Project Summary The thiazolidinediones (TZDs) are powerful anti-diabetic drugs whose use in treating type 2 diabetes is limited by adverse side effects. The goal of this proposal is a biochemical investigation into the mechanism that separates the positive metabolic effects of TZDs to lower glucose from their well-characterized negative side effects. PPARγ, a molecular target of the TZDs, regulates systemic insulin sensitivity by promoting formation of new adipocytes. However, we show that the TZDs have a second biochemical function on PPARγ, to block phosphorylation of serine 273 (pS273). We find that phosphorylation of PPARγ at serine 273 promotes insulin resistance without affecting adipogenesis. Reversing this phosphorylation with pharmacological or genetic inhibition is sufficient to promote insulin sensitivity and increase thermogenic responses to cold. We propose that one of the main mechanisms by which phosphorylation of PPARγ at serine 273 promotes insulin resistance is through increased expression of growth differentiation factor 3 (Gdf3). Gdf3 is a secreted protein in the TGF- /BMP superfamily and a negative regulator of BMP signaling. In adipose tissue, BMP proteins contribute to insulin sensitivity and thermogenesis. We therefore propose that elevated levels of Gdf3 in obesity are the cause of insulin resistance mediated by PPARγ S273 phosphorylation. Indeed, we show that ectopic expression of Gdf3 is sufficient to cause insulin resistance in lean mice. In this proposal we examine the contribution of Gdf3 to the pathogenesis of obesity and insulin resistance. We predict that blocking Gdf3 levels or activity will restore whole-body insulin sensitivity and promote thermogenesis. We will interrogate the role of Gdf3 on glucose homeostasis and obesity using both acute gain-of-function and loss-of-function models. We will utilize innovative new wireless continuous glucose monitoring technology concurrently with indirect calorimetry to achieve high- precision measurements of insulin resistance, energy balance, and circulating nutrient availability. We will determine whether Gdf3 is the mechanism linking PPARγ S273 phosphorylation and insulin resistance. We will also determine whether Gdf3 requires BMP signaling through SMAD1/5/8 proteins. Finally, we will perform an unbiased investigation into the pathways required for Gdf3 signaling using a genome-wide CRISPR/Cas9 knockout screen. This hypothesis suggests a new therapeutic modality which harnesses a specific beneficial aspect of TZD treatment which is independent of TZD-associated side effects. The results from this proposal will definitively determine if Gdf3 is a suitable target for promoting metabolic health.