1. Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells
- Author
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Horinouchi, Takahiro, Hoshi, Akimasa, Harada, Takuya, Higa, Tsunaki, Karki, Sarita, Terada, Koji, Higashi, Tsunehito, Mai, Yosuke, Nepal, Prabha, Mazaki, Yuichi, and Miwa, Soichi
- Subjects
Endothelin-1 ,G-Protein-Coupled Receptor Kinase 2 ,Muscle Fibers, Skeletal ,Cell Differentiation ,Research Papers ,Cell Line ,Rats ,Myoblasts ,Glucose ,RNA, Ribosomal, 18S ,Animals ,Insulin ,Myogenin ,RNA, Messenger ,Phosphorylation ,Muscle, Skeletal ,Proto-Oncogene Proteins c-akt ,MyoD Protein - Abstract
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.
- Published
- 2016