open access

https://hdl.handle.net/2115/64631

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

Mechanisms for ET-1-induced insulin resistance

英語

2016-03

Wiley-Blackwell

British journal of pharmacology

173(6)

1018-1032

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 [3H]-2-deoxy-d-glucose ([3H]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 Thr308 and Ser473, 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 [3H]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [3H]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [3H]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 Thr308 and Ser473 and [3H]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.

学術雑誌論文

Accepted Manuscript

This is the peer reviewed version of the following article: Horinouchi, T., Hoshi, A., Harada, T., Higa, T., Karki, S., Terada, K., Higashi, T., Mai, Y., Nepal, P., Mazaki, Y., and Miwa, S. (2016) Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells. British Journal of Pharmacology, 173: 1018–1032, which has been published in final form at http://dx.doi.org/10.1111/bph.13406. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.

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