Direct Activation of Glucose Transport in Primary Human Myotubes After Activation of Peroxisome Proliferator–Activated Receptor ␦ David Kitz Kra¨mer,1,2 Lubna Al-Khalili,1,2 Sebastio Perrini,1,2 Josefin Skogsberg,3 Per Wretenberg,1 Katja Kannisto,3 Harriet Wallberg-Henriksson,1,2 Ewa Ehrenborg,3 Juleen R. Zierath,1,2 and Anna Krook1,2 Activators of peroxisome proliferator–activated recep- mote gene regulatory responses in cultured human skel- ␥ etal muscle. Moreover, we provide biological validation tor (PPAR) have been studied intensively for their ␦ insulin-sensitizing properties and antidiabetic effects. of PPAR as a potential target for antidiabetic therapy. Recently, a specific PPAR␦ activator (GW501516) was Diabetes 54:1157–1163, 2005 reported to attenuate plasma glucose and insulin levels when administered to genetically obese ob/ob mice. This study was performed to determine whether specific activation of PPAR␦ has direct effects on insulin action ype 2 diabetes is a clinical disorder of glucose in skeletal muscle. Specific activation of PPAR␦ using and lipid metabolism that is partly caused by two pharmacological agonists (GW501516 and GW0742) impaired glucose uptake into adipose tissue and increased glucose uptake independently of insulin in skeletal muscle (1). Although genetic and envi- differentiated C2C12 myotubes. In cultured primary T ronmental (dietary and lifestyle) factors contribute to the human skeletal myotubes, GW501516 increased glucose uptake independently of insulin and enhanced subse- development of insulin resistance and the onset of type 2 quent insulin stimulation. PPAR␦ agonists increased the diabetes (2), the exact mechanism for the metabolic respective phosphorylation and expression of AMP-ac- changes accompanying these disorders has not yet been tivated protein kinase 1.9-fold (P < 0.05) and 1.8-fold completely elucidated. This is partly reflected by the (P < 0.05), of extracellular signal–regulated kinase 1/2 complex interaction between insulin target tissues includ- mitogen-activated protein kinase (MAPK) 2.2-fold (P < ing adipose tissue, an endocrine organ that secretes sev- 0.05) and 1.7-fold (P < 0.05), and of p38 MAPK 1.2-fold eral hormones and cytokines, and skeletal muscle, a major (P < 0.05) and 1.4-fold (P < 0.05). Basal and insulin- site of insulin resistance in type 2 diabetic patients (3,4). stimulated protein kinase B/Akt was unaltered in cells ␦ Peroxisome proliferator–activated receptors (PPARs) preexposed to PPAR agonists. Preincubation of myo- play key roles in defining adipose tissue mass modulating tubes with the p38 MAPK inhibitor SB203580 reduced insulin- and PPAR␦-mediated increase in glucose up- fat and carbohydrate metabolism and storage (5). Three take, whereas the mitogen-activated protein kinase ki- closely related PPAR subtypes have been described: ␣, nase inhibitor PD98059 was without effect. PPAR␦ ␤/␦, and ␥ (6–8). The different PPAR subtypes display agonists reduced mRNA expression of PPAR␦, sterol tissue-specific expression; PPAR␥ is primarily expressed regulatory element binding protein (SREBP)-1a, and in adipocytes (7,9), PPAR␣ is most abundant in liver, and SREBP-1c (P < 0.05). In contrast, mRNA expression of PPAR␦ (also referred to as PPAR␤) is expressed in a wide PPAR␥, PPAR␥ coactivator 1, GLUT1, and GLUT4 was variety of tissues, with high levels in skeletal muscle (9). unaltered. Our results provide evidence to suggest that PPAR␥ plays a key role in adipocyte differentiation and ␦ PPAR agonists increase glucose metabolism and pro- lipid storage (rev. in 7). Thiazolodinediones (TZDs) are a class of insulin-sensitizing drugs that specifically activate PPAR␥ and promote adipocyte differentiation (10,11). From the 1Department of Surgical Science, Karolinska Institute, Stockholm, Sweden; the 2Department of Physiology and Pharmacology, Karolinska Insti- Treatment of type 2 diabetic patients with TZDs improves tute, Stockholm, Sweden; and the 3Gustaf V Research Institute, Karolinska glucose homeostasis (12,13) and alters gene expression in Hospital, Stockholm, Sweden. ␣ Address correspondence and reprint requests to Anna Krook, Integrative adipocytes (8). PPAR is activated by fibrates, a class of Physiology, Department of Physiology and Pharmacology, Karolinska Insti- cholesterol-lowering drugs used in the treatment of lipid tute, 171 77 Stockholm, Sweden. E-mail: [email protected]. disorders, and functions by enhancing ␤-oxidation in the Received for publication 2 June 2004 and accepted in revised form 1 ␣ ␥ January 2005. liver. In contrast to PPAR and PPAR , pharmacological AICAR, 5-aminoimidazol-4-carboxamide-1␤-D-ribonucleoside; AMPK, AMP- treatments to activate PPAR␦ in humans with metabolic activated protein kinase; DMEM, Dulbecco’s minimum essential medium; disease are lacking. ERK, extracellular signal–regulated kinase; FBS, fetal bovine serum; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase Activation of PPAR␦ is linked to oxidative metabolism. kinase; PGC, peroxisome proliferator–activated receptor ␥ coactivator; PKB, The role of PPAR␦ on whole-body glucose homeostasis protein kinase B; PPAR, peroxisome proliferator–activated receptor; SREBP, ␦ sterol regulatory element binding protein; TZD, thiazolodinedione. has been evaluated in muscle-specific PPAR transgenic © 2005 by the American Diabetes Association. mice (14). PPAR␦ transgenic mice are characterized by The costs of publication of this article were defrayed in part by the payment of page enzymatic and gene expression profiles that promote charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. oxidative metabolism in skeletal muscle. Moreover, DIABETES, VOL. 54, APRIL 2005 1157 GW501516 INCREASES GLUCOSE METABOLISM PPAR␦ transgenic mice have reduced body fat mass due to a reduction of adipose cell size (15). Specific activation of PPAR␦ using a pharmacological activator, GW501516 (16,17), has been reported to have positive effects on expression of genes involved in cardiac lipid metabolism in neonatal rat cardiomyocytes (18). When administered to insulin-resistant middle-aged obese rhesus monkeys, GW501516 causes a dramatic dose-dependent rise in se- rum HDL cholesterol, while lowering the levels of small dense LDL and fasting triglycerides and reducing fasting insulin (16). Short-term treatment of obese mice with GW501516 causes dramatic lipid depletion in tissues (15) and attenuates plasma glucose and insulin levels (19). Given the importance of skeletal muscle insulin resis- FIG. 1. Glucose uptake in 3T3-L1 mouse adipocytes incubated for 6 h tance in the development of type 2 diabetes and other with or without 10 nmol/l GW501516 (GW). *P < 0.01 vs. basal or vs. 1 Insulin stimulation .(9 ؍ nmol/l insulin–treated cells as indicated (n metabolic disease, we hypothesized that targeted activa- was included for the last 60 min. tion of PPAR␦ in skeletal muscle could have direct effects on glucose metabolism by improving insulin action. We determined whether direct activation of PPAR␦ using 10% FBS and 1% penicillin/streptomycin. The cells were used ϳ12 days after pharmacological activators alters glucose metabolism, sig- completion of the differentiation protocol, when Ͼ90% of the cells expressed the adipocyte phenotype (i.e., filled with fat droplets). Before experimenta- nal transduction, and gene expression in cultured primary tion, the cells were washed and preincubated with DMEM containing 5 mmol/l human muscle cells. We also elucidated whether similar glucose, 25 mmol/l HEPES (pH 7.4), and 1% penicillin/streptomycin without effects are noted in cultured mouse muscle cell (C2C12) FBS. and adipocyte cell (3T3-L1) lines. C2C12 cell cultures. Mouse C2C12 myoblasts were cultured in DMEM (1,000 mg/l glucose) containing 20% FBS. For initiation of differentiation, the medium was changed to a 2% FBS content when cells were 100% confluent. The cells were grown until days 5–7 for formation of myotubes and then used for RESEARCH DESIGN AND METHODS experimentation. Dulbecco’s minimum essential medium (DMEM), Ham’s F-10 medium, fetal Glucose uptake. Glucose uptake was performed as previously described for bovine serum (FBS), penicillin, streptomycin, and fungizone were obtained primary human muscle cells (21) and 3T3-L1 adipocytes (22), respectively. from GibcoBRL Life Technologies (Stockholm, Sweden). Radiochemicals, When inhibition was performed before glucose uptake experiments, cells 3 Ϫ1 Ϫ1 14 2-[G- H]deoxy-D-glucose (6.0 Ci ⅐ mmol ⅐ l ), and D-[U- C]glucose (310 mCi were preincubated with ERK1/2 and p38 MAPK inhibitors PD98059 (50 Ϫ1 Ϫ1 ⅐ mmol ⅐ l ), were from Amersham (Uppsala, Sweden). All other chemicals ␮mol/l) or SB203580 (10 ␮mol/l). The inhibitors were added to Krebs buffer were analytical grade and from Sigma-Aldrich Sweden AB (Stockholm, with either GW0742 in DMSO or DMSO alone (as basal) for incubation at 37°C 473 Sweden). Phosphospecific antibodies against protein kinase B (PKB) (Ser ) for 15 min, and insulin was added as indicated. and extracellular signal–related kinase (ERK)1/2 mitogen-activated protein Western blot analysis. Cell monolayers were washed once in ice-cold PBS 202 204 kinase (MAPK) (Thr and Tyr ) were from New England Biolabs (Beverly, and harvested directly by scraping into ice-cold lysis buffer (135 mmol/l NaCl, 172 MA), and AMP-activated protein kinase (AMPK) pan ␣-subunit (Thr ) and 1 mmol/l MgCl , 2.7 mmol/l KCl, 20 mmol/l Tris [pH 8.0], 0.5 mmol/l Na VO