Metformin Prevents Hyperglycemia-Associated, Oxidative Stress-Induced Vascular Endothelial Dysfunction: Essential Role for the Orphan Nuclear Receptor Human Nuclear Receptor 4A1 (Nur77)

Vascular pathology is increased in diabetes because of reactive-oxygen-species (ROS)-induced endothelial cell damage. We found that in vitro and in a streptozotocin diabetes model in vivo, metformin at diabetes-therapeutic concentrations (1-50 mM) protects tissue-intact and cultured vascular endothelial cells from hyperglycemia/ROS-induced dysfunction typified by reduced agonist-stimulated endothelium-dependent, nitric oxide-mediated vasorelaxation in response to muscarinic or proteinase-activated-receptor 2 agonists. Metformin not only attenuated hyperglycemia-induced ROS production in aorta derived endothelial cell cultures but also prevented hyperglycemia-induced endothelial mitochondrial dysfunction (reduced oxygen consumption rate). These endothelium-protective effects of metformin were absent in orphan-nuclear-receptor Nr4a1-null murine aorta tissues in accord with our observing a direct metformin-Nr4a1 interaction. Using in silico modeling of metforminNR4A1 interactions, Nr4a1-mutagenesis, and a transfected human embryonic kidney 293T cell functional assay for metformin-activated Nr4a1, we identified two Nr4a1 prolines, P505/P549 (mouse sequences corresponding to human P501/P546), as key residues for enabling metformin to affect mitochondrial function. Our data indicate a critical role for Nr4a1 in metformin's endothelial-protective effects observed at micromolar concentrations, which activate AMPKinase but do not affect mitochondrial complex-I or complex III oxygen consumption rates, as does 0.5 mM metformin. Thus, therapeutic metformin concentrations requiring the expression of Nr4a1 protect the vasculature from hyperglycemia-induced dysfunction in addition to metformin's action to enhance insulin action in patients with diabetes. SIGNIFICANCE STATEMENT Metformin improves diabetic vasodilator function, having cardioprotective effects beyond glycemic control, but its mechanism to do so is unknown. We found that metformin at therapeutic concentrations (1-50mM) prevents hyperglycemia-induced endothelial dysfunction by attenuating reactive oxygen species-induced damage, whereas high metformin (>250 mM) impairs vascular function. However, metformin's action requires the expression of the orphan nuclear receptor NR4A1/Nur77. Our data reveal a novel mechanism whereby metformin preserves diabetic vascular endothelial function, with implications for developing new metformin-related therapeutic agents.