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，它先于2型糖尿病的发展，因此是新出现的糖尿病流行的驱动力。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，而且有大量未得到满足的医疗需求，需要更好的治疗糖尿病的方法，以防止发病率和死亡率。我们最近的工作发现，肥胖会导致肝脏细胞外小泡(EV)miRNAs的分泌发生动态变化，从而对胰岛素产生细胞和外周胰岛素敏感性产生深远影响。肝细胞特异性Rab27KO小鼠肝细胞外miRNAs缺失导致肥胖早期糖耐量和胰岛素敏感性受损。我们进一步证明，4wks高脂饮食(4wks-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，4wks-HFD EV治疗显著提高了胰岛素水平。 体外和体内β细胞的分泌和增殖。相比之下，长期肥胖诱导了致病肝细胞EV miRNAs的分泌，从而削弱了瘦身受体WT小鼠的胰岛素敏感性。一致地，通过敲除肝脏Rab27而没有肝脏细胞外miRNAs的小鼠在16周的HFD喂养后表现出胰岛素抵抗的减少。来自YBX1KO肝细胞的不含miRNA的EVS对受体小鼠的代谢表型影响很小，这表明miRNAs是这些EVS中的关键货物。利用一种新的硫尿嘧啶标记方法，我们发现miR-3075-5P是一种在4wks-HFD EV中高度浓缩的miRNAs，它可以有效地整合到靶细胞中，并通过抑制Fa2h的表达来改善细胞的胰岛素应答。此外，miR-434-3p-Map2k6调节轴在 促进巨噬细胞的促炎性激活，这可能随后加剧组织 炎症和胰岛素抵抗。这些结果表明，肝脏EV miRNAs是调节肥胖中胰岛素产生和靶向细胞功能的重要内分泌分子。这项提议希望建立在这个新发现的肝脏EV miRNAs调控系统的基础上，以揭示肥胖诱导的胰岛素抵抗的潜在细胞和分子机制。我们将进一步确定肝脏EV miRNAs调节β细胞和胰岛素敏化细胞功能以应对肥胖的机制。通过这些实验，我们将开发miR-3075-5P作为胰岛素增敏分子，并探索miR-434-3P在肥胖中的致病作用。该治疗策略可用于糖尿病前期胰岛素抵抗肥胖患者的治疗。这将导致改善血糖控制，在我们的治疗设备中增加一个新的成分，用于治疗这种广泛存在的代谢性疾病。最后，利用硫尿嘧啶标记方法，我们将确定循环中的肝脏细胞外miRNAs作为预测肥胖患者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.