Diabetes-Induced Mitochondrial Dysfunction in the Retina

Diabetes-Induced Mitochondrial Dysfunction in the Retina Renu A. Kowluru and Saiyeda Noor Abbas 4–9 PURPOSE. Oxidative stress is increased in the retina in diabetes, tase are downregulated. We have reported that the long- and antioxidants inhibit activation of caspase-3 and the devel- term administration of antioxidants inhibits the development opment of retinopathy. The purpose of this study was to of retinopathy in diabetic rats and in galactose-fed rats (another investigate the effect of diabetes on the release of cytochrome model of diabetic retinopathy),3 suggesting an important role c from mitochondria and translocation of Bax into mitochon- for oxidative stress in the development of retinopathy in dia- dria in the rat retina and in the isolated retinal capillary cells. betes. Oxidative stress is involved directly in the upregulation ETHODS of vascular endothelial growth factor in the retina during early M . Mitochondria and cytosol fractions were prepared 10 from retina of rats with streptozotocin-induced diabetes and diabetes. Recent studies from our laboratory have shown that from the isolated retinal endothelial cells and pericytes incu- oxidative stress plays an important role, not only in the development of retinopathy in diabetes, but also in the resistance of bated in 5 or 20 mM glucose medium for up to 10 days in the 11 presence of superoxide dismutase (SOD) or a synthetic mi- retinopathy to arrest after good glycemic control is initiated. metic of SOD (MnTBAP). The release of cytochrome c into the Capillary cells and neurons are lost in the retina before other histopathology is detectable, and apoptosis has been cytosol and translocation of the proapoptotic protein Bax into 12–15 the mitochondria were determined by the Western blot tech- implicated as one of the mechanism(s). Apoptosis execu- nique and cell death by caspase-3 activity and ELISA assay. tion enzyme, caspase-3, and nuclear transcriptional factor (NF- ␬B) are activated in the retina when the duration of diabetes in RESULTS. Diabetes of 8 months’ duration in rats increased the rats is such that the capillary cell death and histopathology are release of cytochrome c into the cytosol and Bax into the detectable, and antioxidants inhibit such activations.4,16,17 Ox- mitochondria prepared from the retina, and this phenomenon idative stress is shown to be closely linked to apoptosis in a was not observed at 2 months of diabetes. Incubation of iso- variety of cell types18,19; however, the signaling steps involved lated retinal capillary cells with 20 mM glucose increased in oxidative-stress–induced retinal capillary cell apoptosis are cytochrome c content in the cytosol and Bax in the mitochon- not clear. dria, and these abnormalities were accompanied by increased Mitochondria are the major endogenous source of superox- cell apoptosis. Inclusion of SOD or its mimetic inhibited glu- ides and hydroxyl radicals.20 Reactive oxidant intermediates cose-induced release of cytochrome c, translocation of Bax, can trigger mitochondria to release cytochrome c, resulting in and apoptosis. activation of caspase-3.21–23 Overproduction of superoxides by CONCLUSIONS. Retinal mitochondria become leaky when the mitochondria is considered as a causal link between elevated duration of diabetes is such that capillary cell apoptosis can be glucose and the major biochemical pathways postulated to be observed; cytochrome c starts to accumulate in the cytosol and involved in the development of vascular complications in dia- Bax into the mitochondria. Inhibition of superoxides inhibits betes.24,25 Increasing evidence indicates that mitochondria are glucose-induced release of cytochrome c and Bax and inhibits intimately associated with the initiation of apoptosis. Mito- apoptosis in both endothelial cells and pericytes. Identifying chondrial changes are associated with the activation of apopto- the mechanism by which retinal capillary cells undergo apo- tic pathways resulting in diabetic neuropathy,26,27 impaired ptosis may reveal novel therapies to inhibit the development of kidney function,28 and myocardial abnormalities.29 However, retinopathy in diabetes. (Invest Ophthalmol Vis Sci. 2003;44: the involvement of mitochondria in the development of reti- 5327–5334) DOI:10.1167/iovs.03-0353 nopathy in diabetes is not clear. In the present study the effect of diabetes on mitochondrial iabetes increases oxidative stress, which plays an impor- dysfunction in the retina of rats and in the isolated retinal Dtant role in the development of diabetic complications.1–3 capillary cells was investigated by measuring the release of Oxidative stress is increased in retina in diabetes and in isolated cytochrome c into the cytosol and translocation of Bax into the retinal capillary cells (both endothelial cells and pericytes) mitochondria. The effect of inhibition of mitochondrial oxida- incubated in high-glucose medium.4 The antioxidant defense tive stress on capillary cell death is also determined. system is impaired in the retina in diabetes, GSH levels are decreased, superoxide production is increased, and mRNA levels of superoxide dismutase (SOD) and glutathione reduc- METHODS Rats From the Kresge Eye Institute, Wayne State University, Detroit, Michigan. Wistar rats (male, 200–220 g) were randomly assigned to normal or Supported in part by grants from the Juvenile Diabetes Research diabetic groups. Diabetes was induced with streptozotocin injection Foundation, The Thomas Foundation, and Research to Prevent Blind- (55 mg/kg body weight, intraperitoneal), and insulin was given as ness. needed to allow slow weight gain while maintaining hyperglycemia Submitted for publication April 7, 2003; revised June 9, 2003; (blood glucose levels of 20–25 mM). The rats were weighed two times accepted June 19, 2003. a week, and their food consumption was measured once every week. Disclosure: R.A. Kowluru, None; S.N. Abbas, None Glycated hemoglobin (GHb) was measured at 2 months of diabetes, The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- and every 3 months thereafter, using affinity columns (kit 442-B; ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. Sigma-Aldrich). Diabetic rats and age-matched normal rats were killed Corresponding author: Renu A. Kowluru, Kresge Eye Institute, at 2 and 8 months of diabetes, and the retina was immediately re- Wayne State University, 4717 St. Antoine, Detroit, MI 48201; moved. These experiments conformed to the ARVO Statement for the [email protected]. Use of Animals in Ophthalmic and Vision Research. Investigative Ophthalmology & Visual Science, December 2003, Vol. 44, No. 12 Copyright © Association for Research in Vision and Ophthalmology 5327 Downloaded from iovs.arvojournals.org on 10/01/2021 5328 Kowluru and Abbas IOVS, December 2003, Vol. 44, No. 12 Capillary Cells TABLE 1. Severity of Hyperglycemia in Diabetic Rats Endothelial cells and pericytes were prepared from bovine eyes by a Glycated Body method described by Kennedy et al.30 and routinely used by us.4,31,32 Rats Hemoglobin Urine Volume Weight Endothelial cells were grown to 80% confluence in Petri dishes coated (n) (%) (mL/24 Hours) (g) with 0.1% gelatin in Dulbecco’s modified Eagle’s medium (DMEM) containing heparin, 10% fetal calf serum (heat inactivated), 10% serum Two months Normal 5 4.2 Ϯ 0.3 11 Ϯ 2 375 Ϯ 35 replacement (Nu-serum; BD Biosciences, Lincoln Park, NJ) endothelial Ϯ Ϯ Ϯ ␮ Diabetes 5 11.4 0.7 119 27 272 19 growth supplement (25 g/mL), and antibiotic-antimycotic in an en- Eight months vironment of 95% O2 and 5% CO2. Confluent cells from passages 4 Normal 5 4.5 Ϯ 0.5 14 Ϯ 5 492 Ϯ 53 to 8 were split and incubated under normoglycemic (5 mM glucose) Diabetes 6 11.9 Ϯ 1.7 128 Ϯ 20 313 Ϯ 31 or hyperglycemic (20 mM glucose) conditions for 1 to 10 days in the presence or absence of 20 mU/mL SOD,33,34 200 ␮M MnTBAP Glycated hemoglobin and 24 hours urine volume were measured (Mn(III)tetrakis(4-benzoic acid)porphyrin chloride; a cell-permeable at 2 months of diabetes, and every 3 months thereafter. Values are SOD mimetic; Biomol, Plymouth Meeting, PA35,36), 250 ␮M N-acetyl mean Ϯ SD. cysteine, or 250 ␮M lipoic acid.4 Pericytes were grown in DMEM supplemented with 10% fetal calf serum, antibiotics, and antimycotics, as described by us previ- Translocation of Bax into mitochondria was measured by perform- ously.4,31,32 Pericytes (passages 4–6) were incubated in DMEM con- ing Western blots for Bax in both mitochondrial and cytosolic fractions taining 2.5% fetal bovine serum in 5 or 20 mM glucose in the presence using rabbit polyclonal antibodies (Santa Cruz Biotechnology). and absence of antioxidants. Control incubations containing 20 mM mannitol always were run Cell Death simultaneously to rule out the effect of increased osmolarity. Each Cell death was determined by performing ELISA and by apoptotic DNA experiment was repeated with at least three separate cell preparations. laddering with cell death detection kits (Cell Death Detection ELISA and Apoptotic DNA ladder kits; Roche Diagnostics, Indianapolis, IN). Isolation of Mitochondria and Cytosol Cell death was further confirmed by measuring the activity of the apoptosis executor enzyme caspase-3.4 Mitochondria were isolated from the freshly removed retina or from Relative amounts of mono- and oligonucleosomes generated from cells by centrifugation.37,38 The retina was suspended in the mitochon- apoptotic cells were quantitated with the ELISA kit, using monoclonal dria buffer containing 20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 antibodies directed against DNA and histones, respectively. The cyto- mM MgCl2, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl plasmic fraction of the cells was transferred to a streptavidin-coated fluoride, 10 ␮g/mL leupeptin, 10 ␮g /mL aprotinin, and 250 mM microtiter plate and incubated for 2 hours at room temperature with a sucrose, and gently homogenized with a glass homogenizer.

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