High Glucose-induced Changes in Mitochondrial Membrane Potential, Oxygen Consumption Rate, and Extracellular Acidification Promotes Mitochondrial Dysfunction in Retinal Endothelial and Müller Cells.

Abstract

Purpose : To investigate whether high glucose (HG) promotes mitochondrial dysfunction by altering mitochondrial membrane potential (Δψm), extracellular acidification rate (ECAR), and oxygen consumption rate (OCR) in retinal endothelial and Müller cells.

Methods : Rat retinal Müller cells (rMC-1) and rat retinal endothelial cells (RRECs) were grown in normal medium (N; 5 mM glucose) or HG medium (30 mM) for 7 days. ΔΨm and ADP to ATP phosphorylation in these cells was estimated from changes in Safranin O fluorescence. In parallel, mitochondrial metabolic function under HG was assessed by measuring OCR and ECAR using the XF24 bioenergetic analyzer. Additionally the effects of HG on mitochondrial function were compared between retinal Müller cell primary culture and a retinal Müller cell line.

Results : The Δψm was significantly decreased in rMC-1 grown in HG compared to Δψm in cells grown in normal medium (380±131 vs 559±65 a.u, p<0.05, n=4). Similarly, RRECs grown in HG showed decreased Δψm compared to Δψm in cells grown in N medium (246±47 vs 472±80 a.u, p<0.0001, n=6). The mitochondrial ability to phosphorylate ADP to ATP during this period in HG was significantly decreased in rMC-1 and RRECs by 40% and 42%, respectively, compared to those of control cells. rMC-1 grown in HG showed a significant decrease in steady state (0.05 vs. 0.08 nmol O₂/min/10⁶ cells in N, p<0.05, n=6) and maximum OCR (0.04 vs 0.09 nmol O₂/min/10⁶ cells in N, p<0.05, n=6). ECAR was also significantly decreased in rMC-1 grown under HG condition compared to those grown in N medium (40±4% of control; p<0.05, n=6). A similar decrease in ΔΨm, ECAR, and OCR was observed in primary cultures of retinal Müller cells grown in HG condition compared to those of N cells. RRECs grown in HG showed a significant decrease of steady state (1.55 vs. 1.99 nmol O₂/min/10⁶ cells in N, P = 0.016, n=6) and maximal OCR (1.89 vs. 3.10 nmol O₂/min/10⁶ cells in N, p=0.014, n=6). However, ECAR of RRECs was significantly increased under HG condition (135 ±12% of control, p=0.005, n=6).

Conclusions : In retinal endothelial and Müller cells, HG condition decreases ΔΨm, OCR, and ECAR thereby contributing to mitochondrial dysfunction associated with diabetic retinopathy.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.