Dipartimento di Medicina Clinica, Medicina Clinica, Scienze Cardiovascolari ed Immunologiche, Università Federico II, Via Pansini 5, 80131 Naples, Italy.
Cardiovasc Res. 2009 Dec 1;84(3):407-15. doi: 10.1093/cvr/cvp252. Epub 2009 Jul 20.
Insulin (Ins) resistance (IRES) associates to increased cardiovascular risk as observed in metabolic syndrome. Chronic stimulation of beta-adrenergic receptors (betaAR) due to exaggerated sympathetic nervous system activity is involved in the pathogenesis of IRES. The cellular levels of G protein coupled receptor kinase 2 (GRK2) increase during chronic betaAR stimulation, leading to betaAR desensitization. We tested the hypothesis that GRK2 plays a role in betaAR-induced IRES.
We evaluated Ins-induced glucose uptake and signalling responses in vitro in cell overexpressing the beta(2)AR, the GRK2, or the catalytically dead mutant GRK2-DN. In a model of increased adrenergic activity, IRES and elevated cellular GRK2 levels, the spontaneously hypertensive rats (SHR) we performed the intravenous glucose tolerance test load. To inhibit GRK2, we synthesized a peptide based on the catalytical sequence of GRK2 conjugated with the antennapedia internalization sequence (Ant-124). Ins in human kidney embryonic (HEK-293) cells causes rapid accumulation of GRK2, tyrosine phosphorylation of Ins receptor substrate 1 (IRS1) and induces glucose uptake. In the same cell type, transgenic beta(2)AR overexpression causes GRK2 accumulation associated with significant deficit of IRS1 activation and glucose uptake by Ins. Similarly, transgenic GRK2 overexpression prevents Ins-induced tyrosine phosphorylation of IRS1 and glucose uptake, whereas GRK2-DN ameliorates glucose extraction. By immunoprecipitation, GRK2 binds IRS1 but not the Ins receptor in an Ins-dependent fashion, which is lost in HEK-GRK2 cells. Ant-124 improves Ins-induced glucose uptake in HEK-293 and HEK-GRK2 cells, but does not prevent GRK2/IRS1 interaction. In SHR, Ant-124 infusion for 30 days ameliorates IRES and IRS1 tyrosine phosphorylation.
Our results suggest that GRK2 mediates adrenergic IRES and that inhibition of GRK2 activity leads to increased Ins sensitivity both in cells and in animal model of IRES.
胰岛素（Ins）抵抗（IRES）与代谢综合征中观察到的心血管风险增加有关。由于交感神经系统活动过度兴奋导致β肾上腺素能受体（βAR）的慢性刺激参与了 IRES 的发病机制。在慢性βAR 刺激期间，细胞水平的 G 蛋白偶联受体激酶 2（GRK2）增加，导致βAR 脱敏。我们检验了以下假设：GRK2 在βAR 诱导的 IRES 中起作用。
我们在体外过表达β2AR、GRK2 或催化失活突变体 GRK2-DN 的细胞中评估了 Ins 诱导的葡萄糖摄取和信号转导反应。在肾上腺素能活性增加、IRES 和细胞内 GRK2 水平升高的自发性高血压大鼠（SHR）模型中，我们进行了静脉葡萄糖耐量试验负荷。为了抑制 GRK2，我们合成了一种基于 GRK2 催化序列的肽，与 antennapedia 内化序列（Ant-124）缀合。Ins 在人肾胚胎（HEK-293）细胞中引起 GRK2 的快速积累、Ins 受体底物 1（IRS1）的酪氨酸磷酸化，并诱导葡萄糖摄取。在相同的细胞类型中，转基因β2AR 过表达导致 GRK2 积累，与 IRS1 激活的显著缺陷以及 Ins 诱导的葡萄糖摄取相关。同样，转基因 GRK2 过表达阻止 Ins 诱导的 IRS1 酪氨酸磷酸化和葡萄糖摄取，而 GRK2-DN 改善葡萄糖提取。通过免疫沉淀，GRK2 以 Ins 依赖的方式与 IRS1 结合，但不与 Ins 受体结合，而在 HEK-GRK2 细胞中则丢失。Ant-124 可改善 HEK-293 和 HEK-GRK2 细胞中 Ins 诱导的葡萄糖摄取，但不能阻止 GRK2/IRS1 相互作用。在 SHR 中，Ant-124 输注 30 天可改善 IRES 和 IRS1 酪氨酸磷酸化。
我们的结果表明，GRK2 介导肾上腺素能 IRES，并且抑制 GRK2 活性可导致细胞和 IRES 动物模型中 Ins 敏感性增加。
