C型肝炎病毒非結構性蛋白質NS5A參與正調控糖質新生之分子機制

Abstract

C 型肝炎病毒 (HCV) 是造成慢性肝炎和肝硬化的主要原因，甚至還 可能會發展成肝癌或第二型糖尿病。C 型肝炎病毒的核心蛋白質和NS5A 蛋白質被認為可能會參與HCV 所造成的第二型糖尿病的發生，但是詳細 的機制目前還尚未了解。利用流體力學為基礎之體內轉染法，建立一個以 老鼠肝細胞短暫表現NS5A 蛋白質的系統，進行微陣列 (microarray) 的分 析研究，找出表現受到NS5A 蛋白質影響的基因。結果發現在表現NS5A 蛋白質的老鼠肝臟細胞中，糖質新生的速率限制酵素PEPCK 的表現增加， 結果亦發現在有糖尿病的C 型肝炎患者。PEPCK 是糖質新生過程的重要 酵素，而且PEPCK 的過量表現會造成肝臟葡萄糖的產生增加。在本研究 中也證實NS5A 蛋白質在原代肝細胞中會促進葡萄糖的產生。PEPCK 基因 啟動子的轉錄活化，受轉錄因子CREB (cyclic AMP response element binding protein) 、FoxO1 (forkhead transcription factor O1) 和HNF-4a (hepatocyte nuclear factor 4a) 的調控，其中FoxO1 和HNF-4a 還需要與共 活化因子PGC-1a 結合才能達到活化PEPCK 基因啟動子的作用。過去的 研究指出，PGC-1a 在肝臟糖質新生的過程中扮演重要的調控角色，而且 可能會參與糖尿病的致病機制。在老鼠肝細胞短暫表現NS5A 蛋白質的系 統中，微陣列的分析結果也看到PGC-1a 基因的表現量增加。在表現NS5A 蛋白質的細胞中和HCV 感染及複製系統 (HCV type 1b subgenomic replicon)，藉由即時聚合酶連鎖反應和西方墨點法的分析，都可以看到PEPCK 和 PGC-1a 的表現增加。更進一步的實驗證實，NS5A 蛋白質可 能透過PI3K-Akt 和JNK 之訊息傳遞路徑，正調控PEPCK 和PGC-1a 的 表現。此外，在表現NS5A 蛋白質的細胞中也發現了轉錄因子FoxO1 和 HNF-4a 的表現增加，以及轉錄因子CREB 的磷酸化增加。本研究證實， NS5A 蛋白質可能藉由活化轉錄因子CREB、增加轉錄因子FoxO1 和 HNF-4a 的表現，以及透過PI3K-Akt 和JNK 之訊息傳遞促進PGC-1a 的 表現，進而促進PEPCK 的表現和葡萄糖的產生。推測NS5A 蛋白質可能 藉此參與HCV 所造成的第二型糖尿病的致病機制中。

Hepatitis C virus (HCV) is a major cause of chronic hepatitis, liver cirrhosis, and highly associated with hepatocellular carcinoma and type 2 diabetes mellitus (T2DM). The HCV viral proteins, core and NS5A protein, are thought to be involved in the development of HCV-induced T2DM, but the detailed mechanisms remain unclear. A cDNA microarray analysis with a mouse system with or without NS5A expression following hydrodynamics-based transfection was performed to identify differentially expressed genes. Upregulation of the gluconeogenic rate-limiting enzyme phosphoenolpyruvate carboxykinase (PEPCK) compared with controls was detected in mouse hepatocytes expressing NS5A protein. The result was also found in HCV patients with diabetes. PEPCK is the key enzyme in gluconeogenesis and its over-expression can stimulate hepatic glucose output. Full transcriptional activation of the PEPCK promoter requires transactivation mediated by the cyclic AMP response element binding protein (CREB) and peroxisome proliferator-activated receptor-gamma coactivator-1a (PGC-1a)- mediated coactivation of the glucocorticoid receptor, the forkhead transcription factor O1 (FoxO1), and the hepatocyte nuclear factor 4a (HNF-4a). PGC-1a, a transcriptional coactivator, is a key modulator of hepatic gluconeogenesis likely to be involved in the pathogenesis of diabetes. In this study, an HCV NS5A-mediated dose-dependent increase of glucose production was demonstrated in human primary hepatocytes. Upregulation of PGC-1a compared with controls was also detected in mouse hepatocytes expressing NS5A protein. In addition, the up-regulation of PEPCK and PGC-1a genes were also detected by real-time quantitative polymerase chain reaction and Western blot analysis both in NS5A-expressing cells and in the viral genotype 1b subgenomic replicon system. Further studies demonstrated that the NS5A-mediated upregulation of PEPCK and PGC-1a were resulted from the activation of PI3K-Akt and JNK signaling pathways. In addition, the expression levels of FoxO1 and HNF-4a, and the phosphorylation level of CREB were increased in HCV NS5A-expressing cells. These results suggest that HCV NS5A protein enhances PEPCK expression and stimulates hepatic glucose output by activation of its transactivators FoxO1, HNF-4a, CREB and coactivator PGC-1a mediated by PI3K-Akt and JNK signaling pathways. These data imply that NS5A is involved in the HCV-associated T2DM.