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.

PROJECT NARRATIVE Obesity is one of the most common metabolic disorders in the world and the leading cause of Type 2 Diabetes. Our study aims to understand how the regulation of a microRNA, a small non-coding RNA, from the microvasculature regulates angiogenesis in adipose tissues and whether there is a correlation between improved angiogenesis and adipocyte function and obesity-induced insulin resistance. The results from our studies may have important translational implications and provide a new understanding for adipose tissue dysfunction in obesity induced insulin resistance.