Diabetes Publish Ahead of Print, published online March 31, 2009 Modulation of skeletal muscle insulin signaling with chronic caloric restriction in cynomolgus monkeys Zhong Q. Wang1 Elizabeth Floyd1 Jianhua Qin1 Xiaotuan Liu1 Yongmei Yu1 Xian H Zhang1 Janice D. Wagner2 William T. Cefalu1 Division of Nutrition and Chronic Diseases1, Pennington Biomedical Research Center, Louisiana State University System; Department of Pathology, Wake Forest University School of Medicine 2 Corresponding Author: William T Cefalu, M.D. Email: [email protected] Submitted 18 July 2008 and accepted 19 March 2009. This is an uncopyedited electronic version of an article accepted for publication in Diabetes. The American Diabetes Association, publisher of Diabetes, is not responsible for any errors or omissions in this version of the manuscript or any version derived from it by third parties. The definitive publisher-authenticated version will be available in a future issue of Diabetes in print and online at http://diabetes.diabetesjournals.org. 1 Copyright American Diabetes Association, Inc., 2009 Objective: Caloric restriction (CR) has been shown to retard aging processes, extend maximal life span, and consistently increase insulin action in experimental animals. The mechanism by which CR enhances insulin action, specifically in higher species, is not precisely known. We sought to examine insulin receptor signaling and transcriptional alterations in skeletal muscle of nonhuman primates subjected to caloric restriction over a 4 year period. Research Design: After baseline, 32 male adult cynomolgus monkeys (Macaca fascicularis) were randomized to an Ad libitum diet (AL) or to 30% CR. Dietary intake, body weight and insulin sensitivity were obtained at routine intervals over 4 years. At end of study, hyperinsulinemic-euglycemic clamps were performed and skeletal muscle (vastus lateralis) obtained in the basal and insulin stimulated states for insulin receptor signaling and gene expression profiling. Results: CR significantly increased whole-body insulin mediated glucose disposal compared to AL and increased insulin receptor signaling, i.e. IRS-1 and insulin receptor phosphorylation and IRS-associated PI-3 kinase activity in skeletal muscle (p<0.01, p<0.01 and p<0.01 respectively). Gene expression for insulin signaling proteins, i.e. IRS-1, IRS-2, were not increased with caloric restriction although a significant increase in protein abundance was noted. Components of the ubiquitin-proteasome system, i.e. 20S and 19S proteasome subunit abundance and 20S proteasome activity, were significantly decreased by CR. Conclusion: CR increases insulin sensitivity on a whole body level and enhances insulin receptor signaling in this higher species. CR in cynomolgus monkeys may alter insulin signaling in vivo by modulating protein content of insulin receptor signaling proteins. Abbreviations: AL, Ad libitum; CR, caloric restriction; Glut-4, glucose transporter 4; IL6ST, interleukin 6 signal transducer; IRS-1, IRS-2, insulin substrate-1 and -2; LPL, lipoprotein lipase; PI-3 K, phosphoinositol-3-kinase; STAT3, signal transducer and activator of transcription 3; SGK1, serum- and glucocorticoid-regulated kinase 1 2 Caloric restriction (CR) can improvement in insulin sensitivity appears dramatically extend lifespan in lower as one of the most consistent features of species by retarding aging processes and CR, as observed in both rodent and non- reducing the incidence and severity of human primate models (1-3, 14-18). age-related diseases whether initiated in However, the cellular mechanism by young or old age in mammalian models which CR specifically enhances insulin (1-5). Although longevity studies have not action is not precisely known. been completed in humans, it is well To evaluate the potential documented that lifestyle modification mechanism by which CR enhances that includes CR can significantly delay insulin action would require investigation progression and onset of Type 2 at multiple tissues in vivo as coordinated diabetes. However, the cellular mechanisms from liver, adipocytes and mechanism(s) by which CR exerts its skeletal muscle all contribute to whole effects on longevity and attenuates body insulin action. Yet, analysis of progression to metabolic diseases are not skeletal muscle transcriptional regulation precisely known. CR has been with CR would be an important step given postulated to reduce protein glycation and that skeletal muscle is the major site of glyco-oxidation, scavenge reactive insulin-mediated glucose disposal. In oxygen species, modulate addition, a major metabolic effect thermogenesis, assist in DNA repair, and reported for insulin in muscle is inhibition alter oncogene expression and protein of protein degradation mediated by the degradation (5-10). Recently, SIRT1 (one ubiquitin-proteasome system (19, 20). of the human homologs of the budding Interestingly, insulin resistance was also yeast Sir2) has been attracting great reported to accelerate proteasome- interest as to its role in the anti-aging dependent degradation of muscle effects for CR (11, 12). proteins (21), indicating insulin signaling With specific regard as to exerts control over proteasome function mechanism of action, studying the effects in skeletal muscle. Thus, we sought to of CR on aging processes and age- determine a potential mechanism by related diseases in a long lived which CR enhances insulin action in a nonhuman primate, particularly as it higher specie by evaluating insulin relates to the analysis of the genome, signaling and gene expression in skeletal could provide greater insights into muscle in a non-human primate subjected mechanisms of aging and effects on to chronic CR. aging in humans. Although human studies have only recently been reported METHODS (13), there has been intense study of the Study Design: The effect of metabolic effects of CR in higher species chronic CR to modulate cellular insulin for many years. Specifically, we signaling and transcriptional regulation demonstrated that CR improved insulin was assessed in skeletal muscle obtained sensitivity and reduced intra-abdominal from non-human primates subjected to a fat with aging in a 4 year study in 4 year period of 30% calorie restriction as cynomolgus monkeys (14). An opposed to ad libitum (AL) fed conditions. 3 The metabolic and physiologic changes insulin infusion, repeat biopsies were observed in this cohort with CR were obtained at 5, 20 and 40 minutes of the previously reported (14). Specifically, 32 clamp. Muscle samples (~200 mg wet feral adult male cynolmolgus monkeys weight) were immediately placed into (Macaca fascicularis) [8.2 ± 1.2 years old liquid nitrogen, and then stored in -80 °C. (Mean ± SEM)] were part of a There was no difference between the randomized trial in which the independent steady state plasma glucose (5.38 ± 0.10 effect of CR and its interaction with insulin vs 5.42 ± 0.10 mmol/L) or plasma insulin resistance and atherosclerotic lesion levels observed during the clamp (1472 ± extent and composition were evaluated 145 vs 1535 ± 155 pmol/L) for either the (Figure 1). CR or AL groups, respectively. Animals and Diet: The animals Tissue Preparation: Muscle were acquired directly from Institute tissue lysates were prepared by Pertanian (Bogar, Indonesia) and dissection and homogenized in buffer A quarantined for three months. Animals (25 mM HEPES, pH 7.4, 1% Nonidet P- were housed socially in pairs except 40 (NP-40), 137 mM NaCl, 1 mM PMSF, when separated at mealtime by sliding a 10 µg/ml aprotinin, 1 µg/ml pepstatin, 5 partition to separate them (14). Beginning µg/ml leupeptin) using a PRO 200 in the fourth month and throughout the homogenizer (PRO Scientific Inc, Oxford, pre-trial (months 4-6), all animals were CT). The samples were centrifuged at fed a moderately atherogenic diet (0.25 14,000 g for 20 minutes at 4°C, and mg cholesterol/Cal) containing 30% of protein content of the supernatant calories from fat. Caloric intake for each determined (Bio-Rad protein assay kit, animal was assessed by feeding a known Bio-Rad laboratories, Inc. Hercules, CA). allotment and weighing the uneaten food Supernatants (50 µg) were resolved by (14). After pretrial evaluations, the SDS-PAGE and subjected to animals were assigned to AL or CR diet immunoblotting using chemiluminescence groups using a stratified randomization. reagent (PerkinElmer Life Science Inc, The CR diet was introduced over a 3- Boston, MA) and quantified as described month transition period (90% of AL intake (22). 19S proteasome base anti- during the first month, 80% during the S5A/Rpn10 antibodies were ordered from second month, 70% during the third Calbiochem Inc (Gibbstown, NJ). month and thereafter). Additional vitamin Antibodies for phospho-IRS-1(Tyr612), and mineral mixture, β-sitosterol, and phosph-IR(Tyr1150/1151), phosphoinositol-3- crystalline cholesterol were added to the kinase protein 85 (p85 of PI 3K), phosph- CR diet so that the same amount of these Akt-(ser473), insulin substrate-1 and -2 components was ingested regardless of (IRS-1, IRS-2), 20S proteasome subunit randomized group (14). β2i, Akt, SGK1, STAT3 and SIRT1 Insulin Sensitivity: Insulin antibodies were obtained from Upstate sensitivity was assessed at 6 month Biotech (Lake Placid, NY). Anti-19S intervals with use of modified minimal proteasome lid subunits S9/Rpn6 and model assessment and at the end of S14/Rpn12 antibodies were ordered from study with a hyperinsulinemic-euglycemic Biomol International, Inc (Plymouth