Supplementary Material for: GSK-3β as a potential coordinator of anabolic and catabolic pathways in hepatitis C virus insulin resistance

Introduction: Chronic HCV infection results in insulin resistance (IR) through interaction of pathways for glucose homeostasis, insulin signaling, and autophagy. Not known is how soon the pathways are activated, and how IR is related to the signals generated by catabolic and anabolic conditions occurring in infected cells. We have extended our studies to a cell culture system mimicking acute infection and to downstream pathways involving energy-sensor AMPK and nutrient-sensor mTOR that are active in catabolic and anabolic processes within the infected cells.
Methods: Huh7 liver cells were infected with HCV. We performed proteomics analysis of key proteins in infected cells by western blotting and IP experiments, with or without interferon-α exposure.
Results: We show that IRS-1 Ser312, Beclin-1, protein conjugate Atg12-Atg5 or GS Ser641 are upregulated early in infection by activating the same pathways utilized for persistent infection; Bcl-XL, an inhibitor of both autophagy and apoptosis, is present in a core-complex with IRS-1 Ser312 and Beclin-1 during progression of IR; AMPK level remains the same in infected cells where it is activated by phosphorylation at Thr172 concomitant with increased autophagy, a hallmark of catabolic conditions; an mTOR level that promotes anabolism is increased rather than decreased under an expanded autophagy; hypophosphorylation of translational repressor 4E-BP1 downstream of mTOR is suggestive of reduced protein synthesis; and β-catenin is upregulated but not phosphorylated suggesting indirectly our previous contention that its kinase, GSK-3β, is mostly in an inactive state.
Discussion/Conclusion: We report that in the development of IR following chronic infection, anabolic and catabolic pathways are activated early, and the metabolic interaction occurs possibly in a core-complex with IRS-1 Ser312, Beclin-1 and autophagy inhibitor BcL-XL. Induction of autophagy is usually controlled by a two-edged mechanism acting in opposition under anabolic and catabolic conditions by AMPK/mTOR/4E-BP1 pathways with GSK-3β mediated feed-back loops. However, we observed that an up-regulation of mTOR along with an up-regulation of AMPK caused by HCV infection is a deviation from the normal scenario described above which might be of therapeutic interest.

引言：慢性丙型肝炎病毒（HCV）感染可通过葡萄糖稳态、胰岛素信号传导与自噬通路的相互作用引发胰岛素抵抗（IR）。目前尚不明确这些通路的激活时机，以及IR与感染细胞内分解代谢和合成代谢状态所产生信号的关联机制。本研究将实验体系拓展至模拟急性感染的细胞培养模型，并聚焦感染细胞内参与分解与合成代谢过程的能量感受器腺苷酸活化蛋白激酶（AMPK）与营养感受器哺乳动物雷帕霉素靶蛋白（mTOR）相关的下游通路。
方法：本研究以Huh7肝癌细胞为模型，用HCV感染细胞，并通过蛋白质印迹（Western Blot）与免疫沉淀（IP）实验，结合或不结合干扰素-α干预，对感染细胞内的关键蛋白进行蛋白质组学分析。
结果：本研究发现，感染早期胰岛素受体底物1（IRS-1）Ser312位点、Beclin-1、Atg12-Atg5蛋白结合体以及糖原合酶（GS）Ser641位点的表达上调，其激活通路与持续感染时的通路一致；在IR进展过程中，自噬与凋亡双重抑制剂Bcl-XL可与IRS-1 Ser312及Beclin-1形成核心复合物；感染细胞内AMPK的总蛋白水平未发生改变，但通过Thr172位点磷酸化被激活，伴随自噬水平升高——这是分解代谢状态的标志性特征；在自噬增强的情况下，促进合成代谢的mTOR蛋白水平反而升高而非降低；真核翻译起始因子4E结合蛋白1（4E-BP1）作为mTOR下游的翻译抑制因子，呈低磷酸化状态，提示蛋白质合成受到抑制；而β-连环蛋白（β-catenin）表达上调但未发生磷酸化，间接印证了我们此前的观点：其激酶糖原合成激酶3β（GSK-3β）大多处于失活状态。
讨论/结论：本研究表明，慢性感染后IR的发生过程中，合成与分解代谢通路会被早期激活，且代谢相互作用可能通过由IRS-1 Ser312、Beclin-1与自噬抑制剂Bcl-XL组成的核心复合物实现；通常情况下，自噬的诱导受AMPK/mTOR/4E-BP1通路调控，该通路在合成与分解代谢条件下通过GSK-3β介导的反馈环路发挥双向拮抗作用；但本研究发现，HCV感染引发的mTOR与AMPK同时上调，与上述正常调控模式相悖，这一现象或具有潜在的治疗价值。