December 2003
Volume 44, Issue 12
Free
Retina  |   December 2003
Diabetes-Induced Mitochondrial Dysfunction in the Retina
Author Affiliations
  • Renu A. Kowluru
    From the Kresge Eye Institute, Wayne State University, Detroit, Michigan.
  • Saiyeda Noor Abbas
    From the Kresge Eye Institute, Wayne State University, Detroit, Michigan.
Investigative Ophthalmology & Visual Science December 2003, Vol.44, 5327-5334. doi:10.1167/iovs.03-0353
  • Views
  • PDF
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Renu A. Kowluru, Saiyeda Noor Abbas; Diabetes-Induced Mitochondrial Dysfunction in the Retina. Invest. Ophthalmol. Vis. Sci. 2003;44(12):5327-5334. doi: 10.1167/iovs.03-0353.

      Download citation file:


      © 2016 Association for Research in Vision and Ophthalmology.

      ×
  • Supplements
Abstract

purpose. Oxidative stress is increased in the retina in diabetes, and antioxidants inhibit activation of caspase-3 and the development of retinopathy. The purpose of this study was to investigate the effect of diabetes on the release of cytochrome c from mitochondria and translocation of Bax into mitochondria in the rat retina and in the isolated retinal capillary cells.

methods. Mitochondria and cytosol fractions were prepared from retina of rats with streptozotocin-induced diabetes and from the isolated retinal endothelial cells and pericytes incubated in 5 or 20 mM glucose medium for up to 10 days in the presence of superoxide dismutase (SOD) or a synthetic mimetic of SOD (MnTBAP). The release of cytochrome c into the cytosol and translocation of the proapoptotic protein Bax into the mitochondria were determined by the Western blot technique and cell death by caspase-3 activity and ELISA assay.

results. Diabetes of 8 months’ duration in rats increased the release of cytochrome c into the cytosol and Bax into the mitochondria prepared from the retina, and this phenomenon was not observed at 2 months of diabetes. Incubation of isolated retinal capillary cells with 20 mM glucose increased cytochrome c content in the cytosol and Bax in the mitochondria, and these abnormalities were accompanied by increased cell apoptosis. Inclusion of SOD or its mimetic inhibited glucose-induced release of cytochrome c, translocation of Bax, and apoptosis.

conclusions. Retinal mitochondria become leaky when the duration of diabetes is such that capillary cell apoptosis can be observed; cytochrome c starts to accumulate in the cytosol and Bax into the mitochondria. Inhibition of superoxides inhibits glucose-induced release of cytochrome c and Bax and inhibits apoptosis in both endothelial cells and pericytes. Identifying the mechanism by which retinal capillary cells undergo apoptosis may reveal novel therapies to inhibit the development of retinopathy in diabetes.

Diabetes increases oxidative stress, which plays an important role in the development of diabetic complications. 1 2 3 Oxidative stress is increased in retina in diabetes and in isolated retinal capillary cells (both endothelial cells and pericytes) incubated in high-glucose medium. 4 The antioxidant defense 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 reductase are downregulated. 4 5 6 7 8 9 We have reported that the long-term administration of antioxidants inhibits the development of retinopathy in diabetic rats and in galactose-fed rats (another model of diabetic retinopathy), 3 suggesting an important role for oxidative stress in the development of retinopathy in diabetes. Oxidative stress is involved directly in the upregulation of vascular endothelial growth factor in the retina during early diabetes. 10 Recent studies from our laboratory have shown that oxidative stress plays an important role, not only in the development of retinopathy in diabetes, but also in the resistance of retinopathy to arrest after good glycemic control is initiated. 11  
Capillary cells and neurons are lost in the retina before other histopathology is detectable, and apoptosis has been implicated as one of the mechanism(s). 12 13 14 15 Apoptosis execution enzyme, caspase-3, and nuclear transcriptional factor (NF-κB) are activated in the retina when the duration of diabetes in rats is such that the capillary cell death and histopathology are detectable, and antioxidants inhibit such activations. 4 16 17 Oxidative stress is shown to be closely linked to apoptosis in a variety of cell types 18 19 ; however, the signaling steps involved in oxidative-stress–induced retinal capillary cell apoptosis are not clear. 
Mitochondria are the major endogenous source of superoxides and hydroxyl radicals. 20 Reactive oxidant intermediates can trigger mitochondria to release cytochrome c, resulting in activation of caspase-3. 21 22 23 Overproduction of superoxides by mitochondria is considered as a causal link between elevated glucose and the major biochemical pathways postulated to be involved in the development of vascular complications in diabetes. 24 25 Increasing evidence indicates that mitochondria are intimately associated with the initiation of apoptosis. Mitochondrial changes are associated with the activation of apoptotic pathways resulting in diabetic neuropathy, 26 27 impaired kidney function, 28 and myocardial abnormalities. 29 However, the involvement of mitochondria in the development of retinopathy in diabetes is not clear. 
In the present study the effect of diabetes on mitochondrial dysfunction in the retina of rats and in the isolated retinal capillary cells was investigated by measuring the release of cytochrome c into the cytosol and translocation of Bax into the mitochondria. The effect of inhibition of mitochondrial oxidative stress on capillary cell death is also determined. 
Methods
Rats
Wistar rats (male, 200–220 g) were randomly assigned to normal or diabetic groups. Diabetes was induced with streptozotocin injection (55 mg/kg body weight, intraperitoneal), and insulin was given as needed to allow slow weight gain while maintaining hyperglycemia (blood glucose levels of 20–25 mM). The rats were weighed two times a week, and their food consumption was measured once every week. Glycated hemoglobin (GHb) was measured at 2 months of diabetes, and every 3 months thereafter, using affinity columns (kit 442-B; Sigma-Aldrich). Diabetic rats and age-matched normal rats were killed at 2 and 8 months of diabetes, and the retina was immediately removed. These experiments conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Capillary Cells
Endothelial cells and pericytes were prepared from bovine eyes by a method described by Kennedy et al. 30 and routinely used by us. 4 31 32 Endothelial cells were grown to 80% confluence in Petri dishes coated with 0.1% gelatin in Dulbecco’s modified Eagle’s medium (DMEM) containing heparin, 10% fetal calf serum (heat inactivated), 10% serum replacement (Nu-serum; BD Biosciences, Lincoln Park, NJ) endothelial growth supplement (25 μg/mL), and antibiotic-antimycotic in an environment of 95% O2 and 5% CO2. Confluent cells from passages 4 to 8 were split and incubated under normoglycemic (5 mM glucose) or hyperglycemic (20 mM glucose) conditions for 1 to 10 days in the presence or absence of 20 mU/mL SOD, 33 34 200 μM MnTBAP (Mn(III)tetrakis(4-benzoic acid)porphyrin chloride; a cell-permeable SOD mimetic; Biomol, Plymouth Meeting, PA 35 36 ), 250 μM N-acetyl cysteine, or 250 μM lipoic acid. 4  
Pericytes were grown in DMEM supplemented with 10% fetal calf serum, antibiotics, and antimycotics, as described by us previously. 4 31 32 Pericytes (passages 4–6) were incubated in DMEM containing 2.5% fetal bovine serum in 5 or 20 mM glucose in the presence and absence of antioxidants. 
Control incubations containing 20 mM mannitol always were run simultaneously to rule out the effect of increased osmolarity. Each experiment was repeated with at least three separate cell preparations. 
Isolation of Mitochondria and Cytosol
Mitochondria were isolated from the freshly removed retina or from cells by centrifugation. 37 38 The retina was suspended in the mitochondria buffer containing 20 mM HEPES-KOH (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 0.5 mM EDTA, 0.5 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, 10 μg/mL aprotinin, and 250 mM sucrose, and gently homogenized with a glass homogenizer. The cells were removed from the incubation Petri dishes by trypsin digestion, washed with ice-cold PBS, and homogenized in the mitochondria buffer. The homogenate was centrifuged at 750g for 10 minutes at 4°C to remove nuclei and unbroken cells, and the supernatant was centrifuged at 10,000g for 15 minutes. The resultant mitochondrial pellet was lysed in 50 μL of 20 mM Tris (pH 7.4), 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 μg/mL leupeptin, and 10 μg/mL aprotinin, and the supernatant was centrifuged at 100,000g for 60 minutes to obtain the cytosolic fraction. Protein was determined in both mitochondrial and cytosolic fractions by the bicinchoninic acid assay (Sigma-Aldrich). 
Cytochrome c Release and Bax Translocation
Release of cytochrome c was quantitated by Western blot techniques by measuring the expression of cytochrome c in mitochondrial and cytosolic fractions. Mitochondrial (20 μg) and cytosolic (40 μg) proteins were separated on 15% reducing polyacrylamide gel and then transferred to nitrocellulose membranes. The membranes were blocked in 5% milk, followed by incubation with a polyclonal antibody against cytochrome c (Santa Cruz Biotechnology, Santa Cruz, CA). After washing, the membranes were incubated with anti-rabbit IgG horseradish peroxidase–conjugated antibody in blocking buffer for 1 hour, washed, and developed using a Western blot chemiluminescence detection kit (ECL-Plus; Amersham Biosciences, Arlington Heights, IL). 
To ensure that the same content of mitochondrial or cytosolic protein was loaded in each lane, after they were blotted for cytochrome c, the membrane were incubated in stripping buffer (62.5 mM Tris-HCl [pH 6.8], 100 mM mercaptoethanol, and 2% sodium dodecyl sulfate) at 50°C for 30 minutes and washed (three times for 10 minutes each). The membranes were then incubated with anti-cytochrome c oxidase subunit IV (Cox IV; Molecular Probes, Eugene, OR) or β-actin (Santa Cruz Biotechnology) and developed with the Western blot analysis detection kit. 
Translocation of Bax into mitochondria was measured by performing Western blots for Bax in both mitochondrial and cytosolic fractions using rabbit polyclonal antibodies (Santa Cruz Biotechnology). 
Cell Death
Cell death was determined by performing ELISA and by apoptotic DNA laddering with cell death detection kits (Cell Death Detection ELISA and Apoptotic DNA ladder kits; Roche Diagnostics, Indianapolis, IN). Cell death was further confirmed by measuring the activity of the apoptosis executor enzyme caspase-3. 4  
Relative amounts of mono- and oligonucleosomes generated from apoptotic cells were quantitated with the ELISA kit, using monoclonal antibodies directed against DNA and histones, respectively. The cytoplasmic fraction of the cells was transferred to a streptavidin-coated microtiter plate and incubated for 2 hours at room temperature with a mixture of peroxidase-conjugated anti-DNA and biotin-labeled anti-histone. The plate was then thoroughly washed, incubated with ABTS (2,2′-Azino-di[3-ethylbenzithiazolinesulfonate(6)] disodium salt; Roche Diagnostics), and absorbance was measured at 405 nm against ABTS solution as a blank. After separation of the cytoplasmic fraction, the nuclear pellet was suspended in 50 mM sodium phosphate buffer (pH 7.5) containing 2 mM NaCl and 0.05 mM Na2HPO4 (pH 7.5) and sonicated. DNA was measured in this fraction, and apoptosis was normalized to micrograms of DNA. 
DNA fragmentation was detected with a kit (Apoptotic DNA Ladder Kit; Roche Diagnostics). Briefly, the cells were washed twice with PBS, resuspended in PBS, and incubated for 10 minutes with an equal volume of lysis buffer containing 10 mM Tris-HCl (pH 7.4), 6 M guanidine-HCl, 10 mM urea and EDTA, and 0.2% Triton X-100. The samples were passed through glass fiber fleece by centrifugation and the nucleic acid bound to the glass fibers was eluted. The DNA was applied to a 1.5% agarose gel, and the bands were then visualized by ethidium bromide staining and photographed. 
Statistical Analysis
Data are reported as the mean ± SD, and experimental groups were compared using the nonparametric Kruskal-Wallis test followed by the Mann-Whitney test for multiple-group comparison. Similar conclusions were reached also by using ANOVA with the Fisher or Tukey test. 
Results
Diabetic Rats
Glycated hemoglobin (an index of severity of hyperglycemia) and urine volumes were elevated by two- to threefold in the diabetic rats compared with their age-matched normal rats. The body weights were significantly lower in diabetic rats at 2 and 8 months of diabetes compared with the age-matched normal control (Table 1)
Diabetes of 2 months’ duration in rats had no effect on the release of cytochrome into the cytosol. The cytochrome c content of the cytosolic fraction of the retina was similar in diabetic rats and age-matched normal rats, but 8 months of diabetes resulted in an approximate twofold increase in the cytosolic content of cytochrome c, compared with the age-matched normal rats (Fig. 1A) . However, the expression of β-actin among the lanes did not vary. 
Similarly, the expression of Bax was significantly increased in the mitochondria obtained from the retina of rats diabetic for 8 months compared with the age-matched normal control rats (P < 0.02). However, there was no significant difference in the Bax expression in the mitochondria obtained from the retina of rats diabetic for 2 months compared with that from age-matched normal rats (Fig. 1B)
In the same retina, as reported previously, 4 caspase-3 activity was increased by 40% at 8 months of diabetes compared with age-matched normal control rats, but 2 months of diabetes had no effect on retinal caspase-3 activity. 4  
Isolated Capillary Cells: Studies with Endothelial Cells
The cytosolic content of cytochrome c was similar in the cells incubated in 5 or 20 mM glucose for 3 days, but was increased by more than fourfold when the incubation in 20 mM glucose medium was extended to 5 days. The release of cytochrome c was not further increased when the duration of incubation with 20 mM glucose was further extended to 10 days (Fig. 2A) . Despite the significant differences in the content of cytochrome c among various cytosol preparations, the content of β-actin did not vary. 
As shown in Figure 2B , the expression of Bax in the mitochondria obtained from the same preparations was increased by 40% in the cells incubated with 20 mM glucose for 3 days compared with the cells incubated in 5 mM glucose, but was increased by fourfold when the incubation was extended to 5 days. There was no additional increase in Bax content in the mitochondria when the incubation with glucose was allowed to continue for 10 days. The expression of Cox IV in various lanes was identical. 
Cell Death
Apoptosis levels, as determined by measuring the cytoplasmic nucleosomal DNA, were not changed in the endothelial cells incubated in 20 mM glucose for 3 days compared with the cells incubated in 5 mM, but were increased by 50% when the incubation with 20 mM glucose was increased to 5 days (Fig. 3) . Similarly, increased DNA laddering was observed in these cells at 5 days of incubation with 20 mM glucose (data not shown). 
Inclusion of SOD or MnTBAP in the medium for 5 days inhibited glucose-induced release of cytochrome c from the mitochondria into the cytosol, and in the same mitochondrial fractions, inhibited increased Bax expression. As stated earlier, despite the significant differences in the content of cytochrome c and Bax among various preparations, the content of β-actin and Cox IV did not vary in their respective fractions (Fig. 3) . Other antioxidants, including N-acetyl cysteine and lipoic acid had beneficial effects similar to those of SOD or its mimetic, MnTBAP (data not shown). 
Similarly, the addition of SOD or MnTBAP inhibited glucose-induced increased apoptosis of endothelial cells, and this was confirmed by ELISA assays (Fig. 4) . Both SOD and MnTBAP also inhibited activation of caspase-3 induced by glucose. 
Isolated Retinal Pericytes
Incubation of retinal pericytes with 20 mM glucose showed mitochondrial dysfunction similar to that observed with endothelial cells (Fig. 5) . Cytochrome c release into the cytosol and Bax translocation into the mitochondria were increased by three to fourfold in the cells incubated in 20 mM glucose for 5 days compared with that in 5 mM glucose. This was accompanied by a twofold increase in apoptosis. No significant translocation of cytochrome c or Bax and apoptosis was observed in these cells when the glucose exposure was of 3 days’ or less duration. Addition of MnTBAP inhibited glucose-induced mitochondrial dysfunction and apoptosis in these retinal pericytes (Fig. 5)
Discussion
The results presented herein show for the first time that the retinal mitochondria become leaky and the cytochrome c starts to accumulate in the cytosolic fraction when the duration of diabetes in rats is such that capillary cell apoptosis can be detected in the retina. Further, both in retinal endothelial cells and pericytes, high glucose increases the release of cytochrome c into the cytosol and Bax into the mitochondria, which can be prevented by reducing superoxide levels, thus suggesting that retinal mitochondria experiences dysfunction in diabetes. Retina and its isolated capillary cells experience increased oxidative stress in high-glucose conditions, and increased oxidative stress is postulated to play an important role in the development of diabetic complications. 1 2 3 High glucose can induce apoptosis and activate caspases, including caspase-3, in retinal capillary and Müller cells, 4 15 16 17 39 and we provide data showing that glucose-induced apoptosis can be inhibited by lowering superoxides levels. 
Oxidative stress is closely linked to apoptosis in a variety of cell types. It can alter both signal transduction and genomic processes. 18 19 The mechanism by which oxidative stress can increase apoptosis may involve increased membrane lipid peroxidation, increased oxidative injury to other macromolecules, alterations in signal transduction, change in cellular redox potentials or depletion of glutathione (GSH). 40 41 An altered gene profile of scavenging enzymes is reported in the retinal pericytes obtained from patients with diabetes, and this correlates with the overexpression of the cell death protease gene, suggesting an important role for oxidative stress in the pericyte dropout that occurs in diabetic retinopathy. 10 Increased oxidative stress in diabetes is shown to play a critical role in advanced glycation end-product (AGE)–induced and palmitate-induced apoptosis of retinal capillary cells that can be inhibited by antioxidants. 42 Our previous studies have shown that increased oxidative stress plays an important role in the activation of retinal caspase-3 and in the development of retinopathy in diabetes. 3 4 The results of the present study show that the inhibition of glucose-induced release of cytochrome c into the cytosol and translocation of Bax into the mitochondria in retinal endothelial cells and pericytes by SOD is accompanied by the inhibition of their apoptosis. 
Mitochondria play a key role in regulating apoptosis, reactive oxidant intermediates can trigger mitochondria to release cytochrome c and apoptosis-inducing factor, and increased lipid peroxidation itself can damage mitochondrial membrane potential and provoke apoptosis. 21 22 Once cytochrome c is released from mitochondria, it activates caspase-9, which initiates a cascade of events that activates caspase-3 and results in DNA fragmentation. Cytochrome c can induce apoptosis if it is present in the cytoplasm in the oxidized state, and under normal conditions cytoplasmic GSH maintains cytochrome c in the reduced state. 41 In diabetes, retinal GSH levels are decreased and glutathione redox cycle enzymes are impaired, 5 6 7 which raises the possibility that the reduction of cytochrome c is also impaired, and here we provide data that clearly show that the release of cytochrome c into the cytosol is increased by approximately twofold. The increased release of cytochrome c is seen also when the endothelial cells and pericytes from the retina are incubated in high-glucose medium for 5 days, but not for 3 days. This time course of mitochondrial dysfunction is similar to the activation of caspase-3 that we have reported previously. 6 Thus, the present study provided data to show that the inhibition of mitochondrial changes in retinal endothelial cells and pericytes are accompanied by inhibition of apoptosis in these cells. 
Bax, a proapoptotic protein, enhances the release of cytochrome c by translocating to the mitochondria and by inducing a mitochondrial permeability transition. 43 44 Others have reported that the expression of Bax is increased in the retina in diabetes and in retinal pericytes incubated in high-glucose medium, and this overexpression in retinal pericytes is associated with their apoptosis. 16 In retinal sections, Bax immunostaining is shown to be present in ganglion and vascular cells, the cell types known to undergo accelerated cell death in diabetes. 13 15 16 The results of the present study demonstrate that it is the mitochondrial fraction of the retina where Bax expression is increased in diabetes, and we have confirmed our in vivo results using isolated retinal endothelial cells and pericytes incubated in high-glucose medium for 5 days. This strengthens the possible involvement of mitochondria in the apoptosis of both pericytes and endothelial cells that occurs in diabetes. 12 13 Romeo et al. 39 have suggested a possible involvement of Bax in retinal capillary cell apoptosis in diabetes, but their study did not identify the effect of diabetes on subcellular distribution of Bax in the retina or its capillary cells. Our study is the first to provide data that demonstrate the possible involvement of mitochondrial dysfunction in the apoptosis of both retinal endothelial cells and pericytes in diabetes. 
Release of cytochrome c is considered a key event in the activation of caspase-3, a downstream pivotal step in the initiation of apoptosis. 29 Cells deficient in caspase-3 are resistant to apoptosis, 45 and activation of caspase-3 alone is sufficient to cause cell death in cardiac muscle. 46 The results presented herein demonstrate that caspase-3 activation in diabetes is associated with mitochondrial dysfunction. 
Hyperglycemia-induced overproduction of superoxides by mitochondria is considered as a causal link between elevated glucose and the major biochemical pathways postulated to be involved in the development of vascular complications in diabetes, 25 47 and overexpression of Mn-SOD is reported to suppress glucose-induced collagen accumulation in cultured mesangial cells. 48 We have provided evidence that mitochondrial dysfunction, apoptosis, and caspase-3 activation induced by high glucose in both retinal endothelial cells and pericytes are inhibited by SOD and its mimetic, MnTBAP suggesting that a mitochondria-dependent pathway is operating in both the diabetic retina and its isolated capillary cells, and SOD production is causally involved in the hyperglycemia-induced apoptosis of retinal capillary cells. 
Thus, our data strongly suggest that hyperglycemia-induced retinal capillary cell death most likely is initiated by the mitochondrial cytochrome c–mediated caspase-3 activation pathway. Understanding the signaling pathway(s) involved in the retinal capillary cell death will elucidate important molecular targets for future pharmacological interventions. 
 
Table 1.
 
Severity of Hyperglycemia in Diabetic Rats
Table 1.
 
Severity of Hyperglycemia in Diabetic Rats
Rats (n) Glycated Hemoglobin (%) Urine Volume (mL/24 Hours) Body Weight (g)
Two months
 Normal 5 4.2 ± 0.3 11 ± 2 375 ± 35
 Diabetes 5 11.4 ± 0.7 119 ± 27 272 ± 19
Eight months
 Normal 5 4.5 ± 0.5 14 ± 5 492 ± 53
 Diabetes 6 11.9 ± 1.7 128 ± 20 313 ± 31
Figure 1.
 
Effect of diabetes on the (A) release of cytochrome c in the cytosol and (B) translocation of Bax into mitochondria. The mitochondrial and cytosolic fractions were prepared from retina freshly harvested from rats diabetic for 2 or 8 months and age-matched normal control rats. Cytochrome c and Bax contents were determined by Western blot analyses, and the band intensities were adjusted to the expression of the β-actin or Cox IV in cytosolic and mitochondrial fractions, respectively. The Western blots are representative of five rats in each group, and the retina from each rat was analyzed in duplicate in two separate experiments. *P < 0.05 and # P > 0.05 compared with the age-matched normal control.
Figure 1.
 
Effect of diabetes on the (A) release of cytochrome c in the cytosol and (B) translocation of Bax into mitochondria. The mitochondrial and cytosolic fractions were prepared from retina freshly harvested from rats diabetic for 2 or 8 months and age-matched normal control rats. Cytochrome c and Bax contents were determined by Western blot analyses, and the band intensities were adjusted to the expression of the β-actin or Cox IV in cytosolic and mitochondrial fractions, respectively. The Western blots are representative of five rats in each group, and the retina from each rat was analyzed in duplicate in two separate experiments. *P < 0.05 and # P > 0.05 compared with the age-matched normal control.
Figure 2.
 
Time course of (A) glucose-induced release of cytochrome c and (B) translocation of Bax in retinal endothelial cells. Bovine retinal endothelial cells were incubated in 5 or 20 mM glucose for up to 10 days. At the end of the desired time of incubation, the cells were trypsinized and mitochondrial and cytosolic fractions were isolated by differential centrifugation. The purity of mitochondrial and cytosolic fractions was determined by measuring Cox IV expression. Each measurement was performed in duplicate with three to four separate cell preparations. The results obtained in 5 mM glucose medium did not change with the duration of incubation and are considered as 100%. *P < 0.05 compared with 5 mM glucose.
Figure 2.
 
Time course of (A) glucose-induced release of cytochrome c and (B) translocation of Bax in retinal endothelial cells. Bovine retinal endothelial cells were incubated in 5 or 20 mM glucose for up to 10 days. At the end of the desired time of incubation, the cells were trypsinized and mitochondrial and cytosolic fractions were isolated by differential centrifugation. The purity of mitochondrial and cytosolic fractions was determined by measuring Cox IV expression. Each measurement was performed in duplicate with three to four separate cell preparations. The results obtained in 5 mM glucose medium did not change with the duration of incubation and are considered as 100%. *P < 0.05 compared with 5 mM glucose.
Figure 3.
 
Glucose-induced apoptosis of retinal endothelial cells. Apoptosis was measured by performing ELISA for cytoplasmic histone-associated DNA fragments. Data were obtained from cells incubated with glucose for 5 days, and the values were adjusted to the total DNA. No significant apoptosis was observed in these cells when the glucose exposure was 3 days or less. *P < 0.05 compared with 5 mM glucose; P < 0.05 compared with 20 mM glucose.
Figure 3.
 
Glucose-induced apoptosis of retinal endothelial cells. Apoptosis was measured by performing ELISA for cytoplasmic histone-associated DNA fragments. Data were obtained from cells incubated with glucose for 5 days, and the values were adjusted to the total DNA. No significant apoptosis was observed in these cells when the glucose exposure was 3 days or less. *P < 0.05 compared with 5 mM glucose; P < 0.05 compared with 20 mM glucose.
Figure 4.
 
Effect of SOD on glucose-induced mitochondrial dysfunction. Retinal endothelial cells were incubated with 5 or 20 mM glucose for 5 days in the presence or absence of 20 mU/mL SOD or 200 μM MnTBAP. The cytochrome c and Bax contents were determined in the mitochondrial and cytosolic fractions by Western blot analysis. The experiments were repeated with at least three separate cell preparations, and each measurement was made in duplicate. Similar beneficial effects were observed when the cells were incubated in the presence of 250 μM N-acetyl cysteine or 250 μM α-lipoic acid.
Figure 4.
 
Effect of SOD on glucose-induced mitochondrial dysfunction. Retinal endothelial cells were incubated with 5 or 20 mM glucose for 5 days in the presence or absence of 20 mU/mL SOD or 200 μM MnTBAP. The cytochrome c and Bax contents were determined in the mitochondrial and cytosolic fractions by Western blot analysis. The experiments were repeated with at least three separate cell preparations, and each measurement was made in duplicate. Similar beneficial effects were observed when the cells were incubated in the presence of 250 μM N-acetyl cysteine or 250 μM α-lipoic acid.
Figure 5.
 
Glucose-induced mitochondrial dysfunction and apoptosis in retinal pericytes. Pericytes isolated from bovine retina were incubated in 5 or 20 mM glucose for up to 10 days in the presence or absence of SOD or MnTBAP. Top: release of cytochrome c into the cytosol and Bax into mitochondria after a 5-day incubation with 20 mM glucose. The results are representative of five separate experiments using three different preparations. Bottom: apoptosis measured by ELISA. Results are the mean ± SD obtained from three separate preparations, and each measurement was made in duplicate. *P < 0.05 compared with 5 mM glucose and # P < 0.05 compared with 20 mM glucose.
Figure 5.
 
Glucose-induced mitochondrial dysfunction and apoptosis in retinal pericytes. Pericytes isolated from bovine retina were incubated in 5 or 20 mM glucose for up to 10 days in the presence or absence of SOD or MnTBAP. Top: release of cytochrome c into the cytosol and Bax into mitochondria after a 5-day incubation with 20 mM glucose. The results are representative of five separate experiments using three different preparations. Bottom: apoptosis measured by ELISA. Results are the mean ± SD obtained from three separate preparations, and each measurement was made in duplicate. *P < 0.05 compared with 5 mM glucose and # P < 0.05 compared with 20 mM glucose.
The authors thank Prashant Koppolu for technical assistance. 
Baynes JW. Role of oxidative stress in development of complications in diabetes. Diabetes. 1991;40:405–412. [CrossRef] [PubMed]
Armstrong D, Abdella N, Salman A, et al. Relationship of lipid peroxides to diabetic complications. J Diab Comp. 1992;6:116–122. [CrossRef]
Kowluru RA, Tang J, Kern TS. Abnormalities of retinal metabolism in diabetes and experimental galactosemia. VII. Effect of long-term administration of antioxidants on the development of retinopathy. Diabetes. 2001;50:1938–1942. [CrossRef] [PubMed]
Kowluru RA, Koppolu P. Diabetes-induced activation of caspase-3 in retina: effect of antioxidant therapy. Free Rad Res. 2002;36:993–999. [CrossRef]
Kowluru RA, Kern TS, Engerman RL, Armstrong D. Abnormalities of retinal metabolism in diabetes or experimental galactosemia. III. Effects of antioxidants. Diabetes. 1996;45:1233–1237. [CrossRef] [PubMed]
Kowluru RA, Kern TS, Engerman RL. Abnormalities of retinal metabolism in diabetes or experimental galactosemia. IV. Antioxidant defense system. Free Radic Biol Med. 1996;22:587–592.
Szabo ME, Haines D, Garay E, et al. Antioxidant properties of calcium dobesilate in ischemic/reperfused diabetic rat retina. Eur J Pharmacol. 2001;428:277–286. [CrossRef] [PubMed]
Grant MB, Ellis EA, Wachowski MB, Murray FT. Free radical derived oxidant localization in retinas of BBZ/WOR diabetic rats (Abstract). Diabetes. 1996;45(suppl 2)192A.
Li W, Yanoff M, Jian B, He Z. Altered mRNA levels of antioxidant enzymes in pre-apoptotic pericytes from human diabetic retinas. Cell Mol Biol. 1999;45:59–66. [PubMed]
Obrosova IG, Minchenko AG, Marinescu V, et al. Antioxidants attenuate early up regulation of retinal vascular endothelial growth factor in streptozotocin-diabetic rats. Diabetologia. 2001;44:1102–1110. [CrossRef] [PubMed]
Kowluru RA. Effect of re-institution of good glycemic control on retinal oxidative stress and nitrative stress in diabetic rats. Diabetes. 2003;52:818–823. [CrossRef] [PubMed]
Mizutani M, Kern TS, Lorenzi M. Accelerated death of retinal microvascular cells in human and experimental diabetic retinopathy. J Clin Invest. 1996;97:2883–2890. [CrossRef] [PubMed]
Kern TS, Tang J, Mizutani M, et al. Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia. Invest Ophthalmol Vis Sci. 2000;41:3972–3978. [PubMed]
Barber AJ, Lieth E, Khin SA, et al. Neural apoptosis in the retina during experimental and human diabetes: early onset and effect of insulin. J Clin Invest. 1998;102:783–791. [CrossRef] [PubMed]
Podesta F, Romeo G, Liu WH, et al. Bax is increased in the retina of diabetic subjects and is associated with pericyte apoptosis in vivo and in vitro. Am J Pathol. 2000;156:1025–1032. [CrossRef] [PubMed]
Kowluru RA, Koppolu P, Chakrabarti S, Chen S. Diabetes-induced activation of nuclear transcriptional factor in the retina, and its inhibition by antioxidants. Free Radical Research. .In press
Mohr S, Xi X, Tang J, Kern TS. Caspase activation in retinas of diabetic and galactosemic mice and diabetic patients. Diabetes. 2002;51:1172–1179. [CrossRef] [PubMed]
Baumgartner-Parzer SM, Wagner L, Pettermann M, et al. High-glucose-triggered apoptosis in cultured endothelial cells. Diabetes. 1995;44:1323–1327. [CrossRef] [PubMed]
Du X, Stocklauser-Farber K, Rosen P. Generation of reactive oxygen intermediates, activation of NF-kappaB, and induction of apoptosis in human endothelial cells by glucose: role of nitric oxide synthase?. Free Radic Biol Med. 1999;27:752–763. [CrossRef] [PubMed]
Sandbach JM, Coscun PE, Grossniklaus HE, et al. Ocular pathology in mitochondrial superoxide dismutase (Sod2)-deficient mice. Invest Ophthalmol Vis Sci. 2001;42:2173–2178. [PubMed]
Anuradha CD, Kanno S, Hirano S. Oxidative damage to mitochondria is a preliminary step to caspase-3 activation in fluoride-induced apoptosis in HL-60 cells. Free Radic Biol Med. 2001;31:367–373. [CrossRef] [PubMed]
Phaneuf S, Leeuwenburgh C. Cytochrome c release from mitochondria in the aging heart: a possible mechanism for apoptosis with age. Am J Physiol. 2002;282:R423–R430.
Kristal BS, Koopmans SJ, Jackson CT, et al. Oxidant-mediated repression of mitochondrial transcription in diabetic rats. Free Radic Biol Med. 1997;22:813–822. [CrossRef] [PubMed]
Nishikawa T, Edelstein D, Brownlee M. The missing link: a single unifying mechanism for diabetic complications. Kidney Int (supplement). 2000;77:S26–S30.
Brownlee M. Biochemistry and molecular cell biology of diabetic complications. Nature. 2001;414:813–820. [CrossRef] [PubMed]
Russell JW, Golovoy D, Vincet A, et al. High glucose-induced oxidative stress and mitochondrial dysfunction in neurons. FASEB J. 2002;16:1738–1748. [CrossRef] [PubMed]
Schmeichel AM, Schmelzer JD, Low PA. Oxidative injury and apoptosis of dorsal root ganglion neurons in chronic experimental diabetic neuropathy. Diabetes. 2003;52:165–171. [CrossRef] [PubMed]
Verzola D, Bertolotto MB, Villaggio B, et al. Taurine prevents apoptosis induced by high ambient glucose in human tubule renal cells. J Invest Med. 2002;50:443–451. [CrossRef]
Cai L, Li W, Wang G, et al. Hyperglycemia-induced apoptosis in mouse myocardium: mitochondrial cytochrome C-mediated caspase-3 activation pathway. Diabetes. 2002;51:1938–1948. [CrossRef] [PubMed]
Kennedy A, Frank RN, Sotolongo LB, et al. Proliferative response and macromolecular synthesis by ocular cells cultured on extracellular matrix materials. Curr Eye Res. 1990;9:307–322. [CrossRef] [PubMed]
Kowluru RA, Engerman RL, Case GL, Kern TS. Retinal glutamate in diabetes and effect of antioxidants. Neurochem Int. 2001;38:385–390. [CrossRef] [PubMed]
Kowluru A. Diabetes-induced elevations in retinal oxidative stress, protein kinase C and nitric oxide are inter-related. Acta Diabetol. 2001;38:179–185. [CrossRef] [PubMed]
Valencia A, Moran J. Role of oxidative stress in the apoptotic cell death of cultured cerebellar granule neurons. J Neurosci Res. 2001;64:284–287. [CrossRef] [PubMed]
Anane R, Creppy EE. Lipid peroxidation as pathway of aluminium cytotoxicity in human skin fibroblast cultures: prevention by superoxide dismutase+catalase and vitamins E and C. Hum Exp Toxicol. 2001;20:477–481. [CrossRef] [PubMed]
Szabo C, Day BJ, Salzman AL. Evaluation of the relative contribution of nitric oxide and peroxynitrite to the suppression of mitochondrial respiration in immunostimulated macrophages using a manganese mesoporphyrin superoxide dismutase mimetic and peroxynitrite scavenger. FEBS Lett. 1996;381:82–86. [CrossRef] [PubMed]
Patel M. Inhibition of neuronal apoptosis by a metalloporphyrin superoxide dismutase mimic. J Neurochem. 1998;71:1068–1074. [PubMed]
Tafani M, Cohn JA, Karpinich NO, et al. Regulation of intracellular pH mediates Bax activation in HeLa cells treated with staurosporine or tumor necrosis factor-alpha. J Biol Chem. 2002;277:49569–49576. [CrossRef] [PubMed]
Kowluru A, Tannous M, Chen HQ. Localization and characterization of the mitochondrial isoform of the nucleoside diphosphate kinase in the pancreatic beta cell: evidence for its complexation with mitochondrial succinyl-CoA synthetase. Arch Biochem Biophys. 2002;398:160–169. [CrossRef] [PubMed]
Romeo G, Liu WH, Asnaghi V, et al. Activation of nuclear factor-kappaB induced by diabetes and high glucose regulates a proapoptotic program in retinal pericytes. Diabetes. 2002;51:2241–2248. [CrossRef] [PubMed]
Matsura T, Kai M, Fujii Y, et al. Hydrogen peroxide-induced apoptosis in HL-60 cells requires caspase-3 activation. Free Radic Res. 1999;30:73–83. [CrossRef] [PubMed]
Hancock JT, Desikan R, Neill SJ. Does the redox status of cytochrome C act as a fail-safe mechanism in the regulation of programmed cell death. Free Radic Biol Med. 2001;31:697–703. [CrossRef] [PubMed]
Denis U, Lecomte M, Paget C, et al. Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction. Free Radic Biol Med. 2002;33:236–247. [CrossRef] [PubMed]
Adachi M, Ishii H. Role of mitochondria in alcoholic liver injury. Free Radic Biol Med. 2002;32:487–491. [CrossRef] [PubMed]
Karpinich NO, Tafani M, Rothman RJ, et al. The course of etoposide-induced apoptosis from damage to DNA and p53 activation to mitochondrial release of cytochrome c. J Biol Chem. 2002;277:16547–16562. [CrossRef] [PubMed]
Yang XH, Sladek TL, Liu X, et al. Reconstitution of caspase-3 sensitizes MVF-7 breast cancer cells to doxorubicin- and etoposide-induced apoptosis. Cancer Res. 2001;60:348–354.
Wu W, Lee WL, Wu YY, et al. Expression of constitutively active phosphatidylinositol 3-kinase inhibits activation of caspase-3 and apoptosis of cardiac muscle cells. J Biol Chem. 2000;275:40113–40119. [CrossRef] [PubMed]
Nishikawa T, Edelstein D, Du XL, et al. Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage. Nature. 2000;404:787–790. [CrossRef] [PubMed]
Craven PA, Phillips SL, Melhem MF, et al. Overexpression of manganese superoxide dismutase suppresses increases in collagen accumulation induced by culture of mesangial cells in high-media glucose. Metabolism. 2001;50:1043–1048. [CrossRef] [PubMed]
Figure 1.
 
Effect of diabetes on the (A) release of cytochrome c in the cytosol and (B) translocation of Bax into mitochondria. The mitochondrial and cytosolic fractions were prepared from retina freshly harvested from rats diabetic for 2 or 8 months and age-matched normal control rats. Cytochrome c and Bax contents were determined by Western blot analyses, and the band intensities were adjusted to the expression of the β-actin or Cox IV in cytosolic and mitochondrial fractions, respectively. The Western blots are representative of five rats in each group, and the retina from each rat was analyzed in duplicate in two separate experiments. *P < 0.05 and # P > 0.05 compared with the age-matched normal control.
Figure 1.
 
Effect of diabetes on the (A) release of cytochrome c in the cytosol and (B) translocation of Bax into mitochondria. The mitochondrial and cytosolic fractions were prepared from retina freshly harvested from rats diabetic for 2 or 8 months and age-matched normal control rats. Cytochrome c and Bax contents were determined by Western blot analyses, and the band intensities were adjusted to the expression of the β-actin or Cox IV in cytosolic and mitochondrial fractions, respectively. The Western blots are representative of five rats in each group, and the retina from each rat was analyzed in duplicate in two separate experiments. *P < 0.05 and # P > 0.05 compared with the age-matched normal control.
Figure 2.
 
Time course of (A) glucose-induced release of cytochrome c and (B) translocation of Bax in retinal endothelial cells. Bovine retinal endothelial cells were incubated in 5 or 20 mM glucose for up to 10 days. At the end of the desired time of incubation, the cells were trypsinized and mitochondrial and cytosolic fractions were isolated by differential centrifugation. The purity of mitochondrial and cytosolic fractions was determined by measuring Cox IV expression. Each measurement was performed in duplicate with three to four separate cell preparations. The results obtained in 5 mM glucose medium did not change with the duration of incubation and are considered as 100%. *P < 0.05 compared with 5 mM glucose.
Figure 2.
 
Time course of (A) glucose-induced release of cytochrome c and (B) translocation of Bax in retinal endothelial cells. Bovine retinal endothelial cells were incubated in 5 or 20 mM glucose for up to 10 days. At the end of the desired time of incubation, the cells were trypsinized and mitochondrial and cytosolic fractions were isolated by differential centrifugation. The purity of mitochondrial and cytosolic fractions was determined by measuring Cox IV expression. Each measurement was performed in duplicate with three to four separate cell preparations. The results obtained in 5 mM glucose medium did not change with the duration of incubation and are considered as 100%. *P < 0.05 compared with 5 mM glucose.
Figure 3.
 
Glucose-induced apoptosis of retinal endothelial cells. Apoptosis was measured by performing ELISA for cytoplasmic histone-associated DNA fragments. Data were obtained from cells incubated with glucose for 5 days, and the values were adjusted to the total DNA. No significant apoptosis was observed in these cells when the glucose exposure was 3 days or less. *P < 0.05 compared with 5 mM glucose; P < 0.05 compared with 20 mM glucose.
Figure 3.
 
Glucose-induced apoptosis of retinal endothelial cells. Apoptosis was measured by performing ELISA for cytoplasmic histone-associated DNA fragments. Data were obtained from cells incubated with glucose for 5 days, and the values were adjusted to the total DNA. No significant apoptosis was observed in these cells when the glucose exposure was 3 days or less. *P < 0.05 compared with 5 mM glucose; P < 0.05 compared with 20 mM glucose.
Figure 4.
 
Effect of SOD on glucose-induced mitochondrial dysfunction. Retinal endothelial cells were incubated with 5 or 20 mM glucose for 5 days in the presence or absence of 20 mU/mL SOD or 200 μM MnTBAP. The cytochrome c and Bax contents were determined in the mitochondrial and cytosolic fractions by Western blot analysis. The experiments were repeated with at least three separate cell preparations, and each measurement was made in duplicate. Similar beneficial effects were observed when the cells were incubated in the presence of 250 μM N-acetyl cysteine or 250 μM α-lipoic acid.
Figure 4.
 
Effect of SOD on glucose-induced mitochondrial dysfunction. Retinal endothelial cells were incubated with 5 or 20 mM glucose for 5 days in the presence or absence of 20 mU/mL SOD or 200 μM MnTBAP. The cytochrome c and Bax contents were determined in the mitochondrial and cytosolic fractions by Western blot analysis. The experiments were repeated with at least three separate cell preparations, and each measurement was made in duplicate. Similar beneficial effects were observed when the cells were incubated in the presence of 250 μM N-acetyl cysteine or 250 μM α-lipoic acid.
Figure 5.
 
Glucose-induced mitochondrial dysfunction and apoptosis in retinal pericytes. Pericytes isolated from bovine retina were incubated in 5 or 20 mM glucose for up to 10 days in the presence or absence of SOD or MnTBAP. Top: release of cytochrome c into the cytosol and Bax into mitochondria after a 5-day incubation with 20 mM glucose. The results are representative of five separate experiments using three different preparations. Bottom: apoptosis measured by ELISA. Results are the mean ± SD obtained from three separate preparations, and each measurement was made in duplicate. *P < 0.05 compared with 5 mM glucose and # P < 0.05 compared with 20 mM glucose.
Figure 5.
 
Glucose-induced mitochondrial dysfunction and apoptosis in retinal pericytes. Pericytes isolated from bovine retina were incubated in 5 or 20 mM glucose for up to 10 days in the presence or absence of SOD or MnTBAP. Top: release of cytochrome c into the cytosol and Bax into mitochondria after a 5-day incubation with 20 mM glucose. The results are representative of five separate experiments using three different preparations. Bottom: apoptosis measured by ELISA. Results are the mean ± SD obtained from three separate preparations, and each measurement was made in duplicate. *P < 0.05 compared with 5 mM glucose and # P < 0.05 compared with 20 mM glucose.
Table 1.
 
Severity of Hyperglycemia in Diabetic Rats
Table 1.
 
Severity of Hyperglycemia in Diabetic Rats
Rats (n) Glycated Hemoglobin (%) Urine Volume (mL/24 Hours) Body Weight (g)
Two months
 Normal 5 4.2 ± 0.3 11 ± 2 375 ± 35
 Diabetes 5 11.4 ± 0.7 119 ± 27 272 ± 19
Eight months
 Normal 5 4.5 ± 0.5 14 ± 5 492 ± 53
 Diabetes 6 11.9 ± 1.7 128 ± 20 313 ± 31
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×