ATF4调控肝脏糖异生及酒精性脂肪肝的作用及机制
文献类型：学位论文
| 作者 | 李锴 |
| 学位类别 | 博士 |
| 答辩日期 | 2015-07-01 |
| 授予单位 | 中国科学院上海生命科学研究院营养科学研究所 |
| 授予地点 | 中国科学院上海生命科学研究院 |
| 导师 | 郭非凡 |
| 关键词 | 酒精性脂肪肝 转录激活因子4 腺苷酸活化蛋白激酶 小RNA-214 糖异生 |
| 其他题名 | Novel function of ATF4 in regulating hepatic gluconeogenesis and alcoholic fatty liver |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 中国正在面临慢性代谢性疾病的高发期。我国2 型糖尿病患病人数已从1989 年的2%增加至2007 年的9.7%，全国患病人数已达9200 万，居世界第一。 另一方面，我国因过度饮酒引起的酒精性脂肪肝发病率呈现明显的上升趋势。过去十年间，酒精性肝病的发病率平均达到了4.5%。而与以上疾病密切相关的机体器官是肝脏，肝功能正常对于维持机体糖稳态以及酒精毒性解除并维持肝脏脂质代谢平衡等多方面起到重要的调控作用。转录激活因子4 （activation transcription factor 4，ATF4） 是CREB（cAMP response element-binding protein）蛋白家族的一个重要成员，在调控能量代谢中具有重要作用，但肝脏ATF4在调控糖脂代谢中的作用，依旧有很多未知。在我们的研究中，着重探讨了肝脏ATF4作为miR-214下游调控肝脏糖异生的作用，以及肝脏ATF4在调控酒精性脂肪肝中的作用。 作为二型糖尿病高血糖的原因之一，糖异生近年来得到了人们广泛的关注。但microRNA调控糖异生的作用还有很多未知。在我们的研究中，探索了microRNA中的一员——miR-214作为肝脏糖异生调控因子的新功能。我们发现胰高血糖素可以通过PKA信号通路，抑制肝原代细胞中的miR-214水平，同时在高脂诱导的糖尿病小鼠以及饥饿小鼠肝脏中miR-214也有显著下调。进一步，在肝原代细胞上过表达miR-214可以抑制糖异生，而阻断miR-214也具有相应变化。在体内实验中，我们发现通过小鼠尾静脉注射miR-214过表达腺病毒后，肝脏糖异生水平也显著降低，而在高脂喂养的小鼠中，miR-214也有相同作用。在机制研究中，ATF4作为miR-214的已知下游，能够在体外实验中回复miR-214对糖异生的抑制作用，并且ATF4对糖异生的作用是通过影响FoxO1转录活性而产生的。之后，我们构建了ATF4肝脏特异敲除小鼠，发现该小鼠肝脏糖异生水平也显著降低，而向ATF4肝脏特异敲除小鼠中注射miR-214过表达腺病毒，不能进一步抑制糖异生水平，也说明miR-214确实是通过靶向ATF4而调控糖异生的。最后，我们还构建了miR-214肝脏特异性过表达小鼠，发现该小鼠糖异生水平显著降低。 酒精主要是通过激活脂肪合成信号通路并且同时抑制脂解信号通路而促使肝脏发生脂肪堆积的，但其中的机制依旧有很多未知。在我们前期研究证实：ATF4参与调控高脂和高糖饮食诱导的非酒精性脂肪肝的发展过程，并且ATF4与内质网应激、脂质代谢、氧化应激等过程密切相关。近来有报道称人肝细胞受酒精刺激后诱导ATF4高表达，但ATF4是否直接参与了酒精性脂肪肝的发生、发展，目前尚无报道。在本课题中，我们利用肝脏特异性ATF4敲除小鼠探索肝脏ATF4在酒精性脂肪肝发生发展中的作用及其分子机制。首先，利用肝原代细胞和酒精处理的小鼠肝脏，我们证实酒精能特异性诱导ATF4的高表达；其次，研究发现肝脏特异性ATF4敲除小鼠抵抗酒精诱导的肝脏脂肪堆积和脂质变性；第三，通过细胞和动物试验，我们发现了ATF4缺失抵抗酒精性脂肪肝形成与肝脏AMPK活性密切相关，腺病毒阻断AMPK能有效逆转肝脏特异性ATF4敲除小鼠抵抗酒精性脂肪肝的表型；第四，进一步研究证实ATF4通过其下游靶基因TRB3调控AMPK的活性，TRB3能够直接与AMPK的?亚基结合，进而抑制AMPK被LKB1或者CaMKK?等激酶激活。这一研究揭示了ATF4/TRB3/ATF4这一信号通路在酒精诱导的肝脏脂质变性过程中的重要生理功能，针对这一信号通路的有效的营养干预或者药物治疗将为酒精性肝病的治疗带来曙光。 综上所述，本研究揭示了ATF4具有调控肝脏糖异生及酒精性脂肪肝的新功能，证实了ATF4通过FOXO1影响糖异生、通过TRB3直接结合AMPKa进而抑制AMPK活性参与酒精性脂肪肝形成的重要分子机制，为揭示2型糖尿病和酒精性脂肪肝的发病机理提供了坚实的实验依据和理论基础。 |
| 英文摘要 | China is facing a peak period of chronic metabolic disease. The number of type 2 diabetes in China has increased from 2% in 1989 to 9.7% in 2007, the number of cases has reached 92 million, first in the world. On the other hand, the incidence of alcoholic fatty liver caused by excessive alcohol drinking is obviously rising: the morbidity of alcoholic fatty liver has reached to 4.5% over the past decade. These diseases is closely related with liver, the major metabolic organ in our body. Liver plays a important role in maintainning the blood glucose level and in clearing the alcohol toxicity. Activation transcription factor 4 (ATF4) belongs to the cAMP response element-binding protein (CREB) family, plays important role in regulating metabolism. However, there is little known about liver ATF4 in regulating glucolipid metabolism. In this study, we discusses the role of liver ATF4, as downstream target of miR-214 in regulating gluconeogenesis, and also in regulating the alcoholic fatty liver. While the gluconeogenesis pathway is already a target for the treatment of type 2 diabetes, the potential role of microRNAs (miRNAs) in gluconeogenesis remains unclear. Here, we investigated the physiological functions of miR-214 in gluconeogenesis. The expression of miR-214 was suppressed by glucagon via protein kinase A (PKA) signaling in primary hepatocytes, and miR-214 was down-regulated in the livers of fasted, high-fat diet (HFD)-induced diabetic and leptin receptor-mutated (db/db) mice. The over-expression of miR-214 in primary hepatocytes suppressed glucose production and silencing miR-214 reversed this effect. Gluconeogenesis was suppressed in the livers of mice injected with an adenovirus expressing miR-214 (Ad-miR-214). Additionally, Ad-miR-214 alleviated HFD-induced elevation of gluconeogenesis and hyperglycemia. Furthermore, we found that activating transcription factor 4 (ATF4), a reported target of miR-214, can reverse the suppressive effect of miR-214 on gluconeogenesis in primary hepatocytes, and this suppressive effect was blocked in liver-specific ATF4 knockout mice (LV-ATF4 KO). ATF4 regulated gluconeogenesis via affecting forkhead box protein O1 (FOXO1) transcriptional activity. Finally, Liver-specific miR-214 transgenic mice (LV-miR-214 TG) exhibited suppressed gluconeogenesis and reduced expression of ATF4, phosphoenolpyruvate carboxykinase (PCK) and glucose-6-phosphatase (G6P) in liver. Taken together, our results suggest that the miR-214-ATF4 axis is a novel pathway for the regulation of hepatic gluconeogenesis People consuming excessive amounts of alcohol for prolonged periods suffering form alcoholic fatty liver disease. Activating transcriptional factor 4 (ATF4) is involved in hepatic lipid metabolism under the condition of high fat diet or high carbohydrate diet. Ethanol exposure induces ATF4 expression in human hepatocytes, but the role of ATF4 in alcoholic fatty liver in vivo is not clear. Here, we investigated the role of ATF4 in alcoholic fatty liver and to elucidate the possible mechanisms underlying ATF4 regulating alcoholic fatty liver. We induced alcoholic fatty liver in male liver-specific deletion of ATF4 (ALKO) mice with their LOX littermates (WT). ALKO and WT mice were fed on Lieber-DeCarli ethanol-containing diets for 28 days. Liver and serum samples were collected. The gene expression profiles in livers and serum enzyme activity were performed. We also investigated the effect of ATF4 deficiency on adenosine 5‘-monophosphate (AMP)-activated protein kinase (AMPK) activation and triglyceride (TG) accumulation in AML-12 cell line. Using co-immunoprecipitation and glutathione S-transferase (GST) pull-down assays, we analyzed the interaction between tribbles homolog 3 (TRB3), a direct target of ATF4, and AMPK to regulate its phosphorylation. On the ethanol-containing diet, ATF4LVKO mice were resistant to ethanol-induced liver steatosis; serum of ALKO mice had decreased levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Hepatic deletion of ATF4 alleviates ethanol-mediated liver steatosis, mainly by activating AMPK, a key regulator of lipid metabolism. Overexpression of AMPK dominant negative mutant reverses the effect of ATF4 deletion on ethanol-mediated liver steatosis. In cultured mouse AML-12 hepatocytes, knockdown of ATF4 prevented the decrease of AMPK phosphorylation in response to ethanol exposure, largely by decreasing the expression of TRB3. Overexpression of TRB3 could reverse the effect of ATF4 deletion on AMPK phosphorylation by ethanol. Additionally, TRB3 could directly interact with AMPK to suppress its phosphorylation. These results suggest that ATF4 deficiency resists ethanol-induced fatty liver through its target gene-TRB3, which acts as a partner of AMPK to regulate its phosphorylation. |
| 语种 | 中文 |
| 公开日期 | 2016-02-26 |
| 源URL | [http://202.127.25.144/handle/331004/396]  |
| 专题 | 中国科学院上海生命科学研究院营养科学研究所_代谢的遗传与营养调控研究组 |
| 推荐引用方式 GB/T 7714 | 李锴. ATF4调控肝脏糖异生及酒精性脂肪肝的作用及机制[D]. 中国科学院上海生命科学研究院. 中国科学院上海生命科学研究院营养科学研究所. 2015. |
入库方式： OAI收割
来源：上海营养与健康研究所
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