MiR-409-3p Regulates Angiogensis, Brown Fat Adiposity and Insulin Resistance

项目摘要/摘要 白色和棕色脂肪组织是高度血管化的器官，能够基于 代谢需求和能量消耗。然而，这些组织的不适应调节可能会导致 胰岛素抵抗。在我们对血管生成如何影响脂肪组织的理解中仍然存在关键差距 功能障碍和整体新陈代谢。MicroRNAs(MiRs)参与血管生成的调节。 对病理生理刺激的反应。MIR在调节饮食诱导的血管生成反应中的作用 人们对胰岛素抵抗知之甚少。 使用miRNA-Seq方法，我们发现miR-409-3p在 饮食诱导肥胖(DIO)小鼠和人糖尿病棕色脂肪组织(BAT)内皮细胞的观察 将血浆样本与非糖尿病患者进行比较。MiR-409-3p过表达显著抑制EC 抑制miR-409-3p的作用相反。初步研究表明， MIR-409-3P靶向锌指E盒结合同源框1(ZEB1)的3‘UTRs和丝裂原激活 蛋白激酶3(MAP4K3)。MiR-409-3p过表达降低ZEB1和 MAP4K3在内皮细胞中表达，而抑制则有相反的作用。ZEB1或ZEB1的siRNA敲除 MAP4K3在内皮细胞中的表达可明显抑制miR-409-3p的过表达 减少了EC的增殖和迁移。3T3-L1细胞或人皮肤脂肪类化合物与 高表达miR-409-3p的内皮细胞上清液可降低棕色脂肪细胞的表达 RT-qPCR和Western印迹分析标记(UCP1、CIDA)，而从内皮细胞收获上清液 MiR-409-3p缺失可增加棕色脂肪细胞标志物的表达。全身静脉给药 CD31染色显示，LNA-抗miR-409-3p抑制剂对DIO小鼠血管生成有显著促进作用。 RT-qPCR、Western印迹和免疫组织化学检测BAT和SWAT中UCP-1的表达 分析，同时改善葡萄糖和胰岛素耐量和整体新陈代谢。因此，我们假设 MiR-409-3p在脂肪组织中作为EC生长和血管生成的关键调节因子，可能 改善DIO患者的代谢功能障碍。为了探索这一点，我们首先在Aim1上提出了研究分子 MiR-409-3p调控EC生长和血管生成的机制。在AIM2中，我们将描述 内皮细胞miR-409-3p调节脂肪组织褐变的机制。最后，在Aim3中，我们将 探讨miR-409-3p中和在脂肪组织血管生成和DIO发生中的作用 和小鼠的胰岛素抵抗。这些研究的成功完成将有助于对监管角色的深入了解 MiR-409-3p在饮食诱导肥胖中血管生成受损和脂肪组织功能障碍之间的关系 可用于肥胖诱导的胰岛素抵抗的治疗干预。

PROJECT SUMMARY/ABSTRACT White and brown adipose tissues are highly vascularized organs, capable of plasticity based on metabolic demands and energy expenditure. However maladaptive regulation of these tissues can lead to insulin resistance. Critical gaps remain in our understanding of how angiogenesis impacts adipose tissue dysfunction and overall metabolism. MicroRNAs (miRs) are implicated in the regulation of the angiogenic response to pathophysiological stimuli. The role of miRs in regulating the angiogenic response in diet-induced insulin resistance is poorly understood. Using a miRNA-Seq approach, we identified that miR-409-3p expression was significantly increased in endothelial cells (ECs) of brown adipose tissue (BAT) of diet-induced obese (DIO) mice and in human diabetic plasma samples compared to non-diabetic patients. Overexpression of miR-409-3p markedly inhibited EC growth and migration, whereas miR-409-3p inhibition had the opposite effects. Preliminary studies indicate that miR-409-3p targets the 3’UTRs of Zinc Finger E-box binding Homeobox 1 (ZEB1) and Mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3). Overexpression of miR-409-3p decreased ZEB1 and MAP4K3 expression in ECs, whereas inhibition had the opposite effect. SiRNA knockdown of ZEB1 or MAP4K3 expression in ECs phenocopied the effects of miR-409-3p overexpression and significantly decreased EC proliferation and migration. 3T3-L1 cells or human skin fat organoids co-cultured with supernatant harvested from ECs overexpressing miR-409-3p had decreased expression of brown adipocyte markers (UCP1, Cidea) by RT-qPCR and Western blot analyses, whereas supernatant harvested from ECs deficient in miR-409-3p increased expression of brown adipocyte markers. Systemic intravenous delivery of LNA-anti-miR-409-3p inhibitor to DIO mice significantly increased angiogenesis by CD31 staining, accompanied by higher UCP-1 in BAT and sWAT by RT-qPCR, Western blot, and immunohistochemistry analyses, while improving glucose and insulin tolerance and overall metabolism. Therefore, we hypothesize that miR-409-3p serves as a critical regulator of EC growth and angiogenesis in adipose tissue and may improve metabolic dysfunction in DIO. To explore this, we first propose in Aim1 to investigate the molecular mechanisms by which miR-409-3p regulates EC growth and angiogenesis. In Aim2, we will delineate the mechanisms by which miR-409-3p in ECs regulates browning in adipose tissues. Finally, in Aim3, we will explore the effect of miR-409-3p neutralization in the vasculature of adipose tissues and development of DIO and insulin resistance in mice. Successful completion of these studies will shed insights on the regulatory role of miR-409-3p between impaired angiogenesis in diet-induced obesity and adipose tissue dysfunction, an effect that could be exploited for therapeutic intervention in obesity-induced insulin resistance.