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page. BACKGROUND AND PURPOSE Endothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, leading to the development of insulin resistance. The purpose of this study is to determine molecular mechanisms underlying negative regulation by ET-1 of insulin signaling. EXPERIMENTAL APPROACH Myoblasts of rat L6 skeletal muscle cell line were differentiated into myotubes. Western blotting was employed to analyze changes in the phosphorylation levels of Akt at threonine 308 (Thr) and serine 473 (Ser). Effect of ET-1 on insulin-stimulated glucose uptake was assessed with [H]-labelled 2-deoxy-D-glucose ([H]2-DG). C-terminus region of G protein-coupled receptor kinase 2 (GRK2-ct), a dominant negative GRK2, was overexpressed in L6 cells using adenovirus-mediated gene transfer. GRK2 expression was suppressed by transfection of siRNA. KEY RESULTS In L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr and Ser, which was suppressed by ET-1. The inhibitory effects of ET-1 were counteracted by treatment with a selective ET type A receptor (ETAR) antagonist and a Gq protein inhibitor, overexpression of GRK2-ct, and knockdown of GRK2. Insulin increased [H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [H]2-DG uptake. Blockade of ETAR, overexpression of GRK2-ct and knockdown of GRK2 cancelled the ET-1-induced suppression of insulin-stimulated [H]2-DG uptake. In L6 myotubes overexpressing FLAG-GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2. CONCLUSIONS AND IMPLICATIONS Activation of ETAR with ET-1 suppresses insulin-induced Akt phosphorylation at Thr and Horinouchi et al. 4 Ser and [H]2-DG uptake in a GRK2-dependent manner in skeletal muscle. These findings suggest that ETAR and GRK2 are potential targets for insulin resistance. Abbreviations BSA, bovine serum albumin; DMEM, Dulbecco’s modified Eagle’s medium; ERK1/2, extracellular signal-regulated kinases 1/2; ET-1, endothelin-1; ETAR, endothelin type A receptor; ETBR, endothelin type B receptor; FCS, fetal calf serum; GLUT, glucose transporter; GPCR, G protein-coupled receptor; GRK2, G protein-coupled receptor kinase 2; GRK2-ct, C-terminus region of G protein-coupled receptor kinase 2; GSV, glucose transporter storage vesicle; [H]2-DG, [1,2-H(N)]-2-deoxy-D-glucose; HRP, horseradish peroxidase; HS, horse serum; IR, insulin receptor; IRS, insulin receptor substrate; PDK1, phosphoinositide-dependent kinase 1; PI3K, phosphatidylinositol 3-kinase; PTX, pertussis toxin; qRT-PCR, quantitative reverse transcription-polymerase chain reaction; Rab-GTPase, Rab guanosine triphosphatase; siRNA, short interfering RNA. Horinouchi et al. 5 Introduction Endothelin-1 (ET-1) is a potent vasoconstrictor and pro-inflammatory peptide (Yanagisawa et al., 1988), that has been implicated in the pathophysiology of various diseases, including diabetes mellitus (Pernow et al., 2012), systemic and pulmonary hypertension (Rodríguez-Pascual et al., 2011), and atherosclerosis (Pernow et al., 2012). It is reported that ET-1 levels in the plasma are elevated in patients with these diseases (Ferri et al., 1995; Lerman et al., 1991; Takahashi et al., 1990; Touyz et al., 2003). Excessive production of ET-1 causes a prolonged vasoconstriction mediated mainly through ET type A receptor (ETAR) (Horinouchi et al., 2013), endothelial dysfunction resulting from downregulation of ETBR (Rubin, 2012), and insulin resistance (Shemyakin et al., 2011; Usui et al., 2005). Insulin resistance is a condition where the sensitivity to insulin of the cells expressing insulin receptor (IR) is decreased because of a functional disturbance of insulin-mediated intracellular signaling. The most important tissue responsible for the insulin’s action of decreasing blood glucose levels is skeletal muscle which accounts for approximately 70% of total glucose uptake in healthy subjects (DeFronzo, 1988). Accordingly, disturbance of insulin-stimulated glucose uptake into skeletal muscle is mainly responsible for elevated blood glucose levels in patients with type 2 diabetes (DeFronzo, 1988), where there is no significant change in glucose transporter 4 (GLUT4) protein levels (Schalin-Jäntti et al., 1994). Insulin increases glucose uptake rate in skeletal muscle cells through facilitating translocation of GLUT4 from intracellular GLUT storage vesicle (GSV) to the plasma membrane (Leto et al., 2012). Activation of IR induces phosphorylation of IR substrate (IRS) proteins, resulting in the recruitment and activation of phosphatidylinositol 3-kinase (PI3K) (Leto et al., 2012). PI3K converts phosphatidylinositol-4,5-diphosphate to phosphatidylinositol-3,4,5-trisphosphate which is involved in the recruitment of phosphoinositide-dependent kinase 1 (PDK1) and Akt (Leto et al., 2012; Shisheva, 2008). Akt translocated to the plasma membrane is activated by phosphorylation at threonine 308 (Thr) by PDK1 and at serine 473 (Ser) by mammalian target of rapamycin complex 2 (Laplante et al., 2012; Leto et al., 2012; Sarbassov et al., 2005). Activated Akt facilitates Horinouchi et al. 6 the translocation of GLUT4 to the plasma membrane by phosphorylation and subsequent inactivation of the Rab guanosine triphosphatase (GTPase)-activating protein, AS160 (Leto et al., 2012). The decreased Rab-GTPase activity increases the ratio of the GTP-bound form (active form) of Rab to the GDP-bound form (inactive form), leading to facilitation of GLUT4 translocation and cell membrane fusion (Leto et al., 2012). These data implicate that Akt activation is essential for insulin-stimulated glucose uptake via GLUT4 in skeletal muscle. Several studies have suggested that ET-1 induces insulin resistance through a direct action on skeletal muscle, but not through a reduced insulin delivery to the skeletal muscle resulting from vasoconstriction. That is, in healthy subjects, administration of exogenous ET-1 reduces insulin-stimulated glucose uptake in skeletal muscle without decreasing skeletal muscle blood flow (Ottosson-Seeberger et al., 1997). In addition, prolonged treatment of primary culture of human skeletal muscle cells with ET-1 impairs insulin-stimulated Akt phosphorylation and glucose uptake (Shemyakin et al., 2011). However, the signaling pathways underlying the inhibitory effects of ET-1 on the insulin-induced facilitation of Akt phosphorylation and glucose uptake in skeletal muscle cells are unknown. Growing evidence shows that G protein-coupled receptor (GPCR) kinase 2 (GRK2), a ubiquitous Ser/Thr protein kinase, can function as a negative regulator of insulin signaling in phosphorylation-independent manner, although it was originally identified as a kinase which specifically phosphorylates and desensitizes agonist-stimulated GPCRs (Evron et al., 2012). Several lines of evidence demonstrate that GRK2 binds to numerous signaling molecules including Akt (Liu et al., 2005) and Gβγ subunits (Evron et al., 2012). A direct interaction of GRK2 with Akt impairs Akt activation, causing negative regulation of Akt signaling (Liu et al., 2005). Binding of GRK2 to Gβγ subunits is essential for translocation of GRK2 to the plasma membrane (Evron et al., 2012). These data encouraged us to assume that activation of ETRs in skeletal muscle cells induces insulin resistance through inhibition of Akt signaling: namely, activation of ETRs promotes dissociation of receptor-coupled G protein into its subunits, Gα and Gβγ; the increase in the local concentration of Gβγ subunit in turn recruits GRK2 to the plasma membrane, leading to the augmented interaction of GRK2 with Horinouchi et al. 7 Akt at the plasma membrane and the resulting inhibition of Akt signaling. The purpose of this study was to determine molecular mechanisms underlying negative regulation by ET-1 of insulin signaling in rat L6 myotubes with special attention to possible involvement of GRK2. Since both protein contents of GLUT4 and insulin-stimulated glucose uptake via GLUT4 were increased during myogenesis in L6 cells (Mitsumoto et al., 1992), L6 myoblasts were highly differentiated into myotubes with a modified method to estimate insulin-stimulated glucose uptake. Horinouchi et al. 8 METHODS Materials YM-254890 was kindly provided by Astellas Pharma Inc. (Tokyo, Japan). The following drugs and reagents were used in the present study: synthetic human ET-1, BQ-123 [cyclo(D-trp-D-asp-L-pro-D-val-L-leu)], BQ-788 (N-cis-2,6-dimethyl-piperidinocarbonyl-L-γ-methylleucyl-D-1-methoxycarbonyltryptophanyl -D-norleucine) (Peptide Institute, Osaka, Japan); human insulin (Cell Science and Technology Institute, Inc., Miyagi, Japan); bovine serum albumin (BSA), Hoechst 33342 (Sigma-Aldrich Co., St. Louis, MO, U.S.A.); LY294002 [2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one], pertussis toxin (PTX) (Calbiochem, San Diego, CA, U.S.A.); [1,2-H(N)]-2-deoxy-D-glucose ([H]2-DG; specific activity: 8.0 Ci mmol, 1.0 mCi ml in aqueous solution, PerkinElmer, Inc., Boston, MA, U.S.A.); fetal calf serum (FCS), horse serum (HS), D-glucoseand sodium pyruvate-free Dulbecco’s modified Eagle’s medium (DMEM), EDTA-free, protease inhibitor cocktail (Thermo Fisher Scientific Inc., Waltham, MA, U.S.A.); low-glucose (5.5 mM) DMEM (Wako Pure Chemical Industries, Ltd., Osaka, Japan); GSK2126458 [2,4-difluoro-N-(2-methoxy-5-(4-(pyridazin-4-yl)quinolin-6-yl)pyridin-3-yl)benzenesulfona mide] (Selleck Chemicals, Houston, TX, U.S.A.); NF449 (4,4',4'',4'''-[carbonylbis(imino-5,1,3-benzenetriyl-bis(carbonylimino))]tetrakis-1,3-benzenedi sulfonic acid, octasodium salt) (Tocris Bioscience, Moorend Farm Avenue, Bristol, U.K.); ON-TARGETplus Rat Adrbk1 (25238) siRNA SMARTpool (L-090990-02-0005), ON-TARGETplus Non-targeting Pool (D-001810-10-05) (GE Healthcare Dharmacon Inc., Lafayette, CO, U.S.A.); Lipofectamine RNAiMAX Transfection Reagent, Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific Inc.). Primary antibodies for phospho-Akt (Thr), phospho-Ak