Sex and Leptin control of endothelial cell glycolysis and redox balance in Type 1 Diabetes

PROJECT SUMMARY – PROJECT 2 The goal of Project #2 is to delineate the molecular mechanisms whereby endothelial cell (EC) bioenergetics and, glycolysis, in particular, regulate vascular redox status and vasorelaxation in physiological and pathological conditions with a focus on type 1 diabetes (T1D). Endothelial dysfunction (ED) is a precursor and major contributor to vascular complications which are among the most disabling and life- threatening complications of T1D. Preventing and managing endothelial function as well as reducing the occurrence of cardiovascular events remains a challenge in T1D, notably in female patients who lose the protective effects of female sex and have been poorly studied. Emerging evidence suggests a role for EC bioenergetics, notably glycolysis, in the control of vascular function. In preliminary data, we show that enhanced EC glycolysis is a novel cause of ED in T1D and uncover unexpected roles for female sex and endothelial leptin signaling as regulators of EC metabolism and vascular homeostasis. Our novel data show that ED in T1D involves increases in EC glycolytic flux, increased 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3, a major regulator of glycolysis) expression, and increased expression and activity of the reactive oxygen species (ROS) producing enzyme, NADPH Oxidase 1 (Nox1), in ECs. Functionally, we demonstrate that selective inhibition of PFKFB3 or Nox1 restores EC-dependent relaxation in T1D. Conversely, we show that upregulation of PFKFB3, via ex-vivo adenoviral EC transduction, is alone sufficient to impair EC relaxation in the aorta via mechanisms that include increased EC Nox1 and ROS production and decreased eNOS-derived nitric oxide. Remarkably, in healthy mice we observed better EC function in females which was correlated with lower ECs PFKFB3 levels and reduced ECs glycolytic flux, as compared to males. In T1D-mice, ED is associated with increased EC glycolysis in both sexes. The upstream regulators of EC metabolism remain poorly understood. Herein, we have identified a novel role for the adipokine leptin in regulating EC function and metabolism. Leptin levels are higher in females and leptin treatment restores EC metabolism and function in the aortas of T1D mice. Taken together, these findings inform the core hypothesis of this proposal: EC metabolism shapes EC function via sex-specific, Nox1 and leptin-dependent mechanisms. This hypothesis will be tested in three aims. Aim 1 will investigate whether enhanced EC glycolysis impairs endothelial function by increasing Nox1-derived ROS production and compromising eNOS-derived NO. Aim 2 will test whether sex steroid hormones and/or sex chromosomes regulate EC glycolysis and function through changes in PFKFB3 and Nox1 expression, while Aim 3 will investigate whether EC leptin signaling positively regulates endothelium-dependent relaxation by reducing glycolysis-mediated increases in Nox1 in T1D. With this Project #2, which is highly dependent on the expertise provided by other Projects and Cores, we have the expectation to demonstrate that the leptin- PFKFB3-Nox1 axis is a potential target for the treatment of diabetic vascular complications.