Mitochondrial damage precedes histopathological abnormalities in diabetic retinopathy (DR). Retinal endothelial cells (REC) have the highest level of Acid Sphingomyelinase (ASM) expression in the retina and the increase in ASM activity and ceramide production were shown to promote diabetes-induced pro-inflammatory and pro-apoptotic changes in the retina and REC. The role of diabetes-induced increase in ASM and ceramide production in mitochondrial damage in the retina and REC is not known.
Retinal mitochondria from control, STZ-induced diabetic and ASM-/- rodent models, as well as Bovine REC (BREC) treated with diabetogenic conditions were isolated by differential centrifugation. Sphingolipids were analyzed by eSI-MS. Respiration was measured using a novel custom designed microrespirometer. Mitochondrial sphingomyelin (mSM) decreased (62 ± 1% compared to control 72.5 ± 7%, P<;0.05, n=3) and mitochondrial ceramide (mCer) increased (38 ± 4% compared to control 26.5 ± 6.5%, P<0.05, n=3 in 7 weeks diabetic rat retina. ASM-/- mouse model showed increased mSM (85.0 ± 6.7%) compared to wild type (77.0 ± 2.8%) and decreased mCer (11.5 ± 3.6%) compared to wild type (17.6 ± 3.6%, P<0.05, n=3), confirming that ASM is an essential contributor to mitochondrial ceramide regulation. Pre-treatment of BREC with an ASM inhibitor Desipramine revealed a 1.75-fold increase in maximal respiratory capacity compared to control cells. Diabetes results in decreased mitochondrial sphingomyelin and increased mitochondrial ceramide consistent with ASM-dependent ceramide production. Inhibition of ASM in BREC results in increased maximal, but not basal, respiratory rates. These results support the hypothesis that ASM activation and mitochondrial ceramide accumulation leads to inhibition of oxidative phosphorylation and REC damage in diabetic environment.
D. Pegouske: None. Y. Levitsky: None. S. Hammer: None. T.A. Lydic: None. A. Muchnik: None. D.A. Proshlyakov: None. J.V. Busik: None.
National Institutes of Health (R01EY028049, R01EY016077)