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

Mol Pharmacol. 2021 Nov;100(5):428-455. doi: 10.1124/molpharm.120.000148. Epub 2021 Aug 27.

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 µM) 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 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 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 (1-50µM) prevents hyperglycemia-induced endothelial dysfunction by attenuating reactive oxygen species-induced damage, whereas high metformin (>250 µM) 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Endothelium, Vascular / drug effects*
  • Endothelium, Vascular / metabolism
  • HEK293 Cells
  • Humans
  • Hyperglycemia / metabolism
  • Hyperglycemia / prevention & control*
  • Hypoglycemic Agents / pharmacology
  • Hypoglycemic Agents / therapeutic use*
  • Male
  • Metformin / pharmacology
  • Metformin / therapeutic use*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Knockout
  • Nuclear Receptor Subfamily 4, Group A, Member 1 / biosynthesis*
  • Organ Culture Techniques
  • Oxidative Stress / drug effects*
  • Oxidative Stress / physiology
  • Vasodilator Agents / pharmacology

Substances

  • Hypoglycemic Agents
  • NR4A1 protein, human
  • Nuclear Receptor Subfamily 4, Group A, Member 1
  • Vasodilator Agents
  • Metformin