Insulin resistance plays a major role in the pathogenesis of type 2 diabetes, which is emerging as one of the most critical global health challenges of the 21st century. Despite its significant contributions to whole body glucose production, surprisingly little is known regarding insulin action and insulin resistance in the kidney in vivo. While gluconeogenesis from the renal cortex is responsible for all of the glucose production from the kidney, it remains unknown what role insulin plays in regulating renal cortical metabolic fluxes, such as gluconeogenesis, mitochondrial anaplerosis, and mitochondrial substrate oxidation. Furthermore, it is widely debated if the kidney becomes insulin resistant. As such, the molecular mechanisms of lipid-induced renal cortical insulin resistance have not been well characterized. Recently, our laboratory has generated compelling data demonstrating that increases in plasma membrane sn-1,2-diacylglycerol content leads to activation of PKCe which in turn phosphorylates insulin receptor kinase threonine1150 resulting in reduced insulin receptor kinase activity and insulin resistance in vivo. This mechanism of lipid-induced insulin resistance has now been demonstrated to occur in liver, skeletal muscle and white adipose tissue in mice, rats and humans, raising the possibility of an evolutionarily conserved, whole-body mechanism of lipid-induced insulin resistance that may also extend to the renal cortex. In addition, our group has also recently demonstrated a critical role for acetyl-CoA in the regulation of hepatic gluconeogenesis by insulin and its dysregulation during obesity-associated white adipose tissue dysfunction in vivo. However, the role of acetyl-CoA in the regulation of renal cortical gluconeogenesis in vivo is unknown. This proposal seeks to build upon our strong Preliminary Data demonstrating a key role of plasma membrane sn-1,2-diacylglycerols and PKCe activation in mediating high fat diet (HFD)-induced renal cortical insulin resistance in awake rodents. We will also employ state-of-the-art liquid chromatography-tandem mass spectrometry methods to assess in vivo rates of renal cortical pyruvate dehydrogenase flux, mitochondrial succinate dehydrogenase flux, glutaminase flux and pyruvate carboxylase flux for the first time, in combination with unique transgenic rodent models to thoroughly characterize the pathophysiology and mechanisms of lipid- induced renal cortical insulin resistance and lipid-induced alterations in renal cortical gluconeogenesis and mitochondrial substrate oxidation. This proposal will also assess the effects of a controlled release mitochondrial protonophore (CRMP) in reversing HFD-induced renal cortical insulin resistance. It is anticipated that the results of these studies will provide important new insights into: 1) the molecular mechanisms of lipid-induced renal cortical insulin resistance, 2) insulin regulation of renal cortical gluconeogenesis and renal cortical mitochondri...