Project Summary Type 2 diabetes (T2D) is one of the defining medical challenges of the 21st century, with one in three Americans born in 2000 estimated to develop diabetes in their lifetime. T2D is characterized by multi-organ insulin resistance and perturbed whole-body glucose homeostasis. Over the past three decades, there have been hints that the kidney plays a central pathophysiologic role in dysregulated whole-body glucose homeostasis in diabetes, with a seminal study suggesting renal glucose production to be increased 300%, climbing to 85% that of the liver. Notwithstanding, insulin resistance in the kidney has remained controversial and any potential mechanisms are unknown. It would be of great public health interest to crystallize the mechanisms and precise pathophysiology of insulin resistance in the kidney, as this may have myriad translational implications. In this proposal, we will build upon our strong preliminary evidence that the renal cortex does, indeed, become insulin resistant with high fat diet (HFD) feeding. In further preliminary data, we have observed both diacylglycerol (DAG) accumulation and Protein Kinase Cε (PKCε) translocation in the mouse renal cortex, raising the possibility that diet-induced renal insulin resistance may be mediated by a similar mechanism as in the liver, where high fat feeding leads to DAG accumulation, which activates PKCε. PKCε subsequently phosphorylates insulin receptor (IR) at Thr1160, causing abrogated insulin signaling. In this proposal, we will carefully assess the insulin signaling defects associated with renal insulin resistance and also further characterize if there is aberrant DAG-PKCe-IR axis activation. We will also use two novel 13C isotopic tracer strategies to understanding oxidative and gluconeogenic defects in the insulin resistant renal cortex. Further, we will directly test the hypothesis that the DAG-PKCe-IR axis causes renal insulin resistance by utilizing an already-generated mouse model where the critical Thr1160 residue of IR is mutated to an alanine, which cannot be phosphorylated by PKCε. We predict these mice will be protected from signaling and metabolic flux manifestations of renal insulin resistance when fed a HFD. This proposal represents an integrated scientific approach and new learning experiences that harness techniques of physiology, cell biology, and analytical chemistry to yield novel insights into the mechanisms and pathophysiology or renal insulin resistance.