CR Rats Research Articles

Comparative analysis and validation of posterior cruciate ligament management in mobile-bearing total knee arthroplasty: Meta-analysis and animal study.

Total knee arthroplasty (TKA) is an effective treatment for end-stage knee joint diseases. The debate over preserving or sacrificing the posterior cruciate ligament (PCL) in mobile-bearing TKA (MB TKA) still needs to be solved due to the lack of high-quality evidence, particularly meta-analyses comparing these techniques. This study aims to conduct a meta-analysis to compare the outcomes of PCL retention (CR) and PCL sacrifice (PS) in terms of clinical and functional knee scores, range of motion, complication rates, and revision rates and to validate these findings through animal experiments. A comprehensive search was conducted using MEDLINE, Cochrane, and Embase databases. Relevant studies were selected for the meta-analysis using RevMan 5.3. Additionally, an animal experiment using Sprague-Dawley rats simulated MB TKA to compare the effects of PCL retention and sacrifice surgeries. 12 studies were included in the meta-analysis. No significant differences were found between CR and PS techniques regarding HSS, KSS, KSFS, WOMAC, ROM, and medial/lateral instability. However, CR MB showed slight superiority in NKJS, while PS MB had better outcomes in complication and revision rates. In the animal study, CR rats exhibited significant early postoperative inflammation, but both groups' knee structures gradually normalized. The meta-analysis indicates that PCL retention (CR MB) and sacrifice (PS MB) have similar effects on various clinical and functional knee scores. However, PS MB is significantly better at reducing complications and revision rates. The animal experiment confirms PS MB's advantages in reducing inflammation and promoting joint recovery. Despite the strong evidence, long-term follow-up and larger-scale randomized controlled trials are necessary to confirm these findings.

Independent and combined effects of calorie restriction and AICAR on glucose uptake and insulin signaling in skeletal muscles from 24-month-old female and male rats.

We assessed the effects of two levels of calorie restriction (CR; eating either 15% or 35% less than ad libitum, AL, food intake for 8 weeks) by 24-month-old female and male rats on glucose uptake (GU) and phosphorylation of key signaling proteins (Akt; AMP-activated protein kinase, AMPK; Akt substrate of 160kDa, AS160) measured in isolated skeletal muscles that underwent four incubation conditions (without either insulin or AICAR, an AMPK activator; with AICAR alone; with insulin alone; or with insulin and AICAR). Regardless of sex: (1) neither CR group versus the AL group had greater GU by insulin-stimulated muscles; (2) phosphorylation of Akt in insulin-stimulated muscles was increased in 35% CR versus AL rats; (3) prior AICAR treatment of muscle resulted in greater GU by insulin-stimulated muscles, regardless of diet; and (4) AICAR caused elevated phosphorylation of acetyl CoA carboxylase, an indicator of AMPK activation, in all diet groups. There was a sexually dimorphic diet effect on AS160 phosphorylation, with 35% CR exceeding AL for insulin-stimulated muscles in male rats, but not in female rats. Our working hypothesis is that the lack of a CR-effect on GU by insulin-stimulated muscles was related to the extended duration of the ex vivo incubation period (290 min compared to 40-50 min that was previously reported to be effective). The observed efficacy of prior treatment of muscles with AICAR to improve glucose uptake in insulin-stimulated muscles supports the strategy of targeting AMPK with the goal of improving insulin sensitivity in older females and males.

Early-adulthood caloric restriction is beneficial to improve renal redox status as future anti-aging strategy in rats

AimsCaloric restriction (CR) is an experimental approach proposed to alleviate age-related oxidative damage. In the present study, we investigated the consequences of CR on renal redox homeostasis in rats at a specific time frame in early-adulthood.. MethodsThree groups of male Sprague-Dawley rats; young control at 6-month-old, 2-year-old subjected to 40% CR between 18th-24th months of age, and their non-CR controls were sacrificed, and numerous redox status biomarkers including protein oxidation, glycation, lipid peroxidation, glycation end products, thiol groups, and superoxide dismutase were assayed. It was also ensured that CR rats and their non-CR corresponding rats had similar body weights at the end of the study to decrease the confounding effects of different body weights on redox homeostasis and caloric restriction. ResultsAfter CR, the detrimental effects of the protein oxidation, glycation, and lipid peroxidation were significantly improved in the renal tissue CR rats when compared to their non-CR control group. However, there were no significant difference in thiol fractions between younger controls and both of the elderly groups. ConclusionDetrimental consequences of renal senescence on redox homeostasis are significantly improved via CR especially applied in early-adulthood.

Leptin Intake at Physiological Doses Throughout Lactation in Male Wistar Rats Normalizes the Decreased Density of Tyrosine Hydroxylase-Immunoreactive Fibers in the Stomach Caused by Mild Gestational Calorie Restriction.

Introduction: Gestational under nutrition in rats has been shown to decrease expression of sympathetic innervation markers in peripheral tissues of offspring, including the stomach. This has been linked to lower gastric secretion and decreased circulating levels of ghrelin. Considering the critical role of leptin intake during lactation in preventing obesity and reversing adverse developmental programming effects, we aimed to find out whether leptin supplementation may reverse the above mentioned alterations caused by mild gestational calorie restriction.Methods: Three groups of male rats were studied at a juvenile age (25 days old) and during adulthood (3 and 6 months old): the offspring of ad libitum fed dams (controls), the offspring of dams that were diet restricted (20%) from days 1 to 12 of gestation (CR), and CR rats supplemented with a daily oral dose of leptin (equivalent to 5 times the average amount they could receive each day from maternal milk) throughout lactation (CR-Leptin). The density of TyrOH-immunoreactive (TyrOH+) fibers and the levels of Tyrosine hydroxylase (TyrOH)—used as potential markers of functional sympathetic innervation—were measured in stomach. Plasma leptin and ghrelin levels were also determined.Results: Twenty five-day-old CR rats, but not CR-Leptin rats, displayed lower density of TyrOH+ fibers (−46%) and TyrOH levels (−47%) in stomach compared to controls. Alterations in CR animals were mitigated at 6 months of age, and differences were not significant. Adult CR-Leptin animals showed higher plasma ghrelin levels than CR animals, particularly at 3 months (+16%), and a lower leptin/ghrelin ratio (−28 and −37% at 3 and 6 months, respectively).Conclusion: Leptin intake during lactation is able to reverse the alterations in the density of TyrOH+ fibers in the stomach and normalize the increased leptin/ghrelin ratio linked to a mild gestational calorie restriction in rats, supporting the relevance of leptin as an essential nutrient during lactation.

Effects of Calorie Restriction and Fiber Type on Glucose Uptake and Abundance of Electron Transport Chain and Oxidative Phosphorylation Proteins in Single Fibers from Old Rats.

Calorie restriction (CR; reducing calorie intake by ~40% below ad libitum) can increase glucose uptake by insulin-stimulated muscle. Because skeletal muscle is comprised of multiple, heterogeneous fiber types, our primary aim was to determine the effects of CR (initiated at 14 weeks old) and fiber type on insulin-stimulated glucose uptake by single fibers of diverse fiber types in 23-26-month-old rats. Isolated epitrochlearis muscles from AL and CR rats were incubated with [3H]-2-deoxyglucose ± insulin. Glucose uptake and fiber type were determined for single fibers dissected from the muscles. We also determined CR-effects on abundance of several key metabolic proteins in single fibers. CR resulted in: (a) significantly (p < .05 to .001) greater glucose uptake by insulin-stimulated type I, IIA, IIB, IIBX, and IIX fibers; (b) significantly (p < .05 to .001) reduced abundance of several mitochondrial electron transport chain (ETC) and oxidative phosphorylation (OxPhos) proteins in type I, IIA, and IIBX but not IIB and IIX fibers; and (c) unaltered hexokinase II abundance in each fiber type. These results demonstrate that CR can enhance glucose uptake in each fiber type of rat skeletal muscle in the absence of upregulation of the abundance of hexokinase II or key mitochondrial ETC and OxPhos proteins.

Oral leptin supplementation throughout lactation in rats prevents later metabolic alterations caused by gestational calorie restriction.

Calorie-restriction during gestation in rats has been seen to produce lasting detrimental effects in the offspring, affecting energy balance control and other related metabolic functions. Our aim was to assess whether leptin supplementation throughout lactation may prevent the dysmetabolic phenotype in adulthood associated with gestational calorie restriction. Three groups of male Wistar rats were followed: the offspring of ad libitum fed dams (controls); the offspring of 20% calorie-restricted dams during gestation (CR); and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Pups were weaned with a standard diet (SD) until 4 months of age, and then half of the animals of each group were moved to a Western diet (WD) until 6 months of age. Body weight and food intake were recorded. Energy expenditure, locomotive activity, blood parameters, liver triglycerides (TG), and gene expression and specific proteins in liver and white adipose tissue (WAT) were measured in adulthood. Adult CR rats, but not CR-Leptin rats, displayed greater adiposity index and feed efficiency (both under SD) than controls, along with lower locomotive activity and energy expenditure, higher HOMA-IR index and greater circulating TG and leptin levels. CR animals also exhibited increased values of the respiratory exchange ratio and more severe signs of hepatic steatosis under WD than CR-Leptin animals. Gene expression analysis revealed that CR, but not CR-Leptin, animals displayed indicators of lower capacity for WAT expansion, along with decreased lipogenesis and lipolytic capacity under SD, and impaired lipogenic response of the liver to WD feeding, in accordance with diminished insulin sensitivity and WAT leptin signaling. Oral leptin supplementation in physiological doses throughout lactation in rats prevents most of the detrimental effects on energy homeostasis and metabolic alterations in adulthood caused by inadequate fetal nutrition.

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

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

Transcriptome analysis in calorie-restricted rats implicates epigenetic and post-translational mechanisms in neuroprotection and aging.

BackgroundCaloric restriction (CR) can increase longevity in rodents and improve memory function in humans. α-Lipoic acid (LA) has been shown to improve memory function in rats, but not longevity. While studies have looked at survival in rodents after switching from one diet to another, the underlying mechanisms of the beneficial effects of CR and LA supplementation are unknown. Here, we use RNA-seq in cerebral cortex from rats subjected to CR and LA-supplemented rats to understand how changes in diet can affect aging, neurodegeneration and longevity.ResultsGene expression changes during aging in ad libitum-fed rats are largely prevented by CR, and neuroprotective genes are overexpressed in response to both CR and LA diets with a strong overlap of differentially expressed genes between the two diets. Moreover, a number of genes are differentially expressed specifically in rat cohorts exhibiting diet-induced life extension. Finally, we observe that LA supplementation inhibits histone deacetylase (HDAC) protein activity in vitro in rat astrocytes. We find a single microRNA, miR-98-3p, that is overexpressed during CR feeding and LA dietary supplementation; this microRNA alters HDAC and histone acetyltransferase (HAT) activity, which suggests a role for HAT/HDAC homeostasis in neuroprotection.ConclusionsThis study presents extensive data on the effects of diet and aging on the cerebral cortex transcriptome, and also emphasises the importance of epigenetics and post-translational modifications in longevity and neuroprotection.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-015-0847-2) contains supplementary material, which is available to authorized users.

Leptin intake in suckling rats restores altered T3 levels and markers of adipose tissue sympathetic drive and function caused by gestational calorie restriction

Maternal calorie restriction during gestation in rats has been associated with altered white adipose tissue (WAT) sympathetic innervation and function in offspring. Here, we aimed to investigate whether supplementation with oral leptin (a breast milk component) throughout the lactation period may revert the aforementioned adverse programming effects. Three groups of male and female rats were studied at the postnatal day 25: the offspring of control dams, the offspring of 20% calorie-restricted dams during pregnancy (CR) and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Tyrosine hydroxylase (TH) levels and its immunoreactive area, and mRNA expression levels of lipid metabolism-related genes and of deiodinase iodothyronine type II (Dio2) were determined in WAT. Triiodothyronine (T3) levels were determined in the blood. In CR males, leptin treatment restored the decreased TH levels and its immunoreactive area in WAT, and partially normalized expression levels of genes related to lipolysis and fatty acid oxidation (adipose triglyceride lipase, hormone-sensitive lipase, carnitine palmitoyltransferase 1b and peroxisome proliferator-activated receptor gamma coactivator 1-alpha). Leptin treatment also reverted the decreased T3 plasma levels and WAT lipoprotein lipase mRNA levels occurring in CR males and females, and the decreased Dio2 mRNA levels in CR females. Leptin supplementation throughout the lactation period reverts the malprogrammed effects on WAT structure and function induced by undernutrition during pregnancy. These findings support the relevance of the intake of leptin during lactation, bearing clear characteristics of essential nutrient, and provide a strategy to treat and/or prevent the programmed trend to obesity acquired by inadequate fetal nutrition.

Moderate calorie restriction during gestation programs offspring for lower BAT thermogenic capacity driven by thyroid and sympathetic signaling

Maternal calorie restriction during pregnancy programs offspring for later overweight and metabolic disturbances. Brown adipose tissue (BAT) is responsible for non-shivering thermogenesis and has recently emerged as a very likely target for human obesity therapy. Here we aimed to assess whether the detrimental effects of undernutrition during gestation could be related to impaired thermogenic capacity in BAT and to investigate the potential mechanisms involved. Offspring of control and 20% calorie-restricted rats (days 1-12 of pregnancy) (CR) were studied at the age of 25 days. Protein levels of uncoupling protein 1 (UCP1) and tyrosine hydroxylase (TyrOH); mRNA levels of lipoprotein lipase (LPL), carnitine palmitoyltransferase 1 (CPT1) and deiodinase iodothyronine type II (DIO2) in BAT; and blood parameters including thyroid hormones, were determined. The response to 24-h cold exposure was also studied by measuring body temperature changes over time, and final BAT UCP1 levels. Compared with controls, CR animals displayed in BAT lower UCP1 and TyrOH protein levels and lower LPL and CPT1 mRNA levels; they also showed lower triiodothyronine (T3) plasma levels. CR males, but not females, revealed lower DIO2 mRNA levels than controls. When exposed to cold, CR rats experienced a transient decline in body temperature, but the values were reestablished after 24 h, despite having lower UCP1 levels than controls. These results suggest that BAT thermogenic capacity is diminished in CR animals, involving impaired BAT sympathetic innervation and thyroid hormone signaling. These alterations make animals more sensitive to cold and may contribute to long-term outcomes of gestational calorie restriction in promoting obesity and related metabolic alterations.

Metabolic response of short term calorie restriction and supplementation with Hoodia gordonii

Hoodia gordonii is a supplement of natural origin which is known for its appetite suppressant activity. Owing to its anorectic activity, the aim of this study was to evaluate the effect of H. gordonii supplementation on metabolic changes and appetite regulatory peptides during calorie restriction. Male albino rats were divided into three groups (n=12 in each) — Control, Calorie Restricted (CR, 25% for 5days), Calorie Restricted and H. gordonii supplemented (CR+HG, organic solvent extract given orally for 5days at a dose of 100mg/kg bwt.). The regulatory peptides i.e. ghrelin, leptin, CCK, NPY, insulin, IGF-1, corticosterone, thyroid hormones, adiponectin, serotonin were determined. On comparison with CR rats, modulations were noticed in the appetite regulatory peptides and biochemical variables of the CR+HG rats. A significant decline in ghrelin and increase in CCK was observed. The CR group exhibited a significant decrease in leptin, IGF-1, plasma and whole brain serotonin with a significant increase in the ghrelin and thyroxin levels. These changes indicate altered metabolic responses and hunger suppression which seem to be caused by H. gordonii with CR along with the changes occurring due to CR itself. It is concluded that H. gordonii can modulate hunger during CR and may be used for better adherence to dietary restriction regime.

Wistar rats from different suppliers have a different response in an acute myocardial infarction model

The Wistar rat is a commonly used strain for experimental animal models. Recently it was shown that results vary between studies using Wistar rats of different suppliers. Therefore we studied whether Wistar rats obtained from Harlan Laboratories (Ha, n=24) and Charles River (CR, n=22) had a different outcome in an acute myocardial infarction (AMI) model. AMI was induced in both Ha and CR Wistar rats by one operator. This resulted in a significantly higher survival rate for Ha (79.2±10.2%) compared with CR rats (54.2±10.2%, p<0.05). Furthermore, CR rats had lost significantly more weight after 7days (−5.9±3.1%) compared with Ha rats (−0.8±1.7%; p<0.001), indicating a worse health status of the CR rats. Paradoxically, the induced infarct was smaller in CR rats (7.3±3.6% of the heart) compared with Ha rats (12.1±4.7%, p<0.05). This indicates that CR rats were less sensitive for the cardiomyocyte damage subsequent to AMI induction, but remarkably showed more clinical side effects indicating that Wistar rats from two suppliers had a different response within the same AMI model.

Oral leptin treatment in suckling rats ameliorates detrimental effects in hypothalamic structure and function caused by maternal caloric restriction during gestation.

A poor prenatal environment brings about perturbations in leptin surge and hypothalamic circuitry that program impaired ability to regulate energy homeostasis in adulthood. Here, using a rat model of moderate maternal caloric restriction during gestation, we aimed to investigate whether leptin supplementation with physiological doses throughout lactation is able to ameliorate the adverse developmental malprogramming effects exerted in offspring hypothalamus structure and function. Three groups of male and female rats were studied: the offspring of ad libitum fed dams (controls), the offspring of 20% calorie restricted dams during the first part of pregnancy (CR), and CR rats supplemented with physiological doses of leptin throughout lactation (CR-Leptin). Animals were sacrificed on postnatal day 25. Morphometric and immunohistochemical studies on arcuate (ARC) and paraventicular (PVN) nucleus were performed and hypothalamic expression levels of selected genes were determined. In CR males, leptin treatment restored, at least in part, the number of immunoreactive neuropeptide Y (NPY+) cells in ARC, the total number of cells in PVN, hypothalamic NPY, cocaine- and amphetamine-regulated transcript (CART) and suppressor of cytokine signalling-3 (SOCS-3) mRNA levels, and plasma leptin levels, which were decreased in CR animals. CR-Leptin males showed higher hypothalamic long-form leptin receptor (ObRb) mRNA levels, compared to control and CR animals. In CR females, leptin treatment reverted the increased number of cells in ARC and cell density in ARC and PVN, and reduced hypothalamic SOCS-3 mRNA expression to levels similar to controls. Leptin treatment also reverted the increased relative area of NPY+ fibers in the PVN occurring in CR animals. In conclusion, leptin supplementation throughout lactation is able to revert, at least partly, most of the developmental effects on hypothalamic structure and function caused by moderate maternal caloric restriction during gestation, and hence making this metabolic malprogramming reversible to some extent.

Relative contribution of foetal and post‐natal nutritional periods on feeding regulation in adult rats

The aim of this study was to assess the contribution of both foetal and/or post-natal nutritional periods on feeding regulation in adult rats. Body weight gain, adipose tissue development, food preferences and feeding pattern under regular chow or Western diets were characterized on four experimental groups of rats: pups born from protein-restricted dams (R) and weaned by control (RC) or R dams (RR) and pups born from control dams weaned by C (CC) or R dams (CR). Rats born with intrauterine growth restriction (IUGR) and fed a Western diet at adulthood appeared predisposed to body weight gain and more fat accretion, whereas CR rats, despite their preference for high-fat diet and their hyperphagia for Western diet, did not show significant increase in fat tissue. Daytime food intakes, as well as their speed of ingestion, were found modified in RC and RR. Alterations in the hypothalamic appetite regulatory mechanisms were investigated through neuropeptide expression analysis. IUGR rats showed altered expression of key elements of leptin and NPY signalling, while CR rats exhibited lesser expression of enterostatin, MC4r and HT-1Br mRNA. Altogether, these results indicate that peri-natal nutrition has different lasting effects on feeding pattern and hypothalamic appetite regulation, depending on the time window insult.

Serum myoglobin, but not lipopolysaccharides, is predictive of AMPH-induced striatal neurotoxicity

Determinants of amphetamine (AMPH)-induced neurotoxicity are poorly understood. The role of lipopolysaccharides (LPS) and organ injury in AMPH-induced neurotoxicity was examined in adult male Sprague-Dawley rats that were give AMPH and became hyperthermic during the exposure. Environmentally-induced hyperthermia (EIH) in the rat was compared to AMPH to determine whether AMPH-induced increases in LPS and peripheral toxicities were solely attributable to hyperthermia. Muscle, liver, and kidney function were determined biochemically at 3h or 1 day after AMPH or EIH exposure and histopathology at 1 day after treatment. Circulating levels of LPS were monitored (via limulus amoebocyte coagulation assay) during AMPH or EIH exposure. Blood LPS levels were detected in 40–50% of the AMPH and EIH rats, but the presence of LPS in the serum had no effect on organ damage or striatal dopamine depletions (neurotoxicity). In both CR and NCTR rats, serum bound urea nitrogen and creatinine levels increased at 3h after EIH or AMPH (2- to 3-fold above control) but subsided by 1 day. Alanine transaminase was increased (indicating liver dysfunction) by both AMPH and EIH at 3h (2- to 10-fold above control) in CR rats, but the levels were not significantly different between the control and AMPH groups in NCTR animals. Mild liver necrosis was detected in 1 of 7 rats examined in the AMPH group and in 1 of 5 rats examined in the EIH group (only NCTR rats were examined). Serum myoglobin increased (indicating muscle damage) in both CR and NCTR rats at 3h and was more pronounced with AMPH (≈5-fold above control) than EIH. Our results indicate that: (1) “free” blood borne LPS often increases with EIH and AMPH but may not be necessary for striatal neurotoxicity and CNS immune responses; (2) liver or kidney dysfunction may result from muscle damage; however, it is not sufficient nor necessary to produce, but may exacerbate, neurotoxicity; (3) AMPH-induced serum myoglobin release is a potential biomarker and possibly a factor in AMPH-induced toxicity processes.

Calorie restriction effects on insulin signaling and glucose uptake in multiple insulin‐stimulated rat skeletal muscles in vivo

Calorie restriction (CR; ~60% of ad libitum, AL, food intake) has been shown to enhance whole‐body insulin sensitivity. Skeletal muscle accounts for the majority of insulin‐stimulated glucose disposal, however no previous studies have reported CR effects on both in vivo glucose uptake and insulin signaling in the same skeletal muscles. Accordingly we evaluated the effect of CR (initiated at 14‐wks of age) on the tibialis anterior (TA) and gastrocnemius (GN) muscles from 9‐mo old rats under euglycemic‐hyperinsulinemic clamp conditions. Glucose levels during the clamp were not significantly different for AL (120.7±2.7 mg/dl) versus CR (126.1±2.4 mg/dl) groups. Insulin levels during the clamp were also elevated to similar levels in AL (141.3±9.1 μU/ml) and CR (140.3±6.7 μU/ml) groups. Glucose infusion rates were 78% higher (P&lt;0.05) in CR (31.7±1.6 mg/kg−1 × min−1) versus AL (17.8±0.8 mg/kg−1 × min−1) rats. Glucose uptake (radiolabelled 2‐deoxyglucose) was increased (P&lt;0.05) for CR vs. AL rats in both the TA and GN. Insulin receptorTyr1162/1163 phosphorylation was unaffected by CR in either muscle. AktSer473 phosphorylation was increased (P&lt;0.05) in both muscles of CR vs. AL rats, and there was a strong trend for increased AktThr308 phosphorylation in CR vs. AL rats for both the TA (P=0.052) and GN (P=0.052). These results suggest that CR leads to greater whole‐body insulin sensitivity attributable at least in part to CR effects on in vivo insulin‐stimulated glucose uptake secondary to greater Akt phosphorylation in multiple muscles, including the TA and GN.

Greater insulin-mediated Akt phosphorylation concomitant with heterogeneous effects on phosphorylation of Akt substrates in soleus of calorie-restricted rats

Akt is a serine/threonine kinase that plays a key role in numerous cellular functions including metabolism, growth, protein synthesis, apoptosis, and cell proliferation. The most consistent and robust effect of moderate calorie restriction (CR; ~60% of ad libitum, AL, food consumption) on insulin signaling in rodent muscle has been enhanced insulin-induced phosphorylation of Akt (pAkt). However, there is limited knowledge regarding the mechanism for this enhancement and its consequences in predominantly slow-twitch muscle. Accordingly, in soleus muscle of 9-mo-old rats, we analyzed the effect of CR and insulin on important signaling events that are proximal to Akt activation including: pIR(Tyr1162/1163), pIRS1(Tyr), pIRS1(Ser312), IRS1-associated phosphatidylinositol 3-kinase activity, or pPTEN(Ser380). In addition, we analyzed the effect of CR and insulin on Akt substrates that have established or putative roles in glucose metabolism, cellular growth, maintenance of muscle structure, or protein synthesis including pGSK3α(Ser21), pGSK3β(Ser9), pTSC2(Ser939), pP70S6K(Thr412), pAS160(Thr642), and pFLNc(Ser2213). The current study demonstrated that the CR-induced increase in pAkt in isolated soleus muscles from 9-mo-old rats can occur without concomitant enhancement of several important insulin signaling events that are proximal to Akt activation. These results suggest that the greater pAkt in the soleus muscles from CR rats was attributable to an alternative mechanism. We also observed that the effects of CR were not uniform for phosphorylation of six insulin-regulated Akt substrates in the soleus. The differential response in phosphorylation by Akt substrates likely has important implications for explaining the complex effect of CR diverse cellular functions.

Preventing the calorie restriction-induced increase in insulin-stimulated Akt2 phosphorylation eliminates calorie restriction's effect on glucose uptake in skeletal muscle

Calorie restriction (CR; ~60% of ad libitum, AL, consumption) improves insulin-stimulated glucose uptake in skeletal muscle. The precise cellular mechanism for this healthful outcome is unknown, but it is accompanied by enhanced insulin-stimulated activation of Akt. Previous research using Akt2-null mice demonstrated that Akt2 is essential for the full CR-effect on insulin-stimulated glucose uptake by muscle. However, because Akt2-null mice were completely deficient in Akt2 in every cell throughout life, it would be valuable to assess the efficacy of transient, muscle-specific Akt inhibition for attenuation of CR-effects on glucose uptake. Accordingly, we used a selective Akt inhibitor (MK-2206) to eliminate the CR-induced elevation in insulin-stimulated Akt2 phosphorylation and determined the effects on Akt substrates and glucose uptake. We incubated isolated epitrochlearis muscles from 9-month-old AL and CR (~60–65% of AL intake for 6months) rats with or without MK-2206 and measured insulin-stimulated (1.2nM) glucose uptake and phosphorylation of the insulin receptor (Tyr1162/1163), pan-Akt (Thr308 and Ser473), Akt2 (Thr308 and Ser473), AS160/TBC1D4 (Thr642), and Filamin C (Ser2213). Incubation of isolated skeletal muscles with a dose of a selective Akt inhibitor that eliminated the CR-induced increases in Akt2 phosphorylation prevented CR's effects on insulin-stimulated glucose uptake, pAS160Thr642 and pFilamin CSer2213 without altering pIRTyr1162/1163. These data provide compelling new evidence linking the CR-induced increase in insulin-stimulated Akt2 phosphorylation to CR's effects on insulin-mediated phosphorylation of Akt substrates and glucose uptake in skeletal muscle.

Improved insulin sensitivity with calorie restriction does not require reduced JNK1/2, p38, or ERK1/2 phosphorylation in skeletal muscle of 9-month-old rats

Calorie restriction [CR; ∼40% below ad libitum (AL) intake] improves the health of many species, including rats, by mechanisms that may be partly related to enhanced insulin sensitivity for glucose disposal by skeletal muscle. Excessive activation of several mitogen-activated protein kinases (MAPKs), including JNK1/2, p38, and ERK1/2 has been linked to insulin resistance. Although insulin can activate ERK1/2, this effect is not required for insulin-mediated glucose uptake. We hypothesized that skeletal muscle from male 9-mo-old Fischer 344/Brown Norway rats CR (35-40% beginning at 3 mo old) versus AL rats would have 1) attenuated activation of JNK1/2, p38, and ERK1/2 under basal conditions; and 2) no difference for insulin-induced ERK1/2 activation. In contrast to our hypothesis, there were significant CR-related increases in the phosphorylation of p38 (epitrochlearis, soleus, and gastrocnemius), JNK1 (epitrochlearis and soleus), and JNK2 (gastrocnemius). Consistent with our hypothesis, CR did not alter insulin-mediated ERK1/2 activation. The greater JNK1/2 and p38 phosphorylation with CR was not attributable to diet effects on muscle oxidative stress (assessed by protein carbonyls and 4-hydroxynonenal protein conjugates). In muscles from the same rats used for the present study, we previously reported a CR-related increase in insulin-mediated glucose uptake by the epitrochlearis and the soleus (Sharma N, Arias EB, Bhat AD, Sequea DA, Ho S, Croff KK, Sajan MP, Farese RV, Cartee GD. Am J Physiol Endocrinol Metab 300: E966-E978, 2011). The present results indicate that the improved insulin sensitivity with CR is not attributable to attenuated MAPK phosphorylation in skeletal muscle.

Reduced expression of SIRT1 is associated with diminished glucose-induced insulin secretion in islets from calorie-restricted rats

Alterations in food intake such as caloric restriction modulate the expression of SIRT1 and SIRT4 proteins that are involved in pancreatic β-cell function. Here, we search for a possible relationship between insulin secretion and the expression of SIRT1, SIRT4, PKC and PKA in islets from adult rats submitted to CR for 21 days. Rats were fed with an isocaloric diet (CTL) or received 60% (CR) of the food ingested by CTL. The dose–response curve of insulin secretion to glucose was shifted to the right in the CR compared with CTL islets (EC 50 of 15.1±0.17 and 10.5±0.11 mmol/L glucose). Insulin release by the depolarizing agents arginine and KCl was reduced in CR compared with CTL islets. Total islet insulin content and glucose oxidation were also reduced in CR islets. Leucine-stimulated secretion was similar in both groups, slightly reduced in CR islets stimulated by leucine plus glutamine but higher in CR islets stimulated by ketoisocaproate (KIC). Insulin secretion was also higher in CR islets stimulated by carbachol, compared with CTL islets. No differences in the rise of cytosolic Ca 2+ concentrations stimulated by either glucose or KCl were observed between groups of islets. Finally, SIRT1, but not SIRT4, protein expression was lower in CR compared with CTL islets, whereas no differences in the expression of PKC and PKA proteins were observed. In conclusion, the lower insulin secretion in islets from CR rats was, at least in part, due to an imbalance between the expression of SIRT1 and SIRT4.