PT - JOURNAL ARTICLE AU - Vivek Krishna Pulakazhi Venu AU - Mahmoud Saifeddine AU - Koichiro Mihara AU - Muniba Faiza AU - Evgueni Gorobets AU - Andrew J. Flewelling AU - Darren Derksen AU - Simon Hirota AU - Isra Marei AU - Dana Al-Majid AU - Majid Motahhary AU - Hong Ding AU - Chris R. Triggle AU - Morley D. Hollenberg TI - <strong>Metformin prevents hyperglycaemia-associated, oxidative stress-induced vascular endothelial dysfunction: essential role for the orphan nuclear receptor, Nr4a1 (Nur77).</strong> AID - 10.1124/molpharm.120.000148 DP - 2021 Jan 01 TA - Molecular Pharmacology PG - MOLPHARM-AR-2020-000148 4099 - http://molpharm.aspetjournals.org/content/early/2021/08/27/molpharm.120.000148.short 4100 - http://molpharm.aspetjournals.org/content/early/2021/08/27/molpharm.120.000148.full AB - 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 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 hyperglycaemia-induced dysfunction in addition to metformin's 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(&gt;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.