Betatrophin对肝脏糖异生的影响及其机制研究

Hepatic insulin resistance is a key contributor to the pathogenesis of type 2 diabetes. Inhibition of overactivated hepatic gluconeogenesis is a important therapeutic target for the treatment of type 2 diabetes. Betatrophin, a newly identified secreted protein of 198 amino acids, is enriched in the liver and fat of mice. Recently, it has been showed that insulin receptor antagonist S961 caused dramatic insulin resistance in mice, and S961 infusion-mediated insulin resistance was accompanied with a surge in betatrophin mRNA in liver and fat. Our previous study discovered that betatrophin mRNA expression was significantly up-regulated in the livers of ob/ob and db/db mice. We have demonstrated for the first time that serum betatrophin concentrations were significantly increased in newly diagnosed and untreated type 2 diabetes patients compared with the controls, and serum betatrophin was negatively associated with 1/HOMA-IR (insulin sensitivity index) in type 2 diabetes group. In cultured primary liver cells of mice, cAMP and hexadecadrol induced PEPCK and G6Pase genes transcription to initiate gluconeogenesis, while recombinant betatrophin protein inhibited expression of both genes. Based on the findings metioned above, it showed that betatrophin may improve glycemia by direct inhibition of overactivated gluconeogenesis in liver. In the present study, we aim to comprehensively study the effects of betetrophin on hepatic gluconeogenesis in vivo and vitro, and further clarify the molecular mechanisim of betetrophin regulating gluconeogenesis. This study will provide strong evidence for the new role of betatrophin in regulating hepatic gluconeogenesis, and betatrophin may serve as a potential therapeutic target for the treatment of insulin resistance and type 2 diabetes.

肝脏胰岛素抵抗是2型糖尿病发病机制的主要环节，有效抑制肝脏过度糖异生, 减少内源性葡萄糖生成, 是治疗2型糖尿病的重要靶标之一。Betatrophin，近年来新发现的一种分泌蛋白，主要由肝脏组织表达。本课题组前期动物及临床研究发现，在胰岛素抵抗状态，Betatrophin表达显著增高；在原代培养的小鼠肝脏细胞，cAMP、地塞米松促进糖异生，Betatrophin重组蛋白则明显减弱此作用，同时发现其能抑制糖异生途径限速酶PEPCK等基因的表达；以上研究表明，Betatrophin可能直接调控肝脏糖异生。本研究拟进一步从细胞和整体水平观察Betatrophin对肝脏糖异生的影响，并从信号通路、转录调控等方面入手，探明其调控机制。该课题具有源头创新性，通过本项目的实施，有望加深人们对Betatrophin基因功能和作用的理解，丰富肝脏糖异生的调控机制学说，对胰岛素抵抗、2型糖尿病的防治提供帮助。

肝脏胰岛素抵抗是2型糖尿病发病机制的主要环节，有效抑制肝脏过度糖异生，减少内源性葡萄糖生成，是治疗2型糖尿病的重要靶标之一。Betatrophin，又称ANGPTL8，是近年来新发现的一种含有198个氨基酸的分泌蛋白，在体内主要在肝脏表达。本项目观察了Betatrophin对肝脏糖异生的影响，并探讨了其作用机制。结果发现，在初诊2型糖尿病人群，血清Betatrophin水平显著增加，其与评估肝脏胰岛素敏感性的指标1/HOMA-IR显著负相关。在饥饿状态，Betatrophin在C57BL/6小鼠肝脏低表达，进食后其表达显著增高。db/db糖尿病小鼠肝脏PEPCK、G6Pase等糖异生途径关键催化酶及Betatrophin基因表达均显著增加。在饥饿、进食及胰岛素抵抗状态下，Betatrophin表达的变化趋势与体内胰岛素的变化基本一致，提示其与胰岛素抵抗、肝脏糖异生相关，可能参与肝脏糖异生的调控。进一步利用db/db糖尿病小鼠、高脂喂养联合STZ诱导的2型糖尿病小鼠等动物模型，尾静脉注射Betatrophin过表达腺病毒，分别行葡萄糖耐量试验、胰岛素耐量试验及丙酮酸耐量试验等。结果发现，在这两种糖尿病模型小鼠，Betatrophin过表达后血糖水平均逐渐下降，胰岛素敏感性提高，肝糖异生功能抑制，肝糖原合成增加。分离培养小鼠原代肝细胞，分别予Betatrophin重组蛋白、过表达或敲减腺病毒干预。结果发现，Betatrophin干预可显著抑制肝糖异生，PEPCK、G6Pase基因及蛋白的表达明显下降。同时，研究显示C/EBPα、C/EBPβ等基因的表达显著下降，表明Betatrophin可能通过降低C/EBP等转录因子的表达或活性，抑制糖异生途径关键催化酶的转录。此外，在小鼠原代肝细胞过表达Betatrophin，行转录组测序、Realtime PCR、Western blot 和免疫荧光等实验。结果发现，Betatrophin可能通过调控PI3K/AKT、AMPK等信号通路，抑制肝脏糖异生，改善胰岛素抵抗。本课题增加了人们对Betatrophin基因作用和功能的理解，为胰岛素抵抗、2型糖尿病的防治或治疗药物研发提供帮助。