Molecular mechanism for negative regulation of insulin-stimulated Akt phosphorylation and glucose uptake by endothelin-1 in skeletal muscle cells

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  • Horinouchi Takahiro
    Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
  • Mazaki Yuichi
    Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
  • Hoshi Akimasa
    Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan
  • Terada Koji
    Department of Biochemistry and Molecular Biology, Shiga University of Medical Science, Japan
  • Miwa Soichi
    Department of Cellular Pharmacology, Graduate School of Medicine, Hokkaido University, Japan

Abstract

<p>[Background and purpose] Endothelin-1 (ET-1) is a potent vasoconstrictor and pro-inflammatory peptide that is involved in the onset and development of various diseases, including diabetes mellitus, systemic and pulmonary hypertension, and atherosclerosis. ET-1 is suggested to cause insulin resistance through a direct action on skeletal muscle, but not through a reduced insulin delivery to the skeletal muscle resulting from vasoconstriction. In human skeletal muscle cells, ET-1 impairs insulin-stimulated Akt phosphorylation and glucose uptake. However, molecular mechanism for negative regulation of insulin-stimulated Akt phosphorylation and glucose uptake by ET-1 in skeletal muscle cells remains unclear. The purpose of this study is to determine the signaling pathways underlying the inhibitory effects of ET-1 on the insulin-induced facilitation of Akt phosphorylation and glucose uptake in myotubes of rat L6 skeletal muscle cell line. [Results] mRNA expression levels of differentiation marker genes, MyoD and myogenin, were increased during myoblasts differentiation into myotubes. Some of myotubes possessed the ability to spontaneously constrict. In L6 myotubes, insulin stimulated Akt phosphorylation at Thr380 and Ser473, and [3H]-labelled 2-deoxy-D-glucose ([3H]2-DG) uptake. ET-1 inhibited insulin-stimulated Akt phosphorylation and [3H]2-DG uptake. 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 a dominant negative G protein-coupled receptor kinase 2 (GRK2) construct, and knockdown of GRK2. In L6 myotubes overexpressing FLAG-GRK2, endogenous Akt directly bound to FLAG-GRK2 and their association was enhanced by ET-1. [Conclusions] These findings suggest that activation of ETAR with ET-1 impairs insulin-induced Akt phosphorylation and [3H]2-DG uptake in a GRK2-dependent manner in skeletal muscle, and that ETAR and GRK2 are potential targets for insulin resistance.</p>

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