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Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells.

Authors: Horinouchi, Takahiro  Hoshi, Akimasa  Harada, Takuya  Higa, Tsunaki  Karki, Sarita  Terada, Koji  Higashi, Tsunehito  Mai, Yosuke  Nepal, Prabha  Mazaki, Yuichi  Miwa, Soichi 
Citation: Horinouchi T, etal., Br J Pharmacol. 2016 Mar;173(6):1018-32. doi: 10.1111/bph.13406. Epub 2016 Feb 15.
Pubmed: (View Article at PubMed) PMID:26660861
DOI: Full-text: DOI:10.1111/bph.13406


BACKGROUND AND PURPOSE: 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.
EXPERIMENTAL APPROACH: 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).
KEY 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 AND IMPLICATIONS: 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.

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CRRD Object Information
CRRD ID: 13514041
Created: 2018-03-21
Species: All species
Last Modified: 2018-03-21
Status: ACTIVE



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RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.