Abstract This is a competitive renewal of NIH grant DK082659 entitled “Developmental Genes, miRNAs & Adipose Tissue”. Obesity is epidemic throughout the population and is a major driving force in the pathogenesis of type 2 diabetes, metabolic syndrome and many other disorders. Over the past decade, work supported by this grant has shown that obesity depends not only on the balance between energy intake and utilization, but also on the balance between energy storage in white adipose tissue (WAT) and energy expenditure in brown adipose tissue (BAT). We have shown that both WAT and BAT are heterogeneous, in part due to intrinsic differences between adipocytes in different depots and also through their dependence on insulin/IGF1/FoxO signaling. We have demonstrated heterogeneity at the cellular level by clonal analysis of murine preadipocytes and by single cell analysis of human preadipocytes. In important new studies, we have found that different adipose depots differ in their expression of miRNAs and that there are alterations in miRNAs and miRNA processing in adipose tissue in aging, obesity, and lipodystrophy. Furthermore, we have shown that adipose tissue is a major source of circulating exosomal miRNAs; that these can regulate gene expression in other tissues, forming a novel mode of adipose communication; and that this is, in part, regulated by sequence motifs within miRNAs which govern their cellular retention vs. exosomal secretion. The specific aims for the next 5 years are: Aim 1: Define the interactions between adipocyte heterogeneity, insulin signaling and exosomal miRNA mediated cellular communication. We will define the role of insulin in regulation of adipocyte miRNA expression and exosomal secretion and determine differences between subtypes of WAT and BAT. We will study the effects of BAT activation, obesity and weight loss In both mice and humans. We will determine if the recently defined EXO and CELL motifs and RNA binding proteins involved in miRNA sorting play a role in these processes, and explore the role of exosomal tRNA fragments, as a new class of regulatory small RNAs. Aim 2: Use UPRT labeling and hCD63 to directly track exosomal miRNAs and exosomes in vivo. With these, we will determine the relative contributions of BAT and WAT to the pool of circulating exosomes and exosomal miRNAs and how these change during cold exposure, feeding/fasting, and in obesity. We will also explore the role of insulin in vivo using the euglycemic clamp and tissue specific InsR knockout mice. Aim 3: Enhance potential of miRNA therapeutics by manipulating exosomal sorting of miRNAs in vivo. Taking advantage of our recent identification of motifs that modulate miRNAs cellular retention or exosomal secretion, and using cholesterol/lipoprotein metabolism as a model, we will explore the role of modifying miRNA or their sorting proteins for therapeutic purposes. Together with the studies above, this will allow us to uncover how the exosomal miRN...