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Human Muscle Fiber Type Specific Insulin Signaling – Impact of Obesity and Type 2 Diabetes Peter H. Albers1,2, Andreas J.T. Pe

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Human Muscle Fiber Type Specific Insulin Signaling – Impact of Obesity and Type 2 Diabetes Peter H. Albers1,2, Andreas J.T. Pe Page 1 of 44 Diabetes Human muscle fiber type specific insulin signaling – Impact of obesity and type 2 diabetes Peter H. Albers1,2, Andreas J.T. Pedersen3, Jesper B. Birk1, Dorte E. Kristensen1, Birgitte F. Vind3, Otto Baba4, Jane Nøhr2, Kurt Højlund3, Jørgen F.P. Wojtaszewski1 1Section of Molecular Physiology, Department of Nutrition, Exercise and Sports, August Krogh Centre, University of Copenhagen, Denmark 2Diabetes Research Unit, Novo Nordisk A/S, Maaloev, Denmark 3Diabetes Research Center, Department of Endocrinology, Odense University Hospital, Odense, Denmark 4Section of Biology, Department of Oral Function & Molecular Biology, School of Dentistry, Ohu University, Koriyama, Japan *Corresponding Author: Jørgen F.P. Wojtaszewski, PhD. Universitetsparken 13, DK-2100 Copenhagen Ø, Denmark. Phone no: (+45) 28751625, e-mail: [email protected] Running title: Muscle fiber types and insulin signaling Word count (abstract): 197 Word count (main text): 3979 References: 48 Number of tables+figures: 2+6 1 Diabetes Publish Ahead of Print, published online September 3, 2014 Diabetes Page 2 of 44 ABSTRACT Skeletal muscle is a heterogeneous tissue composed of different fiber types. Studies suggest that insulin-mediated glucose metabolism is different between muscle fiber types. We hypothesized that differences are due to fiber-type specific expression/regulation of insulin signaling elements and/or metabolic enzymes. Pools of type I and II fibers were prepared from biopsies of the vastus lateralis muscles from lean, obese and type 2 diabetic subjects before and after a hyperinsulinemic- euglycemic clamp. Type I fibers compared to type II fibers have higher protein levels of the insulin receptor, GLUT4, hexokinase II, glycogen synthase (GS), pyruvate dehydrogenase (PDH-E1α) and a lower protein content of Akt2, TBC1D4 and TBC1D1. In type I fibers compared to type II fibers, the phosphorylation-response to insulin was similar (TBC1D4, TBC1D1 and GS) or decreased (Akt and PDH-E1α). Phosphorylation-responses to insulin adjusted for protein level were not different between fiber types. Independently of fiber type, insulin signaling was similar (TBC1D1, GS and PDH-E1α) or decreased (Akt and TBC1D4) in muscle from patients with type 2 diabetes compared to lean and obese subjects. We conclude that human type I muscle fibers compared to type II fibers have a higher glucose handling capacity but a similar sensitivity for phosphor-regulation by insulin. 2 Page 3 of 44 Diabetes Keywords Skeletal muscle • Insulin sensitivity • Glucose disposal rate • Indirect calorimetric • Insulin signaling • Myosin heavy chain composition • Glycogen • GLUT4 • Glycogen synthase • TBC1 domain family member • Pyruvate dehydrogenase • Akt Abbreviations BMI Body mass index EDL Extensor digitorum longus GDR Glucose disposal rate GS Glycogen synthase GSK Glycogen synthase kinase HK Hexokinase HRP Horseradish peroxidase mTOR mammalian target of rapamycin mTORC mammalian target of rapamycin complex MHC Myosin heavy chain NDRG N-myc downstream-regulated gene NOGM Non-oxidative glucose uptake PDC Pyruvate dehydrogenase complex PDH-E1α Pyruvate dehydrogenase-E1 alpha subunit RT Room temperature T2D Type 2 diabetic TBC1 Tre-2/USP6, BUB2, cdc16 TBC1D TBC1 domain family member 3 Diabetes Page 4 of 44 INTRODUCTION Skeletal muscle is important for whole body insulin-stimulated glucose disposal (1), and skeletal muscle insulin resistance is a common phenotype of obesity and type 2 diabetes (2). Skeletal muscle is a heterogeneous tissue composed of different fiber types, which can be divided according to myosin heavy chain (MHC) isoform expression. Studies in rodents show that insulin-stimulated glucose uptake in the oxidative type I fiber-dominant muscles is higher than in muscles with a high degree of glycolytic type II fibers (3-6). Whether this phenomenon is due to differences in locomotor activity of individual muscles or a direct consequence of the fiber-type composition is largely unknown. In incubated rat muscle, insulin-induced glucose uptake was higher (~100%) in type IIa (oxidative/glycolytic) compared to IIx and IIb (glycolytic) fibers (7;8), suggesting that insulin-mediated glucose uptake is related to the oxidative capacity of the muscle fiber. In humans, a positive correlation between proportions of type I fibers in muscle and whole-body insulin sensitivity has been demonstrated (9-11). Furthermore, insulin-stimulated glucose transport in human muscle strips was associated with the relative type I fiber content (12). Thus, it is likely that human type I fibers are more important than type II fibers for maintaining glucose homeostasis in response to insulin. Indeed, a decreased proportion of type I fibers has been found in various insulin resistant states such as the metabolic syndrome (9), obesity (13;14), type 2 diabetes in some (10;13;14) but not all (12;15) studies and following bedrest (16) as well as in tetraplegic patients (17) and subjects with an insulin receptor gene mutation (18). Mechanisms for a fiber-type dependent regulation of glucose uptake could involve altered abundance/regulation of insulin signaling elements and/or metabolic enzymes. In rats, insulin receptor content and Akt and GLUT4 protein abundance are higher in type I compared to type II fiber dominated muscles (4;5;19-21). Furthermore, in rats, Akt phosphorylation under insulin stimulationare highest in type I compared to type II fiber dominant muscles (20). In humans, 4 Page 5 of 44 Diabetes GLUT4 protein levels are higher in type I compared to type IIa and IIx muscle fibers (14;22). Overall, these findings suggest that insulin signaling to and effect on glucose transport is highest in type I fibers. Thus, a shift towards reduced type I and hence higher type II fiber content in obesity and type 2 diabetes (10;13;14) could negatively influence muscle insulin action on glucose metabolism. Insulin resistance in obesity and type 2 diabetes is characterized by a decreased ability of insulin to induce signaling proteins proposed to mediate GLUT4 translocation by i.e. phosphorylation/activation of Akt (23-25) and/or TBC1 domain family member (TBC1D) 4 (23;25). Whether this relates to differences in the response to insulin between fiber types is unknown. Intracellular glucose metabolism could also be different between muscle fiber types. Glucose entering the muscle cell is initially phosphorylated by hexokinase (HK) and predominantly stored as glycogen or oxidized in the mitochondria, through processes regulated by glycogen synthase (GS) and the pyruvate dehydrogenase complex (PDC), respectively. HKII content is higher in human soleus muscle (~70% type I fibers) compared to gastrocnemius and vastus lateralis muscle (~50% type I fibers) (26). Also, the content of the PDC subunit PDH-E1α is decreased in muscle of proliferator-activated receptor gamma-coactivator-1α knock-out mice (27), concomitant with a switch towards reduced type I fiber abundance (28). Furthermore, mitochondrial density is higher in human type I compared to type II fibers (29). In contrast, no fiber-type specific expression pattern of GS has been shown (30). All together these observations suggest that glucose phosphorylation and oxidation but not storage rate capacity are enhanced in type I compared to type II fibers. Whether HKII and PDH-E1α abundance as well as GS and PDH-E1α regulation by insulin is different between human muscle fiber types is unknown. We investigated whether proteins involved in glucose metabolism were expressed and/or regulated by insulin in a fiber type specific manner in human skeletal muscle. This was achieved by creating pools of single fibers expressing either MHC I (type I) or II (type II). These fibers were dissected 5 Diabetes Page 6 of 44 from vastus lateralis muscle biopsies obtained from lean and obese normal glucose tolerant subjects as well as type 2 diabetic patients. RESEARCH DESIGN AND METHODS Subjects. 10 lean healthy, 11 obese non-diabetic and 11 obese type 2 diabetic (T2D) subjects were randomly chosen from two studies conducted at Odense University Hospital, Odense, Denmark. One fraction (8 lean, 7 obese, 6 T2D) were from an already published study (31), while the remaining subjects were from an unpublished study, in which subjects were investigated with an identical experimental protocol as previously described (31). Both studies were approved by the regional ethics committee and carried out in accordance with the Declaration of Helsinki II. Subject medication is detailed in supplemental materials. Experimental protocol. Detailed explanation of the in vivo study protocol has been published elsewhere (31). In short, all subjects were instructed to refrain from strenuous physical activity 48 h before the experimental day. After an overnight fast, subjects underwent a 2 h basal tracer equilibration period followed by a 4 h hyperinsulinemic-euglycemic clamp at an insulin (Actrapid, Novo Nordisk, Denmark) infusion rate of 40 mU·m-2·min-1 combined with tracer glucose and indirect calorimetry. A primed-constant [3-3H]glucose infusion was used throughout the 6-h study, and [3-3H]glucose was added to the glucose infusates to maintain plasma specific activity constant at baseline levels during the 4-h clamp period as described in detail previously (32). Vastus lateralis muscle biopsies were obtained before and after the clamp under local anesthesia (1% lidocaine) using a modified Bergström needle with suction. Muscle biopsies were immediately frozen in liquid nitrogen and stored below -80⁰C. Dissection of individual muscle fibers. Muscle fibers were prepared as previously described (33) but with minor modifications. 20-60 mg of muscle tissue were freeze-dried for 48 h before 6 Page 7 of 44 Diabetes dissection of individual muscle fibers in a climate-controlled room (20⁰C, <35% humidity) using a dissection microscope (in total n= 5384 fibers from 64 biopsies). The length of each fiber was estimated under the microscope (1.5±0.4 mm, mean±SD) before being carefully placed in a PCR- tube and stored on dry-ice. On the day of dissection 5 µl of ice-cooled Laemmli sample buffer (125 mM Tris-HCl, pH 6.8, 10% glycerol, 125 mM SDS, 200 mM DTT, 0.004% Bromophenol Blue) was added to each tube.
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