PROJECT SUMMARY Adipose dysfunction is a defining characteristic of metabolically unhealthy obesity. During obesity, excess calories are stored in preexisting adipocytes. This cellular growth, otherwise known as adipocyte hypertrophy, results in a pathological form of fat expansion that leads to inflammation, fibrosis, and insulin resistance. In contrast, there is growing evidence indicating that fat expansion driven by an increase in adipocyte number (hyperplastic growth) promotes metabolically healthy obesity. Our lab and others have recently shown that tilting the balance in favor of hyperplastic fat expansion protects against maladaptive adipocyte hypertrophy and obesity-induced metabolic disease. However, the specific cellular and molecular mechanisms that regulate hyperplastic fat expansion remain unknown. To address this gap in knowledge, our lab has pioneered the combined use of stable isotope tracing and quantitative mass spectrometry to precisely measure in vivo adipogenesis, the process that drives hyperplasia through the differentiation of adipocyte progenitors into new adipocytes, during various stages of development. Using this novel technique, we have characterized a “hyperplastic-to-hypertrophic” adipose growth switch in humans and mice during which adipogenesis precipitously declines. Differential expression analysis shows that this growth switch is accompanied by a stark downregulation in the expression of insulin-like growth factor 2 (Igf2, IGF2) in both subcutaneous adipose tissue and adipocyte progenitors (APs). Follow-up studies in ex vivo primary APs revealed that IGF2 promoted adipogenesis and cell cycle gene expression, leading us to hypothesize that IGF2 stimulates AP proliferation, enhances adipogenesis, and is capable of promoting hyperplastic fat expansion. This proposal will test this hypothesis through the following Aims: (1) Determine whether IGF2 regulates adipocyte progenitor (AP) proliferation ex vivo. We will use stable isotope tracing, EdU flow cytometry assays, and MTT cellular proliferation assays to assess whether recombinant IGF2 treatment enhances proliferation of ex vivo primary APs in basal conditions and during adipogenic differentiation, while also testing the modifying effect of cell cycle inhibition. (2) Determine whether IGF2 regulates adipogenesis and fat expansion in vivo. We will use mouse models of AP-specific IGF2 loss-of-function (AP-IGF2LOF) and gain-of-function (AP-IGF2GOF) to test whether IGF2 regulates hyperplastic fat expansion in vivo during both early postnatal development and in the context of high fat diet-induced obesity. The results of this proposal will provide a better understanding of whether restoring IGF2 action in adipocyte progenitors is a viable strategy for reprogramming hyperplastic fat growth and treating obesity-induced metabolic disease. Completion of this proposal’s aims will also provide excellent training that is consistent with my goal of a career as a cardiologist...