Valverde Angela M, Fabregat Isabel, Burks Deborah J, White Morris F, Benito Manuel
Instituto de Bioquímica/Departamento de Bioquímica y Biología Molecular II, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Hepatology. 2004 Dec;40(6):1285-94. doi: 10.1002/hep.20485.
To assess the role of insulin action and inaction in the liver, immortalized hepatocyte cell lines have been generated from insulin receptor substrate (IRS)-2(-/-) and wild-type mice. Using this model, we have recently demonstrated that the lack of IRS-2 in neonatal hepatocytes resulted in insulin resistance. In the current study, we show that immortalized neonatal hepatocytes undergo apoptosis on serum withdrawal, with caspase-3 activation and DNA laddering occurring earlier in the absence of IRS-2. Insulin rescued wild-type hepatocytes from serum withdrawal-induced caspase-3 activation and DNA fragmentation in a dose-dependent manner, but it failed to rescue hepatocytes lacking IRS-2. In IRS-2(-/-) cells, insulin failed to phosphorylate Bad. Furthermore, in these cells, insulin was unable to translocate Foxo1 from the nucleus to the cytosol. Adenoviral infection of wild-type cells with constitutively active Foxo1 (ADA) induced caspase-8 and caspase-3 activities, proapoptotic gene expression, DNA laddering and apoptosis. Dominant negative Foxo1 regulated the whole pathway in an opposite manner. Prolonged insulin treatment (24 hours) increased expression of antiapoptotic genes (Bcl-xL), downregulated proapoptotic genes (Bim and nuclear Foxo1), and decreased caspase-3 activity in wild-type hepatocytes but not in IRS-2(-/-) cells. Infection of IRS-2(-/-) hepatocytes with adenovirus encoding IRS-2 reconstituted phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/Foxo1 signaling, restored pro- and antiapoptotic gene expression, and decreased caspase-3 activity in response to insulin, thereby blocking apoptosis. In conclusion, IRS-2 signaling is specifically required through PIP3 generation to mediate the survival effects of insulin. Epidermal growth factor, via PIP3/Akt/Foxo1 phosphorylation, was able to rescue IRS-2(-/-) hepatocytes from serum withdrawal-induced apoptosis, modulating pro- and anti-apoptotic gene expression and downregulating caspase-3 activity.
为了评估胰岛素在肝脏中的作用及非作用机制，已从胰岛素受体底物（IRS）-2基因敲除（-/-）小鼠和野生型小鼠中构建了永生化肝细胞系。利用该模型，我们最近证实新生肝细胞中IRS-2的缺失会导致胰岛素抵抗。在本研究中，我们发现永生化新生肝细胞在血清撤除后会发生凋亡，在缺乏IRS-2的情况下，半胱天冬酶-3激活和DNA梯状条带出现得更早。胰岛素能以剂量依赖的方式挽救野生型肝细胞，使其免受血清撤除诱导的半胱天冬酶-3激活和DNA片段化，但无法挽救缺乏IRS-2的肝细胞。在IRS-2基因敲除细胞中，胰岛素无法使Bad磷酸化。此外，在这些细胞中，胰岛素无法将Foxo1从细胞核转运至细胞质。用组成型激活的Foxo1（ADA）对野生型细胞进行腺病毒感染会诱导半胱天冬酶-8和半胱天冬酶-3活性、促凋亡基因表达、DNA梯状条带形成及凋亡。显性负性Foxo1则以相反的方式调节整个通路。延长胰岛素处理时间（24小时）可增加野生型肝细胞中抗凋亡基因（Bcl-xL）的表达，下调促凋亡基因（Bim和核内Foxo）的表达，并降低半胱天冬酶-3活性，但在IRS-2基因敲除细胞中则无此作用。用编码IRS-2的腺病毒感染IRS-2基因敲除肝细胞可重建磷脂酰肌醇3激酶（PI3激酶）/Akt/Foxo1信号通路，恢复促凋亡和抗凋亡基因表达，并降低胰岛素刺激下的半胱天冬酶-3活性，从而阻止凋亡发生。总之，IRS-2信号通路通过生成磷脂酰肌醇-3,4,5-三磷酸（PIP3）来介导胰岛素的存活效应是特异性必需的。表皮生长因子通过PIP3/Akt/Foxo1磷酸化，能够挽救IRS-2基因敲除肝细胞免受血清撤除诱导的凋亡，调节促凋亡和抗凋亡基因表达并下调半胱天冬酶-3活性。
