Hyperglycemia impairs mitochondrial respiration in human brain microvascular endothelial cells

Abstract

ObjectiveDiabetes is an important risk factor for the stroke. Hyperglycemia‐induced changes in the cerebral vasculature play critical role in adverse effects of diabetes on stroke. Previously, we reported the regulation of vascular function by mitochondria. In this study, we characterized for the first time the effect of hyperglycemia on mitochondrial respiratory function in human brain microvascular endothelial cells (HBMECs) in vitro using the Seahorse XFe24 instrument.MethodsHBVECs were plated on seahorse cell pates and exposed to endothelial cell media containing normal glucose (5 mmol/L), high glucose (25 mmol/L) and normal glucose (5 mmol/L) with mannitol (20 mmol/L) for 24 hours. Basal respiration, ATP production and maximal respiration were measured along with the spare respiratory capacity, proton leak and non‐mitochondrial respiration. Parameters were reported as oxygen consumption rate normalized to the protein concentration of cell lysates in each well (OCR, picomoles/L of O2 per microgram protein). Experiments were repeated three times with 7 to 8 technical replicates for each treatment in each experiment.ResultsBasal respiration was not significantly altered by hyperglycemia, whereas maximal respiration was decreased by 27.5 % when compared to HBMECs treated with normal glucose (7.31±0.32 vs 10.1±0.29, p=0.0002). Spare respiratory capacity was decreased by 47.5% (2.20±0.30 vs 4.25±0.24, p=0.0003) by hyperglycemia, whereas no significant changes were observed in ATP production and non‐mitochondrial respiration. Proton leak showed a strong trend towards decrease (1.09±0.07 vs 1.45±0.03, p=0.06) in high glucose‐treated HBMECs when compared to normal glucose‐treated cells. The observed respiratory changes were due to hyperglycemia but not due to elevated osmotic pressure, as HBMECs treated with mannitol did not exhibit similar respiratory changes.ConclusionsHyperglycemia diminishes the ability of endothelial cells to meet the increased energy demands placed by cellular stress such as neurovascular coupling (physiological) stroke (pathological). Thus, mitochondrial respiratory dysfunction in HBMECs is a novel link between hyperglycemia and cerebrovascular dysfunction.Support or Funding InformationSupport: National Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.