Abstract Wed124: Metabolic Bifurcation of O-GlcNAcylation Determines Functional versus Pathological Angiogenesis in Diabetes

Introduction: Peripheral arterial disease (PAD) causes limb ischemia and amputation, particularly in diabetes. Angiogenesis—the formation of new blood vessels from pre-existing vasculature—is a potential therapeutic approach in PAD; however, clinical trials therapeutically inducing angiogenesis have failed in PAD patients. In contrast, supervised exercise is the only intervention shown to induce functional angiogenesis and improve outcomes. We previously showed that glucosamine, which bypasses glutamine fructose-6-phosphate amidotransferase-1 (GFAT1), induces endothelial O-GlcNAcylation and exercise-like functional angiogenesis. Conversely, diabetes induces GFAT1-dependent O-GlcNAcylation yet impairs angiogenesis and outcomes. Hypothesis: We hypothesize that GFAT1-independent O-GlcNAcylation is adaptive, whereas GFAT1-dependent O-GlcNAcylation is pathological. Methods: Endothelial cells were treated under hypoxia and serum starvation (HSS) with either glucosamine or high glucose, with or without silencing of GFAT1, OGT, or YAP/TAZ. Angiogenesis, oxidative phosphorylation, and barrier function were assessed. Glycoproteomics and OGT-IP defined O-GlcNAcylated proteins. Hindlimb ischemia (HLI) was performed in control and high-fat diet (HFD) mice. Results: High glucose induced GFAT1-dependent O-GlcNAcylation comparable to glucosamine yet failed to activate ATF4, impaired mitochondrial metabolism, and suppressed angiogenesis. Glucosamine induced GFAT1-independent O-GlcNAcylation, activated ATF4, enhanced oxidative phosphorylation, and promoted angiogenesis, but lost efficacy in diabetic conditions. HFD increased O-GlcNAcylation after HLI but impaired perfusion recovery, and glucosamine failed to improve perfusion recovery. Glycoproteomics identified Hippo signaling, with LATS1 O-GlcNAcylation, reduced YAP phosphorylation, and YAP/TAZ-dependent ATF4 induction. Furthermore, proteomic analysis on OGT-IP in HUVECs showed distinct O-GlcNAcylation profile high glucose vs. glucosamine. GFAT1 silencing restored metabolism, angiogenesis, and barrier function under high glucose. Conclusion: GFAT1-independent O-GlcNAcylation drives exercise-like, functional angiogenesis, whereas GFAT1-dependent O-GlcNAcylation mediates diabetic vascular dysfunction. Targeting GFAT1 may enable therapeutic angiogenesis in diabetic PAD.