§The Key Laboratory of Regenerative Biology, Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
Biochem J. 2013 Oct 15;455(2):207-16. doi: 10.1042/BJ20130760.
Insulin inhibits hepatic glucose production through activation of the protein kinase Akt, and any defect in this pathway causes fasting hyperglycaemia in Type 2 diabetes. APPL1 [adaptor protein, phosphotyrosine interaction, PH (pleckstrin homology) domain and leucine zipper containing 1] sensitizes hepatic insulin action on suppression of gluconeogenesis by binding to Akt. However, the mechanisms underlying the insulin-sensitizing actions of APPL1 remain elusive. In the present study we show that insulin induces Lys63-linked ubiquitination of APPL1 in primary hepatocytes and in the livers of C57 mice. Lys160 located within the BAR (Bin/amphiphysin/Rvs) domain of APPL1 is the major site for its ubiquitination. Replacement of Lys160 with arginine abolishes insulin-dependent ubiquitination and membrane localization of APPL1, and also diminishes membrane recruitment and activation of Akt, thereby abrogating the effects of APPL1 on alleviation of hepatic insulin resistance and glucose intolerance in obese mice. Further analysis identified TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) as an E3 ubiquitin ligase for APPL1 ubiquitination. Suppression of TRAF6 expression attenuates insulin-mediated ubiquitination and membrane targeting of APPL1, leading to an impairment of insulin-stimulated Akt activation and inhibition of gluconeogenesis in hepatocytes. Thus TRAF6-mediated ubiquitination of APPL1 is a vital step for the hepatic actions of insulin through modulation of membrane trafficking and activity of Akt.
胰岛素通过激活蛋白激酶 Akt 抑制肝葡萄糖生成，而该途径的任何缺陷都会导致 2 型糖尿病患者空腹高血糖。APPL1（衔接蛋白，磷酸酪氨酸相互作用，PH（pleckstrin homology）结构域和富含亮氨酸拉链 1）通过与 Akt 结合，使肝胰岛素作用对糖异生的抑制作用敏感。然而，APPL1 的胰岛素增敏作用的机制仍不清楚。在本研究中，我们发现在原代肝细胞和 C57 小鼠肝脏中，胰岛素诱导 APPL1 的 Lys63 连接泛素化。APPL1 的 BAR（Bin/amphiphysin/Rvs）结构域内的 Lys160 是其泛素化的主要位点。用精氨酸替换 Lys160 可消除胰岛素依赖性泛素化和 APPL1 的膜定位，也可减少 Akt 的膜募集和激活，从而消除 APPL1 对肥胖小鼠肝胰岛素抵抗和葡萄糖耐量异常的影响。进一步的分析确定了 TRAF6（肿瘤坏死因子受体相关因子 6）是 APPL1 泛素化的 E3 泛素连接酶。TRAF6 表达的抑制减弱了胰岛素介导的 APPL1 泛素化和膜靶向，导致胰岛素刺激的 Akt 激活受损和肝细胞中糖异生抑制。因此，TRAF6 介导的 APPL1 泛素化是胰岛素通过调节膜运输和 Akt 活性发挥肝作用的关键步骤。
