Ceramide-induced Mitochondrial Damage in Diabetic Retinopathy
Yan Levitsky1, 2,3; Sandra S.Hammer1; Todd Lydic1; Artem Muchnik1; Anand Saripalli2; David Pegouske2,1; Denis A.Proshlyakov2; Julia V.Busik1
1Physiology, 2Chemistry, 3COM Michigan State University, E. Lansing, MI, USA
Mitochondrial damage precedes histopathological abnormalities in sight threatening complication of diabetes, diabetic retinopathy (DR). Mitochondria contain sphingolipids, as well as enzymes of sphingolipid pathway, including Acid Sphingomyelinase (ASM). Increase in ASM activity and ceramide production were shown to promote diabetes-induced pro-inflammatory and pro-apoptotic changes in the retina and RPE cells. This study addressed the role of diabetes-induced increase in ASM and ceramide in mitochondrial damage in the retina and RPE cells.
Retinal mitochondria from control, STZ-induced diabetic and ASM-/- rodent models were isolated by differential centrifugation. Sphingolipids were analyzed by eSI-MS. Human (H)RPE cells were isolated from control and diabetic donors. Respiration was measured using a novel custom designed microrespirometer.
Mitochondrial ceramide (mCer) increased in short term (7 wks) diabetic rat retina (38 ± 4%) compared to control (26.5 ± 6.5%, P<0.05, n=3). Mitochondrial sphingomyelin (mSM) decreased in diabetic retina (62 ± 1%) compared to control (72.5 ± 7%, P<0.05, n=3). Long term (36 wks) diabetic (n=4) rat retina showed significant increases in short chain (C16:0, C18:0) mCer compared to control (n=3, P<0.05). Wildtype and ASM-/- mouse retinas were used to determine if mCer changes are due to ASM. ASM-/- retina showed increased mSM (85.0 ± 6.7%) compared to wildtype (77.0 ± 2.8%) and decreased mCer (11.5 ± 3.6%) compared to wildtype (17.6 ± 3.6%, P<0.05, n=3). HRPE cells, a mitochondrial rich cell population missing from retina preparations, isolated from diabetic donor eyes showed increased IL1ß, IL6 and ASM expression and a 2- fold increase in Cer compared to control (P<0.05, n=3). Average mitochondrial length decreased in diabetic HRPE (1.2 ± 0.57 μm) compared to control (3.4 ± 0.78 μm, P<0.05, n=3). Whole cell respiratory control ratio was decreased greater than 2-fold in diabetic HRPE compared to control.
Diabetes-induced elevation of ASM leads to increased ceramide in mitochondria isolated from diabetic retina and RPE cells. This increase is associated with upregulation of inflammatory markers and mitochondrial dysfunction in diabetic retina and RPE cells.