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

Abstract

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 aortaderived 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 metformin- NR4A1 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 inmetformin'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.