serum hormone levels). Therefore, our findings suggest that excessive protein supplementation using L-Leucine should be used with caution among those at high risk for developing pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 809. doi:10.1158/1538-7445.AM2011-809

Calorie Restriction Reduces Oxidative Stress by SIRT3-Mediated SOD2 Activation

A major cause of aging and numerous diseases is thought to be cumulative oxidative stress, resulting from the production of reactive oxygen species (ROS) during respiration. Calorie restriction (CR), the most robust intervention to extend life span and ameliorate various diseases in mammals, reduces oxidative stress and damage. However, the underlying mechanism is unknown. Here, we show that the protective effects of CR on oxidative stress and damage are diminished in mice lacking SIRT3, a mitochondrial deacetylase. SIRT3 reduces cellular ROS levels dependent on superoxide dismutase 2 (SOD2), a major mitochondrial antioxidant enzyme. SIRT3 deacetylates two critical lysine residues on SOD2 and promotes its antioxidative activity. Importantly, the ability of SOD2 to reduce cellular ROS and promote oxidative stress resistance is greatly enhanced by SIRT3. Our studies identify a defense program that CR provokes to reduce oxidative stress and suggest approaches to combat aging and oxidative stress-related diseases.

Serum Insulin-like Growth Factor-I and Mammary Tumor Development inAd libitum–Fed, Chronic Calorie–Restricted, and Intermittent Calorie–Restricted MMTV-TGF-α Mice

The effect of chronic (CCR) and intermittent (ICR) caloric restriction on serum insulin-like growth factor (IGF)-I levels and mammary tumor (MT) development was investigated. Ten-week-old MMTV-TGF-alpha female mice were assigned to ad libitum-fed (AL; AIN-93M diet), ICR [3-week 50% caloric restriction using AIN-93M-mod diet, 2x protein, fat, vitamins, and minerals followed by 3 weeks of daily 100% AL consumption of AIN-93M ( approximately 75% of AL for each 6-week cycle)], and CCR (calorie and nutrient intake matched for each 6-week ICR cycle) groups. Half of the mice from each group were sacrificed at 79 (end of restriction) or 82 (end of refeeding) weeks of age. Serum was obtained at euthanasia and in cycles 1, 3, 5, 8, and 11. MT incidence was 71.0%, 35.4%, and 9.1% for AL, CCR, and ICR mice. ICR-Restricted mice had significantly lower terminal serum IGF-I and IGF-I/IGF binding protein-3 (IGFBP-3) ratio than CCR, ICR-Refed, and AL mice. There were no differences in terminal IGFBP-3. Final body, internal, and mammary fat pad weights correlated positively with IGF-I and negatively with IGFBP-3. Few changes were found for protein expression of IGF-IRalpha and IGFBP-3 in mammary tissue and MTs. During the study, IGF-I levels of ICR-Restricted mice were reduced, whereas refeeding allowed partial recovery. For all groups, elevated IGF-I levels preceded MT detection, although not all values were significant versus mice without MTs. However, the specific role of IGF-I in the protective effect of calorie restriction remains to be determined. These results confirm that ICR prevents MT development to a greater extent than CCR.

Molecular mechanisms of calorie restriction's protection against age-related sclerosis

The current knowledge on the molecular mechanisms of the protective effect of calorie restriction (CR) against age-related fibrosclerosis is tentatively reviewed with specific reference to the role of oxidative stress in aging. The effects of oxidative stress are often mediated by its own final products. Of these, 4-hydroxy-2,3-nonenal (HNE) induces the expression and synthesis of transforming growth factor beta1 (TGFbeta1) and activates nuclear binding of transcription factor activator protein 1 (AP-1) thus stimulating fibrogenesis. Several studies have shown that, as well as extending mean and maximum life span in a variety of species, CR delays the onset and slows the progression of a variety of age-associated diseases, including diabetes, cardiovascular diseases and neoplasia. However, the anti-aging mechanisms of CR are still not clearly understood. Of the numerous hypotheses put forward, one that still remains popular is protection against the age-associated increase of oxidative stress and consequent cell damage. CR protects the rat aorta from the age-related increase of both oxidative damage and fibrosis; as regards the possible mechanism/s of CR's protection against fibrosclerosis, it is conceivable that, by decreasing oxidative stress, CR reduces HNE levels and consequently TGFbeta1 expression and collagen deposition, likely by down-regulating the activation of Jun-N terminal kinase and of AP-1. Through the modulation of reactive oxygen species and oxidative stress CR may also attenuate the age-associated increase in the inflammatory milieu, thus preserving vascular functional integrity by suppressing the age-associated increase in inflammatory enzyme activities and prostanoids.