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 due to 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 to 50 µM) protects tissue-intact and cultured vascular endothelial cells from hyperglycaemia/ROS-induced dysfunction, typified by reduced agonist-stimulated endothelium-dependent, NO-mediated vasorelaxation in response to muscarinic or Proteinase-activated-receptor-2 (PAR2) agonists. Metformin not only attenuated hyperglycaemia-induced ROS production in aorta-derived endothelial cell cultures, but also prevented hyperglycaemia-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 modelling of metformin-NR4A1 interactions, Nr4a1-mutagenesis and a transfected HEK 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 metformin9s 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 hyperglycaemia-induced dysfunction in addition to metformin9s action to enhance insulin action in diabetics. Significance Statement Metformin improves diabetic vascular vasodilator function, having cardioprotective effects beyond its glycemic control; but its mechanism to do so is unknown. We found that metformin, at therapeutic concentrations(1-50µM), prevents hyperglycaemia-induced endothelial vasodilator dysfunction by attenuating reactive oxygen-species-induced damage, whereas high metformin(>250 µM) impairs vascular function. However, this action of metformin requires the expression of the orphan nuclear receptor, NR4A1/Nur77. Our data reveal a novel mechanism whereby metformin improves diabetic vascular endothelial function, with implications for developing new metformin-related therapeutic agents.