Mechanisms by which hepatocyte extracellular miRNAs mediate peripheral insulin sensitivity

肥胖诱导的胰岛素抵抗是代谢综合征的主要决定因素，代谢综合征先于2型糖尿病的发展，因此是新兴糖尿病流行病背后的驱动力。目前的抗糖尿病疗法是可用的，但不足以控制大多数患者的疾病，并且对于治疗糖尿病以预防发病率和死亡率的更好方法存在大量未满足的医疗需求。我们最近的工作已经导致发现肥胖诱导肝细胞外囊泡（EV）miRNA分泌的动态变化，其对胰岛素产生细胞和外周胰岛素敏感性产生深远影响。在肝细胞特异性Rab 27 KO小鼠中，肝细胞外miRNAs的消耗导致肥胖早期葡萄糖耐量和胰岛素敏感性受损。我们进一步证明了4 wks-HFD诱导的肝脏EV miRNAs可以降低肥胖受体小鼠的胰岛素抵抗。此外，4周-HFD EV治疗显著增强了胰岛素分泌。 在体外和体内β细胞的分泌和增殖。相比之下，长期肥胖诱导致病性肝细胞EV miRNA的分泌，其减弱瘦受体WT小鼠的胰岛素敏感性。一致地，通过敲除肝脏Rab 27而没有肝脏细胞外miRNAs的小鼠在16周HFD喂养后显示胰岛素抵抗降低。来自YBX 1 KO肝细胞的无miRNA EV对受体小鼠的代谢表型的影响最小，表明miRNA是这些EV中的关键货物。使用一种新的硫氧嘧啶标记方法，我们鉴定了miR-3075- 5 p，一种在4 wks-HFD EV中高度富集的miRNAs，可以有效地掺入靶细胞并通过抑制Fa 2 h表达来改善细胞胰岛素应答。此外，miR-434- 3 p-Map 2k 6调节轴在以下方面起关键作用： 促进巨噬细胞的促炎性激活，随后可加重组织 炎症和胰岛素抵抗。这些结果导致的结论是，肝EV miRNA是重要的内分泌分子调节功能的胰岛素产生和靶向细胞在肥胖症。本研究基于新发现的肝脏EV miRNAs调控系统，揭示了肥胖诱导胰岛素抵抗的细胞和分子机制。我们将进一步确定肝脏EV miRNA调节β细胞和胰岛素敏感细胞功能的机制。通过本实验，我们将开发miR-3075- 5 p作为胰岛素增敏剂分子，并探索miR-434- 3 p在肥胖中的致病作用。该治疗策略可用于治疗肥胖伴胰岛素抵抗的糖尿病前期患者。这将导致改善血糖控制，在我们的治疗设备中添加一个新的组成部分，用于治疗这种广泛的代谢疾病。最后，使用硫氧嘧啶标记方法，我们将确定循环中的肝细胞外miRNAs作为预测肥胖症胰岛素抵抗状态的生物标志物。

Obesity-induced insulin resistance is the major determinant of metabolic syndrome, which precedes the development of Type 2 diabetes mellitus and is thus the driving force behind the emerging diabetes epidemic. Current anti-diabetic therapeutics are available, but are inadequate to control the disease in most patients and there is a large unmet medical need for better methods of treating diabetes to prevent morbidity and mortality. Our recent work has led to the discovery that obesity induces a dynamic change in secretion of hepatic extracellular vesicle (EV) miRNAs that exert profound impacts on insulin producing cells and peripheral insulin sensitivity. Depletion of hepatic extracellular miRNAs in the hepatocyte-specific Rab27KO mice resulted in impaired glucose tolerance and insulin sensitivity at the early onset of obesity. We further demonstrated that 4wks high-fat diet feeding (4wks-HFD)-induced hepatic EV miRNAs can reduce the insulin resistance of obese recipient mice. In addition, the 4wks-HFD EV treatment significantly enhanced the insulin secretion and proliferation of beta cells in vitro and in vivo. By contrast, prolonged obesity induced secretion of pathogenic hepatocyte EV miRNAs that blunted insulin sensitivity of lean recipient WT mice. Consistently, the mice without hepatic extracellular miRNAs by knockout of hepatic Rab27 showed a reduction in insulin resistance after 16 weeks HFD feeding. miRNA-free EVs derived from YBX1KO hepatocytes had minimal effects on the metabolic phenotypes of recipient mice, suggesting miRNAs as key cargoes within these EVs. Using a novel thiouracil tagging method, we identified that miR-3075-5p, a highly enriched miRNAs in 4wks-HFD EVs, can be efficiently incorporated into target cells and improves cellular insulin responses through repressing Fa2h expression. In addition, the miR-434-3p-Map2k6 regulatory axis plays a critical role in promoting proinflammatory activation of macrophages, which can subsequently exacerbate tissue inflammation and insulin resistance. These results lead to the conclusion that hepatic EV miRNAs are important endocrine molecules regulating functions of insulin-producing and -targeting cells in obesity. This proposal sees to build on this newly identified hepatic EV miRNAs regulatory system to reveal the underlying cellular and molecular mechanisms of obesity-induced insulin resistance. We will further determine the mechanisms by which hepatic EV miRNAs modulate functions of beta cell and insulin sensitizing cells in response to obesity. With the proposed experiments, we will develop miR-3075-5p as an insulin sensitizer molecule and explore the pathogenic effect of miR-434-3p in obesity. This therapeutic strategy could be used for the treatment of obese patients with insulin resistance pre-diabetic state. This would lead to improved glycemic control adding a new component in our therapeutic armamentarium for the treatment of this widespread metabolic disease. Finally, using the thiouracil tagging method, we will identify the hepatic extracellular miRNAs in circulation as biomarkers predicting the insulin resistance state in obesity.