Modulation of Mitochondrial Bioenergetics in Human Brain Microvascular Endothelial Cells by Telomerase Reverse Transcriptase

Abstract

<b>Abstract ID 22482</b> <b>Poster Board 362</b> Human brain microvascular endothelial cells (HBMECs) play a critical role in preserving brain function by regulating blood flow and blood-brain-barrier (BBB) integrity. HBMECs use both glycolysis and oxidative phosphorylation as sources of energy. The catalytic subunit of telomerase, telomerase reverse transcriptase (TERT), is known to translocate to mitochondria during stress and regulate the generation of reactive oxygen species (ROS). This study tested the hypothesis that TERT modulates bioenergetics in HBMECs. Cultured HBMECs were used at passage 8. Agilent Seahorse (XFp) Bioanalyzer was used for determining glycolysis and oxidative phosphorylation (OXPHOS) by analyzing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR). 2-Deoxy-d-glucose (2-DG) (50 mM) and rotenone/antimycin A (R/A) (0.5 μM), and Mito Stress Test kit (Agilent), respectively, were used to evaluate different metabolic indices that are indicators of glycolysis and OXPHOS. OCR determined basal and maximal respiration, ATP production, proton leak, spare respiratory capacity, and non-mitochondrial respiration, whereas ECAR defined basal and compensatory glycolysis, post-2-DG acidification, and glycolytic proton efflux rate (glycoPER). Pharmacological activator or inhibitor of TERT, cycloastragenol (CAG) or BIBR1532, respectively, was used to determine the role of TERT in mitochondrial bioenergetics. BIBR1532 (1 μM) increased basal by 120 ± 5% (<i>p</i> &lt; 0.05) and maximal respiration (determined by FCCP (1.1 μM) -uncoupling) by 157 ± 7% (<i>p</i> &lt; 0.05) (n=5). The spare respiratory capacity and nonmitochondrial respiration were unaltered by BIBR1532 (n=5). No change was observed in the basal glycolysis by BIBR1532 (n=3) however, the compensatory glycolysis, an indicator of the maximum glycolytic capacity, was increased (n=3). Post-2DG acidification, an indicator of proton secretion from the non-glycolytic sources, was increased in the presence of BIBR1532 (n=3). However, glycoPER was unaltered. CAG showed no effect on any of the parameters of OXPHOS in the concentration range of 0.3-30 μM (n=3). This study shows that physiological levels of TERT negatively regulate and that acute inhibition of TERT increases OXPHOS and glycolysis.