胎盘来源的NADPH氧化酶通过外泌体传递导致胰岛素外周靶器官能量及节律基因调控异常在妊娠期糖尿病发病中的作用

Insulin insensitivity is the major etiology of gestational diabetes mellitus (GDM). The circadian oscillation associated genes CLOCK/BMAL regulate insulin sensitivity via Sirt1-dependent fashion. If Sirt1 unable to function effectively, energy metabolism and circadian homeostasis feedback loop will be disrupted in insulin peripheral target organs. Our previous studies demonstrated that exosomes isolated from GDM placenta contained an abnormal higher level of NADPH oxidase. When co-cultured these exosomes with cells resulted in Sirt1 and CLOCK/BMAL downregulation as well as ROS accumulation. Therefore, we hypothesized that GDM placental-derived exosomes comprise an irregular level of NADPH enzyme. Once these exosomes reach insulin peripheral target organs via maternal circulation, the following phenomena will be induced; promoting oxidative stress, increase NAD+ consumption, a decline of Sirt1 biosynthesis, compromised CLOCK gene acetylation resistance, and instability of CLOCK/BMAL1 complex. Consequently, the disruption of peripheral NAMPT/NAD+, Sirt1, CLOCK/BMAL1 feedback loop and imbalance of insulin sensitivity homeostasis lead to GDM complication. The implementation of molecular biology, subcellular proteomics, and metabolomics provides novel ideas for GDM mechanism and explaining the pathogenesis of disease in a new perspective.

胰岛素敏感性降低是妊娠期糖尿病（GDM）发病关键点。外周生物钟节律基因CLOCK/BMAL1，以Sirt1依赖方式参与调控胰岛素敏感性。若未有效激活Sirt1，将打破胰岛素外周靶器官能量代谢与节律基因反馈调控环稳态。课题组前期证实GDM胎盘外泌体中NADPH氧化酶含量异常增高；且与其共培养的细胞Sirt1与CLOCK/BMAL1表达下调，ROS堆积。推测：GDM胎盘源性外泌体中NADPH氧化酶含量异常，随循环达胰岛素外周靶器官，促进氧化应激，NAD+大量消耗，Sirt1合成下降，无法拮抗CLOCK基因乙酰化，CLOCK/BMAL1复合体稳定性降低，进而打破外周NAMPT/NAD+、Sirt1 和 CLOCK/BMAL1反馈调控环的稳态，胰岛素敏感性的节律性无法维持，导致GDM。项目用分子生物学、亚细胞器蛋白质组学及代谢组学等方法，以全新角度解释GDM的病生过程，为研究其发病机制提供新思路。

细胞外囊泡（EVs）是由脂质双分子层包裹的纳米大小的微粒，最初被认为只是细胞碎片。近年来，EVs被认为是正常和病理条件下细胞间通讯的重要媒介，包括妊娠期糖尿病（GDM）。胰岛素抵抗是GDM的基本病理改变，而肝脏作为胰岛素靶器官对胰岛素敏感性的高低具有重要决定作用。研究发现GDM循环EVs与肝细胞共培养后会引起细胞葡萄糖消耗能力和能量代谢降低，胰岛素通路关键蛋白表达下调，并改变肝细胞代谢组。通过对循环EVs的miRNA谱分析，发现GDM循环EVs中miR-1299含量升高，并预测到STAT3为miR-1299的靶基因。STAT3 作为一种转录因子可调节多种组织中的胰岛素信号，其下游效应因子FAM3A可改善代谢状况，促进肝细胞内ATP的产生和释放。实验发现GDM循环EVs可导致肝细胞STAT3，FAM3a和P-AKT丰度显著降低。并在给予STAT3激活剂后，可明显改善GDM循环EVs降低的细胞葡萄糖摄取和相关蛋白的表达。故推测：GDM循环EVs中miR-1299含量异常升高，随循环到达肝脏，抑制STAT3/FAM3A信号通路，造成胰岛素抵抗，糖代谢异常，导致GDM。项目运用分子生物学、亚细胞器转录组学、细胞生物学和代谢组学等手段，将EVs中的miRNA与肝脏组织有机的联系在一起，为进一步认识GDM发病机制提供新的思路。