SPHINGOLIPID METABOLISM CONTRIBUTES TO MITOCHONDRIAL DYSFUNCTON INDUCED BY DIABETOGENIC CONDITION IN RETINAL EPITHELIAL CELLS

Abstract

Purpose : Diabetic Retinopathy (DR) is a sight-threatening complication of diabetes and is the leading cause of vision loss in working age adults. Mitochondrial damage is well accepted to precede histopathological abnormalities in DR, and significantly contribute to DR pathogenesis. Recent studies identified sphingolipid metabolism and mitochondrial ceramide content as significant factors in mitochondrial damage. This study sought to determine the role of acid sphingomyelinase (ASMase) activation and ceramide accumulation in diabetes-induced mitochondrial damage in retinal pigment epithelial cells (RPEs).

Methods : Human RPEs were isolated from control and diabetic donors and cultured according to published protocols. ARPE-19 cells were treated with high-glucose (HG) to mimic diabetic conditions. Mitochondria were isolated using differential centrifugation followed by ultracentrifugation or a Magnetic-Activated Cell Sorting (MACS) kit. Mitochondrial sphingolipid composition was assessed by electrospray ionization high resolution mass spectrometry (nESI-MS). Fragmentation was assessed by morphometric analysis of fluorescently labelled mitochondria. Mitochondrial respiration was measured using a novel custom designed microrespirometer. Inflammatory markers were measured by qPCR.

Results : Mitochondria isolated from the HRPEs of diabetic donors demonstrated a statistically significant increase in inflammatory markers IL1B and IL6, and RPEs from control donors treated under HG conditions showed similar elevation of VEGF (n=3, *p<0.01). Purified mitochondria from ARPE-19 cells treated with high glucose condition showed statistically significant increases in short-chain ceramides compared to control cells, this increase was abrogated by ASMase inhibition (*p<0.05). HRPEs obtained from diabetic donors showed an RCR of 3.9 (95% CI 2.81 to 4.99) compared to 11.1 (95% CI 9.91 to 12.3) in control donors. Mitochondria from diabetic donors showed a decrease in the average mitochondrial length of 1.2 ± 0.57 µm compared to 3.4 ± 0.78 µm in control donors.

Conclusions : These results suggest that diabetes-induced mitochondrial functional changes may form a part of the diabetic sequelae that leads to DR development and progression.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.