Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells
Endothelin-1 suppresses insulin-stimulated Akt phosphorylation and glucose uptake via GPCR kinase 2 in skeletal muscle cells
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DOI：
10.1111/bph.13406
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发表时间：
2016-03-01
影响因子：
7.3
通讯作者：
Miwa, Soichi
中科院分区：
文献类型：
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作者：
Horinouchi, Takahiro;Hoshi, Akimasa;Miwa, Soichi
背景和目的内皮素-1 (ET-1) 会减少骨骼肌中胰岛素刺激的葡萄糖摄取，从而诱导胰岛素抵抗。在这里，我们确定了 ET-1 对胰岛素信号负向调节的分子机制。实验方法我们使用完全分化为肌管的大鼠 L6 骨骼肌细胞。通过蛋白质印迹评估 Akt 磷酸化的变化。用[H-3]-2-脱氧-d-葡萄糖([H-3]2-DG)评估ET-1对胰岛素刺激的葡萄糖摄取的影响。 GPCR 激酶 2 (GRK2-ct) 的 C 末端区域（GRK2 显性失活）通过腺病毒介导的基因转移在 L6 细胞中过表达。 GRK2 表达通过转染相应的短干扰 RNA (siRNA) 受到抑制。 主要结果 在 L6 肌管中，胰岛素在 Thr(308) 和 Ser(473) 处引发持续的 Akt 磷酸化，而这种磷酸化被 ET-1 抑制。通过选择性 ETA 受体拮抗剂和 G(q) 蛋白抑制剂治疗、GRK2-ct 过表达和 GRK2 敲低，可防止 ET-1 的抑制作用。胰岛素以浓度依赖性方式增加[H-3]2-DG 摄取率。 ET-1 非竞争性拮抗胰岛素刺激的 [H-3]2-DG 摄取。 ETA 受体的阻断、GRK2-ct 的过度表达和 GRK2 的敲除可防止 ET-1 诱导的胰岛素刺激的 [H-3]2-DG 摄取抑制。在过表达 FLAG 标记的 GRK2 的 L6 肌管中，ET-1 促进内源性 Akt 与 FLAG-GRK2 的相互作用。结论和意义用 ET-1 激活 ETA 受体可抑制骨骼肌细胞中胰岛素诱导的 Akt Thr(308) 和 Ser(473) 磷酸化以及 [H-3]2-DG 摄取，以 GRK2 依赖性方式进行。这些发现表明 ETA 受体和 GRK2 是克服胰岛素抵抗的潜在靶标。
Background and PurposeEndothelin-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 ApproachWe 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 [H-3]-2-deoxy-d-glucose ([H-3]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 ResultsIn 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 G(q) protein inhibitor, overexpression of GRK2-ct and knockdown of GRK2. Insulin increased [H-3]2-DG uptake rate in a concentration-dependent manner. ET-1 noncompetitively antagonized insulin-stimulated [H-3]2-DG uptake. Blockade of ETA receptors, overexpression of GRK2-ct and knockdown of GRK2 prevented the ET-1-induced suppression of insulin-stimulated [H-3]2-DG uptake. In L6 myotubes overexpressing FLAG-tagged GRK2, ET-1 facilitated the interaction of endogenous Akt with FLAG-GRK2.Conclusions and ImplicationsActivation of ETA receptors with ET-1 suppressed insulin-induced Akt phosphorylation at Thr(308) and Ser(473) and [H-3]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.