AM-09: Caloric restriction

Calorie Restriction in the Elderly People

Obesity is associated with an increased risk of chronic, low-grade systematic inflammation for which exercise training (EX) and caloric restriction (CR) are potential treatments. We therefore performed a systematic meta-analysis to compare the effect of EX vs. CR and EX + CR vs. CR on inflammation markers in overweight and obese individuals. PubMed, Scopus, Web of Science and the Cochrane were searched up to April 2020 for EX vs. CR or EX + CR vs. CR interventions studies on inflammatory makers i.e. CRP, IL-6 and TNF-α in overweight and obese individuals. Standardized mean differences and 95% confidence intervals were calculated. Thirty two articles (reporting 38 trials) involving 2108 participants were included in the meta-analysis. Based on studies that directly compared EX and CR, there were no evidence for an effect of EX on IL-6 (p = 0.20) and TNF-α (p = 0.58), when compared with a CR. However, when compared to EX, CR has a statistically greater benefit on CRP (p = 0.01). In those studies, directly comparing EX + CR and CR, EX + CR caused a larger decrease in TNF-α (p = 0.002) and IL-6 (p = 0.02) and tended to decrease CRP (p = 0.06) when compared with CR. These results suggest that a combination of EX and CR may be more effective than CR alone at reducing inflammatory cytokines and CRP in overweight and obese individuals.

Caloric restriction (CR) extends lifespan through a reduction in oxidative stress, delays the onset of morbidity and prolongs lifespan. We previously reported that long-term CR hastened clinical onset, disease progression and shortened lifespan, while transiently improving motor performance in G93A mice, a model of amyotrophic lateral sclerosis (ALS) that shows increased free radical production. To investigate the long-term CR-induced pathology in G93A mice, we assessed the mitochondrial bioenergetic efficiency and oxidative capacity (CS – citrate synthase content and activity, cytochrome c oxidase - COX activity and protein content of COX subunit- I and IV and UCP3- uncoupling protein 3), oxidative damage (MDA – malondialdehyde and PC – protein carbonyls), antioxidant enzyme capacity (Mn-SOD, Cu/Zn-SOD and catalase), inflammation (TNF-α), stress response (Hsp70) and markers of apoptosis (Bax, Bcl-2, caspase 9, cleaved caspase 9) in their skeletal muscle. At age 40 days, G93A mice were divided into two groups: Ad libitum (AL; n = 14; 7 females) or CR (n = 13; 6 females), with a diet equal to 60% of AL. COX/CS enzyme activity was lower in CR vs. AL male quadriceps (35%), despite a 2.3-fold higher COX-IV/CS protein content. UCP3 was higher in CR vs. AL females only. MnSOD and Cu/Zn-SOD were higher in CR vs. AL mice and CR vs. AL females. MDA was higher (83%) in CR vs. AL red gastrocnemius. Conversely, PC was lower in CR vs. AL red (62%) and white (30%) gastrocnemius. TNF-α was higher (52%) in CR vs. AL mice and Hsp70 was lower (62%) in CR vs. AL quadriceps. Bax was higher in CR vs. AL mice (41%) and CR vs. AL females (52%). Catalase, Bcl-2 and caspases did not differ. We conclude that CR increases lipid peroxidation, inflammation and apoptosis, while decreasing mitochondrial bioenergetic efficiency, protein oxidation and stress response in G93A mice.

Multiple mouse models of pancreatic, colon, prostate, skin and breast cancer have shown that calorie restriction (CR) prevents obesity and suppresses tumor development and growth. In normal muscle, studies have shown that mild CR attenuates the age-related impairment of autophagy, cellular damage, and cell death. Autophagy is a survival response in solid tumors that co-localizes with hypoxic regions, allowing tolerance to metabolic stress. Autophagy also protects cells from genome damage and limits both inflammation and cell death as possible means to tumor suppression. Thus, we hypothesized that autophagy plays a major role in CR-mediated tumor inhibition. To test this hypothesis, we utilized immortalized baby mouse kidney epithelium (iBMK) tumor cell lines. iBMK cells were first immortalized by E1A and dominant negative p53 (p54DD) transfection, and rendered apoptosis-deficient by Bcl-2 overexpression. They were then transfected with oncogenic Ras and EGFP-LC3 plasmids. Cell lines were either autophagy deficient through knockout of the essential autophagy gene Atg5 (Atg5 -/-) or were autophagy competent (Atg5 +/+). Female nude mice were fed either control AIN-76A diet ad-libitum or a CR regimen (isonutrient; 30% reduction in calories relative to control group) for 8 weeks prior to subcutaneous flank injection of cell lines (n=15 per group). CR mice were significantly lighter, had decreased body fat percentage, and had lower fasting glucose levels (p<0.001 for all). Additionally, CR mice had lower serum levels of IGF-1 (p<0.001), insulin (p<0.001), leptin (p<0.001), and resistin (p=0.01), and higher serum adiponectin (p<0.001). We found that autophagy competent tumors, transplanted into female nude mice (5x10^4 cells per mouse), grew at significantly different growth rates depending on the diet treatment (Control > CR; p<0.001), while autophagy deficient tumors were unaffected by CR (no difference between control and CR tumors). In order to determine the effect of CR on LC3 localization and activation of autophagy, we repeated the injections in a separate cohort of control and CR mice (n=2 per timepoint). Tumors were collected at 1, 3, 8, and 15 days post-injection. In CR mice injected with autophagy competent cells, we saw LC3 translocate to the autophagosomes, demonstrated by punctate perinuclear localization, as early as 1d post-injection. In contrast, in control mice injected with autophagy competent cells, and in all mice injected with autophagy deficient cells, we saw a diffuse cytoplasmic LC3 localization. In conclusion, we found that CR is capable of activating autophagy as a mechanism of tumor growth suppression and increases LC3 autophagosome localization in tumors. Taken together, these findings suggest that autophagy may play a key role in the anticancer effects of CR and may be an important intervention target for the prevention and treatment of obesity-related cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4104. doi:1538-7445.AM2012-4104

The only non-pharmacological intervention known to date to alleviate the age-related deleterious conditions is caloric restriction (CR). We have previously reported that CR induces metabolic and signaling changes that affect the tumor microenvironment preventing mammary tumors growth and metastases. Despite the beneficial effects of CR, it is not feasible to maintain long-term dietary restriction in humans. Based on this, investigators have been focused on the discovery of CR mimetics that evoke some of the benefits of CR without an actual reduction in the calorie intake. Metformin (MET) is an anti-diabetic and a well-known CR mimetic; while Orlistat (OR) is an anti-obesity drug which has also antitumor properties. In this study we used 4T1, a highly metastatic breast cancer, and the B16F10 melanoma mouse models. At first, we evaluated the effects of CR and MET alone. Then, we investigated whether the combination of MET+OR will enhance the CR mimetic properties of MET alone. Our overall hypothesis is that CR mimetics decrease tumor progression by affecting extracellular matrix, epithelial-to-mesenchymal transition (EMT) and inflammation. Briefly, 8-weeks old female mice were fed with normal diet (ND) or 40% CR and received vehicle, MET (in drinking water, 3mg/ml), OR i.p. injection (240mg/kg/d) or MET+OR. After 5 weeks on diets, 4T1 or B16F10 cells were injected into BALB/c or C57BL6 mice respectively; diets and treatments continued for the remainder of the experiment. CR and Mimetics treated-mice had a reduction in body weight (p<0.01) and adiposity (p<0.05) without any signs of toxicity. CR and mimetics treatments reduced tumor growth (p<0.01), microvessel density, total vessel length (p<0.01) and decreased the number and size of spontaneous lung metastases (p<0.01). Also, CR and mimetics tumor bearing mice had significantly lower levels of intra-tumor uPA, MMP-9 and TGF-β. Moreover, CR and mimetics-treated mice had lower levels of circulating insulin, leptin and increased levels of adiponectin and fibroblast growth factor 21 (FGF21) (p<0.01). In a cell invasion assay, we also showed that MET+OR resulted in a significantly reduction on invasion (p<0.01). It is known that the loss of E-Cadherin promotes metastasis. We observed that CR and treated-mice had greater expression of E-Cadherin compared to the controls; in particular MET+OR had higher expression compared to either agent alone. NFĸB activation, the linker between inflammation and cancer, induces the expression of genes that promote a mesenchymal phenotype. We found that the MET+OR decreased phosphorylated P65S536 in 4T1 tumors, which corresponded with a decreased expression in COX-2 and Bcl-2; and an increased expression of P53 and cleaved caspase-3. Our results suggest that CR and MET+OR reduce tumor growth and metastases may in part be due to NFĸB inhibition and by remodeling the ECM, preventing the loss of E-Cadherin and the local invasion. Citation Format: Anirudh Goyal, Shakir M. Saud, Jessica D. Bedele, Mariana S. De Lorenzo. Caloric restriction mimetics prevent tumor progression by tumor microenvironment remodeling [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 77.

Tumor-associated macrophages (TAMs) are the most abundant inflammatory cells in the breast cancer (BC) tumor microenvironment (TME) and suppress antitumor immune function, contributing to treatment resistance. These M2-polarized macrophages are characterized by fatty acid oxidation as opposed to the glycolytic pathways associated with M1-polarization. MiR-21 is known to affect several metabolic pathways relevant to macrophage polarization. We have previously shown that caloric restriction (CR) can decrease miR-21 expression and increase response to radiation therapy (RT). We propose CR as a means of metabolically reprogramming TAMs toward M1 phenotype to increase efficacy of radiation therapy (RT). To assess the effects of CR in vivo on the composition of the TME in breast cancer, 40 female Balb/c mice were orthotopically injected at 12 weeks old with 4T1 cells and assigned to one of four cohorts: ad lib (AL) fed, 8 Gy RT to the primary tumor (RT), 30% reduction in caloric intake (CR) or CR+RT. MiR-21 levels were significantly decreased in both the CR and CR+RT arms; however, miR-21 was increased in the RT arm. Increased expression of M2 markers was noted in tumors in the RT group as compared to the CR and CR+RT groups, which favored M1 markers. On microarray analysis, the top pathways affected by CR discovered by gene ontology pathway analysis were: inflammation mediated by chemokine and cytokine signaling (31/509 hits), Wnt signaling (21/509 hits) and apoptotic signaling (18/509 hits). Multiple genes related to macrophage polarization were altered by CR including Ccl2 and IL12A. IL-12 is a known target of miR-21, and CR decreases miR-21 expression, therefore we sought to determine if miR-21 may be the mechanism behind the shift in chemokine and cytokine expression in the TME. Cohorts of MMTV-PyMT mice with spontaneously developed breast tumors were bred with a miR-21-/- mouse to generate breast tumors that lack expression of miR-21. These tumors exhibited high levels of IL-12 as well as IL-23 and Cxcl10, all of which are associated with M1 phenotype. MiR-21+/+ tumors exhibited high levels of IL-4 and TGF-β, both associated with M2 phenotype. In addition, peripheral monocytes (PBMCs) were co-cultured with sera from each of the following mice: miR-21-/-, miR-21+/+, AL, RT, CR and CR+RT. M1-polarization was induced by sera from miR-21-/- mice as well as mice receiving CR alone and CR+RT. Ionizing radiation seemed to induce M2-polarization in this manner, as did the miR-21+/+ serum. Taken together, these data suggest that CR can counteract the M2-polarization caused by RT in the TME. This is likely due, in part, to miR-21 downregulation leading to enhanced M1 response. By increasing M1-polarized macrophages and enhancing antitumor immunity, we believe CR is a viable adjunct to standard cytotoxic therapies that could help overcome resistance to chemotherapy and immunotherapy. Citation Format: Brittany A. Simone, Tiziana DeAngelis, Ajay Palagani, Kevin Ko, Larry Harshyne, D. Craig Hooper, Adam P. Dicker, Nicole L. Simone. Caloric restriction promotes antitumor immunity through polarization of intratumoral macrophages to M1 phenotype via downregulation of miR-21 [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 71.

Caloric restriction, gene expression and aging

Caloric restriction and IGF-I administration promote rabbit fecundity: Possible interrelationships and mechanisms of action

Calorie restriction (CR) is promoted to increase longevity, yet this regimen could lead to bone loss and fracture and therefore affect quality of life. Forty-six individuals were randomized to 4 groups for 6 months: (1) healthy diet (control group); (2) 25% CR from baseline energy requirements (CR group); (3) 25% energy deficit by a combination of CR and increased aerobic exercise (CR + EX group); and (4) low-calorie diet (890 kcal/d; goal, 15% weight loss) followed by weight maintenance (LCD group). Bone mineral density (total body and hip by dual-energy x-ray absorptiometry) and serum bone markers (bone-specific alkaline phosphatase, osteocalcin, cross-linked C-telopeptide of type I collagen, and cross-linked N-telopeptide of type I collagen) were measured at baseline and after 6 months. Mean +/- SE body weight was reduced by -1.0% +/- 1.1% (control), -10.4% +/- 0.9% (CR), -10.0% +/- 0.8% (CR + EX), and -13.9% +/- 0.7% (LCD). Compared with the control group, none of the groups showed any change in bone mineral density for total body or hip. Bone resorption by serum cross-linked C-telopeptide of type I collagen was increased in all 3 intervention groups, with the largest change observed in the LCD group (CR, 23% +/- 10%; CR + EX, 22% +/- 9%; and LCD, 74% +/- 16% vs control, 4% +/- 10%). Serum levels of cross-linked N-telopeptide of type I collagen were also increased in the LCD group. With regard to bone formation, bone alkaline phosphatase levels were decreased in the CR group (-23% +/- 10%) but were unchanged in the CR + EX, LCD, and control groups. Moderate CR, with or without exercise, that preserves calcium intake for 6 months leads to large changes in body composition without significant bone loss in young adults. Longer studies with assessments of bone architecture are needed to confirm that CR nutrient-dense diets have no deleterious effect on bone health. clinicaltrials.gov Identifier: NCT00099151.

Backgrounds: It's well known that calorie restriction (CR) brings striking benefits in metabolic improvement with complex and unclear mechanism. In this study, we aimed to investigate the specific manifestation and possible mechanisms of metabolism in CR mice. Methods: B6 mice were randomly assigned to three groups: (1) chow diet (control) , (2) chow diet with 40% calorie restriction (CR) , (3) high-fat diet (HFD) . The metabolism status and related examination were performed after 12 weeks of feeding. Results: In this study, we found that CR increased fasting blood glucose and plasma insulin, as well as reduced insulin sensitivity reflected by insulin tolerance test. Comparing to the control mice, the decrease of phosphorylation levels of IRS-1 including serine307 and tyrosine632 residues, and AKT (threonine308) after insulin rejection further confirmed that CR induced insulin resistance. Different from insulin resistance induced by HFD, CR improved glucose tolerance and reduced gluconeogenesis. Meanwhile, CR led to absolute lipid reduction, manifested in decreased plasma lipid, liver lipid deposition and fat mass. In contrast, liver glycogen increased dramatically. Fasting glycogen was twice as much as that of HFD group and 20 times as that of control group. Furthermore, the CR increased p-PYGL, p-GSK3β and decreased p-GS notably indicating active glycogen synthesis and decomposition. Subsequently, we found that CR increased plasma corticosterone and decreased plasma c-reactive protein (CRP) compared with control and HFD groups, while plasma corticosterone and CRP in HFD were higher than those in control group. Additionally, CR lowered levels of orexigenic neuropeptides and elevated levels of anorexigenic neuropeptides. Conclusions: Our study suggested that CR induced lipid insufficiency and stress from starvation, resulting in physiological insulin resistance and enhanced glycogen metabolism, including increased glycogen synthesis, glycogenolysis and glycogen content. Disclosure L.Hu: None. X.Xia: None. Y.Gu: None. J.Yin: None. Funding National Natural Science Foundation of China (No. 82070885) ,Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support (No. 20172025)

Hungry for life

Influence of long-term caloric restriction on myocardial and cardiomyocyte contractile function and autophagy in mice

Caloric Restriction Impairs Regulatory T cells Within the Tumor Microenvironment After Radiation and Primes Effector T cells.

Calorie restriction (CR) has been shown to delay the onset of age‐related diseases and prolong life spans in a variety of species. CR reduces mitochondrial reactive oxygen species production, attenuates oxidative damage and alters membrane composition towards saturation. This has led to a theory that changes in membrane saturation are central to the actions of CR. As a first step towards testing this theory, C57BL/6 mice were assigned to 3 dietary groups (control and two CR groups) and fed AIN‐93G diets at either 95% (control) or 60% (CR) of ad libitum. To manipulate membrane composition, the primary dietary fat for the CR groups were either soybean oil (also used in the control diet) or fish oil. The diets were fed for 1 month and then liver mitochondria were isolated for measures of mitochondrial lipid composition, proton leak, and H2O2 production. Results indicate that changes in mitochondrial lipids in the CR mice reflected the dietary lipid source. Body weight was lower (p<0.01) in the soy compared to fish CR mice. Mitochondrial H2O2 production was decreased (p<0.01) in the CR fish group compared to the control and CR soy groups regardless of the substrate used for respiration. The results of this study indicate that decreases in long chain unsaturated fatty acids are not necessary for reduced ROS production with CR. Diets containing fish oil may further decrease ROS production with CR. (Supported by NIH RO1 AG028125)

Caloric restriction (CR), energy intake reduced below ad libitum (AL) intake, increases life span in many species. The implications for humans can be clarified by randomized controlled trials of CR. To determine CR's feasibility, safety, and effects on predictors of longevity, disease risk factors, and quality of life in nonobese humans aged 21-51 years, 218 persons were randomized to a 2-year intervention designed to achieve 25% CR or to AL diet. Outcomes were change from baseline resting metabolic rate adjusted for weight change ("RMR residual") and core temperature (primary); plasma triiodothyronine (T3) and tumor necrosis factor-α (secondary); and exploratory physiological and psychological measures. Body mass index averaged 25.1 (range: 21.9-28.0 kg/m(2)). Eighty-two percent of CR and 95% of AL participants completed the protocol. The CR group achieved 11.7±0.7 %CR (mean ± standard error) and maintained 10.4±0.4% weight loss. Weight change in AL was negligible. RMR residual decreased significantly more in CR than AL at 12 months (p = .04) but not 24 months (M24). Core temperature change differed little between groups. T3 decreased more in CR at M12 and M24 (p < .001), while tumor necrosis factor-α decreased significantly more only at M24 (p = .02). CR had larger decreases in cardiometabolic risk factors and in daily energy expenditure adjusted for weight change, without adverse effects on quality of life. Sustained CR is feasible in nonobese humans. The effects of the achieved CR on correlates of human survival and disease risk factors suggest potential benefits for aging-related outcomes that could be elucidated by further human studies.