title: Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells.
abstract: OBJECTIVE
  Endothelin-1 (ET-1) reduces insulin-stimulated glucose uptake in skeletal muscle, inducing insulin resistance. Here, we have determined the molecular mechanisms underlying negative regulation by ET-1 of insulin signalling.
  
  
  METHODS
  We used the rat L6 skeletal muscle cells fully differentiated into myotubes. Changes in the phosphorylation of Akt was assessed by Western blotting. Effects of ET-1 on insulin-stimulated glucose uptake was assessed with [(3) H]-2-deoxy-d-glucose ([(3) H]2-DG). The C-terminus region of GPCR 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 the corresponding short-interfering RNA (siRNA).
  
  
  RESULTS
  In L6 myotubes, insulin elicited sustained Akt phosphorylation at Thr(308) and Ser(473) , which was suppressed by ET-1. The inhibitory effects of ET-1 were prevented by treatment with a selective ETA receptor antagonist and a Gq protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [(3) H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [(3) H]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [(3) H]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2.
  
  
  CONCLUSIONS
  Activation of ETA receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr(308) and Ser(473) and [(3) H]2-DG uptake in a GRK2-dependent manner in skeletal muscle cells. These findings suggest that ETA receptors and GRK2 are potential targets for overcoming insulin resistance.
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Horinouchi
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Akimasa
authors.initials: A
authors.lastname: Hoshi
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Harada
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Tsunaki
authors.initials: T
authors.lastname: Higa
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Sarita
authors.initials: S
authors.lastname: Karki
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Koji
authors.initials: K
authors.lastname: Terada
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Tsunehito
authors.initials: T
authors.lastname: Higashi
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Mai
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Prabha
authors.initials: P
authors.lastname: Nepal
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Mazaki
authors.affiliation: Department of Cellular Pharmacology, Hokkaido University Graduate School of Medicine, Sapporo City, Japan.
authors.forename: Soichi
authors.initials: S
authors.lastname: Miwa
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D019332:Endothelin-1 / Q000494:pharmacology*; D054769:G-Protein-Coupled Receptor Kinase 2 / Q000235:genetics / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology*; D018485:Muscle Fibers, Skeletal / Q000187:drug effects* / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology; D017570:MyoD Protein / Q000235:genetics; D032446:Myoblasts / Q000166:cytology; D018008:Myogenin / Q000235:genetics; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D051381:Rats
chemical_list: D019332:Endothelin-1; D007328:Insulin; D017570:MyoD Protein; C503803:Myog protein, rat; D018008:Myogenin; D012333:RNA, Messenger; D012337:RNA, Ribosomal, 18S; D051057:Proto-Oncogene Proteins c-akt; C496569:Grk2 protein, rat; D054769:G-Protein-Coupled Receptor Kinase 2; D005947:Glucose