oa Metformin Reverses Glucose Starvation Induced Endoplasmic Reticulum Stress And Autophagy In The Microvascular Endothelium

Background and Objective: Diabetes Mellitus is associated with a higher incidence of myocardial infarction (MI) and poor prognosis in affected individuals; however, treatment with the oral hypoglycemic drug, metformin, is associated with a reduction in cardiovascular morbidity and mortality. A MI reduces oxygen delivery and results in nutrient/glucose starvation (GS) with micro-vascular endothelial cells (MECs) receiving the initial impact, the effect being more profound in a diabetic individual as glucose levels drop from hyperglycemia to GS. It is, however, not known whether GS triggers endoplasmic reticulum (ER) stress and autophagy in MECs and whether metformin has any effect on GS associated ER stress and autophagy. Thus, protocols were designed to determine whether GS-induced ER stress and autophagic events could be rescued. Methods— Mouse Microvasuclar Endothelial Cells (MMECs) were subjected to GS for 24h in the presence and absence of sodium-4-phenylbutyrate (chemical chaperone, 4PBA, 10mM) or metformin (50µM and 2mM) and western blot analysis was performed to assess the induction of ER stress and autophagy. Immunofluorescence staining was also done to assess LC3B punctae staining in cells, indicating autophagy. Results- GS caused ER stress as evidenced by significant increase in the levels of ER stress markers such as GRP78, ATF4 and CHOP. Significant increases in the levels of LC3A-II and LC3B-II and an increase in the level of LC3B punctae staining in glucose starved cells confirmed autophagic activation. Treatment with 10mM 4PBA significantly reversed GS induced ER stress and subsequent autophagy. The effects of varying concentrations of metformin (10µM-2mM) were studied. Metformin (2mM) significantly reversed ER stress and autophagy in glucose starved endothelial cells while lower concentrations that were in the therapeutic range had no effect. Markedly decreased LC3B punctae staining in metformin treated glucose deprived cells also ascertained the reversal of autophagy. Conclusions— This study demonstrates for the first time that GS induced ER stress and autophagy in the microvascular endothelium can be reversed by metformin, albeit only at mM concentrations. This work has been supported by QNRF grants JSREP- 3- 016-3-009 and NPRP 4-910-3-244.