miR199a-5p通过Caveolin-1调控肝脏葡萄糖代谢的作用及机制研究

Dysregulation of hepatic glucose metabolism could induce diabetes , and both fatty liver and diabetes are important components of metabolic syndrome and are closely related. Our previous research has demonstrated that miR199a-5p and its target gene Caveolin-1(Cav1) were unregulated and downregulated in the livers of fatty liver mice, respectively;, further study found that hepatic overexpression of miR199a-5p in obese and diabetic mice induced the elevation of serum glucose. Mitochondrion is the primary organelle of glucose oxidative phosphorylation. Previous study has found that Cav1 defect may lead to mitochondrial dysregulation and affect glucose metabolism, however, whether Cav1 regulates glucose oxidative phosphorylation in hepatic mitochondria remains to be determined. Our project will aim hepatic mitochondria as the entry point and will use whole genome chip and primary hepatocyte cell culture to address questions: : what is the role of miR199a-5p in the regulation of hepatic glucose metabolism, how Cav1, as the bona fide target of miR199a-5p, regulates hepatic glucose metabolism, how miR199a-5p/Cav1 axis influences mitochondrial oxidative phosphorylation signaling, and whether aberrant Cav1 induces mitochondrial complex dysfunction via directly regulating oxidative phosphorylation pathways. Our project could potentially elucidate the molecular mechanism of dysregulated hepatic glucose metabolism in fatty liver, and provide scientific basis for the treatment and prevention of the glucose and lipid metabolic diseases.

肝脏糖代谢异常能引起糖尿病，脂肪肝和糖尿病是代谢综合征的重要组成部分并联系密切。我们前期研究结果已证实，miR199a-5p在脂肪肝小鼠肝脏中上调，其靶基因Caveolin-1(Cav1)随之下调，进一步的实验通过在肥胖糖尿病小鼠肝脏中过表达miR199a-5p，发现其可导致小鼠血糖升高。线粒体是糖氧化磷酸化的主要细胞器， Cav1缺失可导致线粒体功能紊乱并影响代谢，但Cav1在肝脏线粒体中是否调控糖氧化磷酸化仍不明确。本项目将以肝脏线粒体为切入点，通过全基因组芯片、肝脏原代细胞分离等技术，研究miR199a-5p在调控肝脏糖代谢中的作用，观察Cav1在肝脏糖代谢中的调控情况，探讨miR199a-5p/ Cav1如何影响线粒体氧化磷酸化通路，分析Cav1是否通过影响线粒体复合体功能来调控氧化磷酸化通路。本课题不仅有利于阐明肝脏糖代谢的调节机制，也将为糖脂代谢性疾病的预防和治疗提供科学依据。

已知微小RNA（miRNA）会导致许多代谢疾病，包括糖尿病。在本课题中，我们研究了miR199a-5p在调节肝脏胰岛素敏感性中的作用。将Ad-anti-miR199a-5p腺病毒注射到高脂饮食喂养的雄性C57BL / 6J WT小鼠中来抑制miR199a-5p的表达，继而评估小鼠中葡萄糖和胰岛素抵抗水平。同样，我们将Ad-miR199a-5p腺病毒注射到雄性C57BL / 6J WT小鼠中以过表达miR199a-5p，继而研究其葡萄糖和胰岛素抵抗水平。为了研究自噬相关蛋白14（ATG14）和miR199a-5p在调节胰岛素敏感性中的作用，我们在进行相关功能实验之前将Ad-miR199a-5p和（或不和）Ad-ATG14病毒一起注射到WT C57BL / 6J小鼠中。此外，我们用Ad-anti-miR199a-5p或Ad-miR199a-5p病毒感染HepG2细胞或肝脏原代细胞，以测定miR199a-5p对体外胰岛素抵抗的影响。最后，我们通过检测来自糖尿病患者的肝脏样本中miR199a-5p的表达水平，来探索miR199a-5p的临床相关性。在本研究中，我们首先证明沉默miR199a-5p导致体内葡萄糖耐量降低、胰岛素耐量降低，而miR199a-5p的过表达则产生相反的效果。我们进一步将ATG14鉴定为miR199a-5p的靶点，并且发现ATG14能部分改善miR199a-5p引起的的葡萄糖水平升高和胰岛素耐受性下降。此外，关于ATG14，LC3和BECLIN1的透射电子显微镜数据和蛋白质印迹实验数据，说明了miR199a-5p通过ATG14调节自噬。敲除原代肝细胞和HepG2细胞中的miR199a-5p，可抑制胰岛素刺激产生的胰岛素受体β，糖原合成酶激酶3β和蛋白激酶B磷酸化水平升高，而miR199a-5p的过表达可进一步增强磷酸化。最后，我们检测到上调的miR199a-5p水平，与从糖尿病患者肝标本中的ATG14 mRNA水平降低和自噬下调相关。我们的研究揭示了miR199a-5p通过ATG14介导的自噬调节肝脏胰岛素敏感性的生物学作用。