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Muscle-Specific Overexpression of PGC-1A Does Not Augment

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Muscle-Specific Overexpression of PGC-1A Does Not Augment 1532 Diabetes Volume 64, May 2015 Kari E. Wong,1 Catherine R. Mikus,1 Dorothy H. Slentz,1 Sarah E. Seiler,1,2 Karen L. DeBalsi,1,2 Olga R. Ilkayeva,1 Karen I. Crain,3 Michael T. Kinter,4 C. Lawrence Kien,3,5 Robert D. Stevens,1 and Deborah M. Muoio1,2,6 Muscle-Specific Overexpression of PGC-1a Does Not Augment Metabolic Improvements in Response to Exercise and Caloric Restriction Diabetes 2015;64:1532–1543 | DOI: 10.2337/db14-0827 This study used mice with muscle-specific overexpression risk of numerous comorbidities, including cardiovascular of PGC-1a, a transcriptional coactivator that promotes disease, impaired blood glucose control, and type 2 mitochondrial biogenesis, to determine whether increased diabetes (1). Importantly, even moderate levels of weight oxidative potential facilitates metabolic improvements loss can improve risk factors for these diseases (2). The in response to lifestyle modification. MCK-PGC1a mice most effective, noninvasive strategies for achieving weight and nontransgenic (NT) littermates were fed a high-fat control are based on lifestyle modifications that promote diet (HFD) for 10 weeks, followed by stepwise exposures balanced nutrition and physical activity. Moreover, habit- to voluntary wheel running (HFD+Ex) and then 25% calo- ual exercise imparts metabolic benefits independent of ric restriction with exercise (Ex/CR), each for an additional weight loss, including improvements in insulin sensitivity, 10 weeks with continued HFD. Running and CR improved glucose tolerance, and blood lipid profiles. Whereas lifestyle a weight and glucose control similarly in MCK-PGC1 and factors clearly influence disease risk, enthusiasm for behav- NT mice. Sedentary MCK-PGC1a mice were more sus- fi METABOLISM ioral modi cation approaches has been dampened by psy- ceptible to diet-induced glucose intolerance, and insulin chological, economical, or physical barriers to long-term action measured in isolated skeletal muscles remained patient compliance. lower in the transgenic compared with the NT group, even after Ex/CR. Comprehensive profiling of >200 metabolites While the need for novel therapeutics continues to and lipid intermediates revealed dramatic group-specific grow, efforts to develop drugs that facilitate weight responses to the intervention but did not produce a lead loss or improve weight-loss maintenance have thus far candidate that tracked with changes in glucose tolerance met limited success. The compelling association be- irrespective of genotype. Instead, principal components tween routine physical activity and favorable health analysis identified a chemically diverse metabolite cluster outcomes has fueled strong interest in the develop- that correlated with multiple measures of insulin respon- ment of exercise-mimetic compounds. Although the siveness. These findings challenge the notion that in- concept of “exercise in a pill” is attractive, the molec- creased oxidative capacity defends whole-body energy ular strategy to meet this end remains uncertain be- homeostasis and suggest that the interplay between mito- cause the precise mechanisms responsible for the chondrial performance, lipotoxicity, and insulin action is health benefits of physical activity are poorly under- more complex than previously proposed. stood (3). Prominent among the many molecular and cellular adaptations that occur in response to aerobic exercise training are increased mitochondrial biogene- The escalating obesity pandemic presents a serious threat sis and enhanced potential for oxidative metabolism to global health and economic stability. Obesity increases (4). One prevailing view in this field posits that 1Sarah W. Stedman Nutrition and Metabolism Center, Duke Molecular Physiology Corresponding author: Deborah M. Muoio, [email protected]. Institute, Durham, NC Received 24 May 2014 and accepted 18 November 2014. 2Department of Pharmacology and Cancer Biology, Duke University, Durham, NC This article contains Supplementary Data online at http://diabetes 3Department of Medicine, University of Vermont, Burlington, VT .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0827/-/DC1. 4Free Radical Biology & Aging Research Program, Oklahoma Medical Research Foundation, Oklahoma City, OK © 2015 by the American Diabetes Association. Readers may use this article as 5Department of Pediatrics, University of Vermont, Burlington, VT long as the work is properly cited, the use is educational and not for profit, and 6Department of Medicine, Duke University, Durham, NC the work is not altered. diabetes.diabetesjournals.org Wong and Associates 1533 remodeling of skeletal myofibers toward a more oxida- Exercise Performance Studies tive phenotype confers protection against disease by Mice were acclimated to the treadmill (Exer-3/6 treadmill; promoting fat catabolism and preventing tissue accu- Columbus Instruments) 3 days prior to the experiments mulation of toxic lipid intermediates such as diacylgly- by running for 5 min/day at 5 m/min and 10 m/min cerol (DAG), ceramides, and long-chain acyl-CoAs. This followed by 15 m/min for 1 min. The grade increased by model stems from numerous human and animal stud- 5% each day, reaching a 10% grade on day three. During ies reporting that severe obesity and glucose intoler- the endurance protocol, mice ran for 30 min at 10 m/min. ance associate with increased muscle content of lipid Speed was increased 2 m/min every 15 min until reaching intermediates and reduced respiratory function (5–7). 28 m/min and then every 10 minutes until exhaustion. Additionally, investigators have suggested that individ- During the VO2 peak test, mice ran at 6 m/min, and speed uals with an inherently high content of oxidative myo- was increased 3 m/min every 3 min until exhaustion, de- fibers fare better during a weight-loss intervention fined as remaining on the shock grid for 10 consecutive than those with more glycolytic fibers (8–10). seconds. Respiratory exchange ratio (RER) and VO2 were Although a large body of correlational data supports monitored using the Oxymax Modular Treadmill System a link between oxidative potential and health outcomes, (Columbus Instruments). direct experimental evidence of cause and effect remains sparse (11). The principal goal of the current study was Mitochondrial Oxidation to test the causal relation between respiratory capacity, Mitochondria were isolated from the gastrocnemius muscle energy balance, and glucose control using a transgenic of MCK-PGC1a and NT mice fed an HFD for 6 weeks mouse model with muscle-specific overexpression of (18). Oxidation studies were performed using 0.2 mmol/L 14 6 14 PGC-1a (MCK-PGC1a), a transcriptional coactivator [1- C]palmitate 1.0 mmol/L sodium pyruvate. CO2 14 that functions as a master regulator of oxidative metab- trapped in NaOH and C-labeled acid-soluble metabolites olism (12). The MCK-PGC1a model was selected as a ge- (ASMs) were assessed by liquid scintillation counting in netically engineered mimic of exercise training because Uniscint BD (National Diagnostics) (21). 1)PGC-1a expression and activity increase with exercise Citrate Synthase Activity training (13,14), 2) muscle-specific overexpression of Ground tibialis anterior muscles were resuspended in this coactivator augments mitochondrial biogenesis, fat CelLytic MT (Sigma-Aldrich). Assays were performed on oxidation, and exercise endurance (15–17), and 3)acti- unclarified homogenates at 25°C in a reaction buffer con- vation of PGC-1a is often credited as a unifying mecha- taining acetyl-CoA, DTNB, and tris-HCl as previously de- nism that mediates favorable metabolic responses to scribed (22). Reactions were initiated with oxaloacetate. various behavioral, pharmacological, and genetic maneu- Data were normalized to ground tissue weight. vers (18,19). Herein, MCK-PGC1a mice and nontransgenic control Glucose Tolerance Tests littermates (NT) (20) were fed a high-fat diet (HFD) Mice were fasted 6 h before an intraperitoneal injection of administered alone or in combination with weight-loss glucose (1.5 g/kg lean body wt). Plasma glucose levels were interventions that encompassed exercise and caloric re- measured at the indicated times. striction. The animals were monitored longitudinally, and primary outcomes included body weight, glucose Glucose Oxidation in Isolated Muscle Strips tolerance, and comprehensive metabolic profiling of Whole soleus and extensor digitorum longus (EDL) muscles muscle specimens. Our findings challenge the notion were dissected from mice and placed in KHB buffer for that enhanced oxidative potential protects against met- reoxygenation and then incubated with 5 mmol/L 14C- abolic disease and raise new and important questions glucose 6 100nmol/Lporcineinsulinfor2hat37°C. regarding the roles of mitochondrial function and lipo- Reactions were terminated with 70% perchloric acid. 14 fi toxicity in the pathogenesis of obesity and insulin CO2 was trapped in 1 N NaOH and quanti ed by liquid resistance. scintillation counting in Uniscint BD (National Diagnostics). Flash-frozen muscles were used for measurement of gly- RESEARCH DESIGN AND METHODS cogen synthesis (23). Animal studies were approved by the Duke University Institutional Animal Care and Use Committee. Male mice Targeted Proteomics were housed in a temperature-controlled environment with Proteomic analysis was performed as previously described a 12:12 h light:dark cycle and weaned on standard chow (5% (24). Briefly, mitochondria isolated from the quadriceps fat, 72% carbohydrate) (5001; Harlan Teklad). At 13–16 wererunonanSDS-PAGEgelandunderwentin-gel
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