OBJECTIVE—Mitochondrial superoxide levels are elevated in the retina in diabetes, and manganese superoxide dismutase overexpression prevents the development of retinopathy. Superoxide inhibits glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which activates major pathways implicated in diabetic complications, including advanced glycation end products (AGEs), protein kinase C, and hexosamine pathway. Our aim is to investigate the role of GAPDH in the development and progression of diabetic retinopathy and to elucidate the mechanism.

RESEARCH DESIGN AND METHODS—Rats with streptozotocin-induced diabetes were in a state of poor control (GHb >11%) for 12 months, good control (GHb <7) soon after induction of diabetes, or poor control for 6 months with 6 months’ good control. Retinal GAPDH, its ribosylation and nitration, AGEs, and PKC activation were determined and correlated with microvascular histopathology.

RESULTS—In rats with poor control, retinal GAPDH activity and expressions were subnormal with increased ribosylation and nitration (25–30%). GAPDH activity was subnormal in both cytosol and nuclear fractions, but its protein expression and nitration were significantly elevated in nuclear fraction. Reinstitution of good control failed to protect inactivation of GAPDH, its covalent modification, and translocation to the nucleus. PKC, AGEs, and hexosamine pathways remained activated, and microvascular histopathology was unchanged. However, GAPDH and its translocation in good control rats were similar to those in normal rats.

CONCLUSIONS—GAPDH plays a significant role in the development of diabetic retinopathy and its progression after cessation of hyperglycemia. Thus, therapies targeted toward preventing its inhibition may inhibit development of diabetic retinopathy and arrest its progression.

Retinopathy is a multifactorial sight-threatening complication of diabetes. It is a progressive disease associated with chronic hyperglycemia (1). Although many glucose-induced retinal metabolic abnormalities are postulated to contribute to its development, the exact mechanism remains elusive (25). We have shown that in diabetes, retinal mitochondria experience increased oxidative damage and the mitochondrial enzyme that scavenges superoxide (manganese superoxide dismutase [MnSOD]) prevents vascular histopathology that is characteristic of diabetic retinopathy (68). Increased mitochondrial superoxide production inactivates glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vascular endothelial cells, and inhibition of GAPDH is postulated to activate some of the key pathways that are associated with the development of diabetes complications, including increased formation of advanced glycation end products (AGEs) and activation of protein kinase C (PKC) and hexosamine pathway (9,10).

GAPDH is a glycolytic enzyme that catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bis-phosphoglycerate. Recent studies have shown that GAPDH is a protein with multiple cytoplasmic, membrane, and nuclear functions and is a major intracellular messenger mediating apoptosis of cells (11,12). GAPDH translocation to the nucleus is considered an important step in glucose-induced apoptosis of retinal Muller cells (13). The mechanism that initiates its translocation is not well understood; covalent modification by nitration/ribosylation is considered a likely possibility (1416). How GAPDH contributes to the pathogenesis of diabetic retinopathy remains to be established.

Good glycemic control attenuates the development/progression of retinopathy in diabetic patients, but its effects on the progression of retinopathy are not immediate, and it takes years for retinopathy to halt progression after the reestablishment of good control. The imprinted effects of prior glycemic control either produce the long-lasting benefits of good control or resist the arrest of progression of diabetic retinopathy after reinstitution of good control. Reinstitution of good control after a profound period of poor glycemic control does not immediately benefit the progression of retinopathy. This suggests a “metabolic memory” phenomenon (1720). Metabolic memory phenomenon is observed also in animal models of diabetic retinopathy (2126); the formation of acellular capillaries, characteristic of early signs of diabetic retinopathy, does not stop for at least 6 months when good control is initiated 6 months after induction of diabetes in rats, and nitrotyrosine levels and oxidative stress remain elevated (24,26). These abnormalities are, however, partially inhibited if the duration of poor control is reduced to 2 months, suggesting the role of oxidative stress in the metabolic memory phenomenon (24). The role of GAPDH in metabolic imprinting remains to be elucidated.

In the present study, we investigated how GAPDH inhibition contributes to the development of retinopathy in diabetes and the mechanism(s) that could result in its inactivation. We have also explored the role of GAPDH in the metabolic memory phenomenon in diabetic rats by maintaining them in a state of poor control before initiation of a state of good control.

Animals and glycemia.

Lewis rats (male, 200 g) were randomly assigned to normal or diabetic groups. Diabetes was induced with streptozotocin (55 mg/kg body wt), and rats were divided at random among three groups according to intended degree of glycemic control. Rats in group 1 remained in poor control for 12 months; in group 2, rats were in poor control for 6 months followed by good control for 6 additional months (poor control–good control); and in group 3, rats were subjected to good control soon after induction of diabetes. Some of the same rats had been used by us in our previous studies (26,27).

The degree of glycemia was achieved by adjusting the dose and frequency of insulin (NPH) administration. The rats in the poor-control group received a single injection of insulin (1–2 units) four to five times a week to prevent ketosis and weight loss, and the rats in good control group received insulin twice daily (7–8 units total) to maintain their blood glucose levels below 150 mg/dl and a steady gain in body weight (24,26). The entire rat colony was housed in metabolism cages: 24-h urine samples were measured daily and tested for glycosuria. Blood glucose was measured once a week using Elite Glucometer (Bayer, Tarrytown, NY), and GHb was measured every 2 months. The entire rat colony received a powdered diet (Purina 5001; TestDiet, Richmond, IN); their food consumption was measured once every week, and body weights were measured two to three times every week. These experiments conformed to the ARVO Resolution on Treatment of Animals in Research and to the specific institutional guidelines. The experiment was terminated 12 months after initiation of diabetes, and the animals were killed by an overdose of pentobarbital. Eyes were enucleated immediately; one eye was fixed in 10% buffered formalin, and from the other eye, the retina was immediately isolated by gently separating sensory retina from choroid using a microspatula.

Preparation of subcellular fractions.

Retina was gently homogenized in a glass homogenizer in 50 mmol/l glycyl glycine buffer (pH 7.0) containing 10 mmol/l EDTA, 100 mmol/l sodium fluoride, 0.5 mmol/l dithiolthreitol, and protease inhibitors. The homogenate was centrifuged at 750g for 5 min, and the supernatant was centrifuged at 5,000g for 15 min. The nuclear pellet was resuspended in 50 mmol/l HEPES buffer (pH 7.5) containing 1% Triton X-100, 150 mmol/l sodium chloride, 1 mmol/l EDTA, and protease inhibitors. The supernatant was further centrifuged at 105,000g for 90 min to separate the cytosolic fraction (13,28). The purity of the fractions was determined by measuring the expressions of Histone 2-B (nuclear marker) and lactate dehydrogenase (LDH; cytosolic marker).

GAPDH enzyme activity.

The enzyme activity was measured spectrophotometrically in a final assay volume of 100 μl containing 50 mmol/l triethanolamine buffer (pH 7.6), 50 mmol/l arsenate, 2.4 mmol/l glutathione, 250 μmol/l NAD, and cytosolic/nuclear protein (0–10 μg protein). The assay mixture was preincubated for 5 min at 37°C, and the reaction was initiated by 100 μg/ml glyceraldehyde-3-phosphate. Increase in NADH production was monitored at 340 nm. GAPDH activity was expressed as difference in absorbance in the presence/absence of glyceraldehyde-3-phosphate (28).

Quantitative real-time PCR.

RNA was isolated from the retina using TRIzol reagent, and 1 μg RNA was converted to single-stranded cDNA and quantified spectrophotometrically. Gene expression was measured using 90- to 300-ng cDNA templates in 96-well plates in ABI-7500 sequence detection system (29,30). Each sample was analyzed in triplicate, and the data were normalized to β2-microglobulin (B2M) expression in each sample. GenBank accession numbers for the ABI TaqMan assays for GAPDH and B2M used were NM_017008.3 and NM_012512.1, respectively. The fold change in gene expression relative to normal was calculated using the delta delta cycles to threshold (ddCT) method.

Protein expression.

Protein (15–30 μg) was separated by SDS-PAGE on a 4–16% gradient gel and blotted to nitrocellulose membrane. The membranes were blocked in 5% nonfat milk, incubated with the target primary antibody, washed, and followed by incubation with appropriate horseradish peroxidase–coupled secondary antibody. The target proteins were enhanced by ECL reagent and determined by autoradiography. The membranes then were stripped and reprobed with β-actin (Sigma-Aldrich, St. Louis, MO). The band intensity was quantified using Un-Scan-It Gel digitizing software (Silk Scientific, Orem, UT), and protein expression levels were calculated relative to β-actin in the same sample. Activity of poly(ADP-ribose) polymerase (PARP) was determined by measuring poly(ADP-ribosyl)ation of retinal proteins by separating them on SDS-PAGE gel.

Ribosylation and nitration of GAPDH.

Covalent modification of GAPDH was determined by first immunoprecipitating protein (75–100 μg) with polyclonal anti-GAPDH antibody. Protein A/G plus agarose beads were used to collect GAPDH complexed with the antibody and analyzed by Western blot technique using monoclonal antibodies against poly(ADP-ribose) (PAR; Alexis Biochemicals, San Diego, CA) and nitrotyrosine (Upstate Biotechnology, Lake Placid, NY). To normalize for equal loading in each lane, the membranes were reprobed for GAPDH.

Quantification of GAPDH-mediated pathways.

Because inhibition of GAPDH activates major pathways that are implicated in the development of diabetes complications, we investigated the effect of reversal of hyperglycemia on AGEs, PKC, and hexosamine pathways in the retina from the same set of animals used for GAPDH. Total AGEs formation was determined by Western blot using anti-AGE antibody (Wako Chemicals, Richmond, VA). PKC activation was determined by quantifying the expression of PKC βII, the isoform that is activated in diabetes, as we previously described (23).

Because addition of single O-linked ß-N-acetylglucosamine (O-GlcNAc) monosaccharides to serine or threonine residues on proteins is one of the processes coupled to the hexosamine pathway, we assessed hexosamine pathway by quantifying O-linked N-acetylglucosamine–modified proteins in the retina by Western blot using monoclonal antibody against O-GlcNAc (Covance, Princeton, NJ). BSA was used as a blocking medium (30).

Isolation of retinal microvessels and quantification of acellular capillaries.

Microvessels were prepared from formalin-fixed eyes by trypsin digestion. The retina was isolated, rinsed in water overnight, and incubated with 3% crude trypsin containing 0.2 mmol/l sodium fluoride for 90 min at 37°C. Nonvascular cells were removed by gentle brushing, and the isolated vasculature was dried onto a microscope slide. The slides were stained with hematoxylin and periodic acid Schiff. The number of acellular capillaries (representing basement membrane tubes lacking cell nuclei) was counted in a masked manner in multiple midretinal fields with one field adjacent to each of the five to seven retinal arterioles radiating out from the optic disc and expressed as per square millimeter of retinal area examined (5,8,26).

Results are presented as means ± SD and analyzed statistically using the nonparametric Kruskal-Wallis followed by Mann-Whitney U test for multiple group comparison. Similar conclusions were achieved by using ANOVA with Fisher or Tukey tests.

Severity of hyperglycemia.

Hyperglycemia, as reported previously (26), was severe in the rats in the poor-control group; GHb values were >11% throughout the entire duration of the experiment (12 months). The rats in the good-control group maintained their GHb values, which were similar to those in normal rats. In the poor control–good control group, GHb values before initiation of good control were not different from the poor-control group (GHb >11%) but became similar to those in the normal group after initiation of good control (GHb <7%) (Table 1). Average body weight and 24-h urine volumes were similar in good-control rats and normal rats.

Effect of diabetes on retinal GAPDH.

Twelve months of poor control had a marginal but statistically significant effect on the expression of GAPDH in the retina; protein expression was decreased by 15–20% (Fig. 1) and gene expression by ∼20% (Fig. 2) in diabetic rats.

Effect of diabetes on activation of retinal PARP.

As shown in Fig. 3A, 12 months of poor control significantly increased poly(ADP-ribosyl)ation of retinal proteins compared with that in normal rats, suggesting an increase in PARP activity. We did not, however, identify these ribosylated proteins (other than GAPDH, please see below), and that is beyond the focus of this study.

Subcellular translocation of GAPDH and its covalent modification.

Because retinal cell apoptosis precedes the development of diabetic retinopathy (31,32), we investigated the effect of diabetes on subcellular translocation of GAPDH in the retina. In normal rat retina, the protein expression of GAPDH was 35% higher in the cytosolic fraction compared with the nuclear fraction, but 12 months of poor control in rats resulted in reduction in its expression in the cytosol fraction, with a concomitant increase in the nuclear fraction (Fig. 4). The ratio of GAPDH expression in cytosolic and nuclear fraction was almost 4:1 in normal rat retina and decreased to 1.2:1 in diabetic rat retina. In the same diabetic rats, despite increased expression of GAPDH, the glycolytic activity was decreased by ∼70% in the nuclear fraction (Fig. 5A). The good-control group had expressions similar to those of the normal control rats (data not shown).

Ribosylation and nitration have been shown to inhibit GAPDH activity (15,33). We determined the levels of its ribosylation and nitration of retinal GAPDH. Twelve months of poor control resulted in an ∼25% increase in ribosylation of GAPDH (Fig. 3B) and a 30% increase in nitration compared with the normal rat retina (Fig. 3). The rats in the good-control group had expressions similar to those of the normal-control rats (data not shown). The ratio of nitrated GAPDH was 2.5:1 in normal rat retina and increased to almost 1:1 in the poor-control group. However, in the nuclear fraction, nitrated GAPDH was about twofold higher in poor-control rats compared with normal rats (Fig. 5B), strongly suggesting that the enzyme in nuclear fraction is mainly in its covalently modified and inactivated form.

Effect of reversal of hyperglycemia on GAPDH.

When diabetic rats were allowed to remain in poor control for 6 months before institution of good control, the protein and gene expressions of retinal GAPDH remained subnormal, and PARP remained activated; values obtained in poor-control and poor control–good control groups were not different from each other (Figs. 1, 2, and 3A). In addition, both ribosylation and nitration of GAPDH were also not different in the retina from the poor-control group and poor control–good control group, suggesting that 6 months of good control had no beneficial effect on the covalent modification of the enzyme (Fig. 3B and C). The expression and activity of GAPDH in nuclear fraction of retina from the poor control–good control group were significantly different from those in normal rats, and the enzyme remained nitrated (Figs. 4 and 5). However, these values were similar to those obtained from the poor-control group, suggesting that 6 months of good control did not prevent the enzyme from translocating to the nucleus and thus failed to protect the retina from apoptosis. But when the rats were maintained in good control soon after induction of diabetes (good-control group), GAPDH gene expression was similar to that obtained from normal rat retina (Fig. 2).

Effect of reversal of hyperglycemia on GAPDH-mediated pathways.

As shown in Fig. 6A, multiple protein bands with increased AGEs were observed in the retina from rats in the poor-control group compared with those from normal rats, and protein staining from the rats in the poor control–good control group was similar to those obtained from the rats in the poor-control group. We, however, did not identify the retinal proteins that had increased AGEs. In the same retina samples, reversal of hyperglycemia had no effect on increased PKCβII (Fig. 6B); the enzyme expression remained significantly elevated in both the poor-control and the poor control–good control groups compared with that in the normal group of rats (P < 0.05). Similarly, 6 months of good control did not produce any reduction in O-GlcNAcylation of retinal proteins (data not shown), confirming that reversal of hyperglycemia had no beneficial effect on GAPDH-mediated downstream and upstream signaling pathways.

Effect of reversal of hyperglycemia on the retinal histopathology.

Poor glycemic control in rats for 12 months increased the number of acellular capillaries in the retinal vasculature (Fig. 7) by about fourfold compared with that in normal rats. The 6 months of good control that followed 6 months of poor control failed to provide any protection; the number of acellular capillaries was similar in poor-control and poor control–good control rats (average number of acellular capillaries per millimeter squared of retina in rats in the normal, poor-control, and poor control–good control groups: 1.5, 6.1, and 6.8, respectively).

GAPDH, a classic glycolytic enzyme, is implicated in diverse cytoplasmic, membrane, and nuclear activities and has been shown to play a significant role in cell death (12), and its inhibition is considered to activate major pathways of endothelial cell damage, including activation of PKC, hexosamine pathway flux, and AGE formation (9,10). Here, we show that diabetes inhibits GAPDH activity in the retina, and its expression becomes subnormal. The enzyme is translocated from cytosol to the nucleus, and GAPDH-mediated downstream and upstream signaling pathways (AGEs, PKC, and the hexosamine pathway) are activated. Our data suggest that the enzyme translocated to the nuclear fraction is covalently modified. Furthermore, we provide exciting data demonstrating that reinstitution of good control after 6 months of poor control does not produce any beneficial effects on the inactivation of retinal GAPDH. The enzyme remains inactive with its expression elevated in the nucleus, suggesting that inhibition of GAPDH and its subcellular translocation resist reversal after reinstitution of good control. Reinstitution of good control also fails to provide any benefit to the covalent modification; the elevated levels of both ribosylation and nitration are sustained for at least 6 months after reversal of poor control. In the same animals the signaling pathways that are in direct control of GAPDH remain activated in the retina, and the number of acellular capillaries remain elevated after reestablishment of good control. These novel and exciting observations strongly suggest a role for GAPDH in the development and progression of diabetic retinopathy.

Although others have reported decreased retinal GAPDH activity at 3 months of diabetes duration in rats (34), ours is the first report showing that the enzyme remains inhibited at duration of diabetes in rats when signs of retinopathy can be detected. High glucose decreases GAPDH in vascular cells, putatively because of overproduction of superoxide by mitochondrial electron transport chain (9). Retinal mitochondria are dysfunctional in diabetes, and superoxide levels are elevated (7,8,35); and complex III is considered one of the sources of increased superoxide (8). Overexpression of MnSOD that is shown to prevent glucose-induced inhibition of GAPDH in vascular cells (9) also prevents the development of retinopathy in diabetic mice (8). Thus, taken together, data strongly implicate the role for GAPDH in the pathogenesis of retinopathy in diabetes.

Mitochondrial superoxide break DNA strand and activate PARP, and PARP-mediated poly(ADP-ribosyl)ation of GAPDH is considered one of the mechanisms in the inhibition of GAPDH activity in hyperglycemic conditions (14). Our data show that PARP activity is significantly increased in the poor-control group, and this is in accordance with another published report (36). Increased PARP activity plays an important role in diabetes-induced retinal capillary cell death, and inhibitors of PARP prevent retinal leukostasis, oxidative stress, and retinopathy in diabetic rats (3639). Here, we show that increased ribosylation of retinal GAPDH could be one of the mechanisms responsible for its inactivation.

GAPDH is susceptible to nitration by peroxynitrite (formed by reaction between nitric oxide and superoxide); steady-state exposure of GAPDH to low doses of peroxynitrite in rat astrocytes results in its inhibition (15,16). GAPDH is also a target for inactivation by nitric oxide in endothelial cells (40), which are some of the microvascular cells in the retina that present pathology of diabetic retinopathy. Nitric oxide and peroxynitrite levels are elevated in the retina and its capillary cells, and these levels remain elevated at duration when vascular histopathology characteristic of retinopathy is developing in diabetic rats (5,6,25,4143). Our results clearly show that diabetes increases nitration of retinal GAPDH, implying that nitration is associated with its inhibition. In support, covalent modification of GAPDH by nitration is also observed in other pathological conditions associated with inflammation (44), and diabetic retinopathy is believed to be a low-grade chronic inflammatory disease (4548). Peroxynitrite itself damages DNA and triggers activation of PARP (49); we clearly show that ribosylation of GAPDH is also increased in diabetes. Furthermore, reduction in retinal GAPDH expression (gene and protein) suggests that, in addition to its covalent modification, the gene transcript of GAPDH is decreased in diabetes.

GAPDH is also a major intracellular messenger that mediates cell death via apoptosis. Covalent modification of GAPDH is suggested to trigger its translocation from cytosol to the nuclear fraction (12,13). During nuclear translocation, its activity is lost (50), and an increase in hydrophobicity due to its nitration is being postulated as one of the mechanisms (16). Nuclear translocation of GAPDH is suggested to play a role in retinal glial cell apoptosis in hyperglycemic conditions (13), and increased apoptosis of retinal capillary cells precedes the development of retinal pathology associated with diabetic retinopathy (32). Our results show that the expression of GAPDH is increased in nuclear fraction in the retina from diabetic rats compared with normal rats; however, the enzyme in the nuclear fraction appears to be in a more nitrated and inactivated state compared with the cytosol fraction. This implies that although diabetes increases GAPDH translocation to the nucleus, it is covalently modified before being translocated from the cytosol and is in an inactivated state.

Reinstitution of good control after 6 months of poor control failed to produce any significant beneficial effects on GAPDH, but if good control was initiated soon after induction of diabetes in rats and allowed to continue for 12 months, GAPDH remained similar to that obtained from age-matched normal rats. This strongly supports that in addition to being a central player in the development of diabetic retinopathy, GAPDH is important in the metabolic memory phenomenon. Reversal of hyperglycemia failed to provide any benefit to the activation of PARP; the enzyme remained activated even after 6 months of good control. Sustained increases in nitration and ribosylation of retinal GAPDH after reversal of hyperglycemia in rats suggests that good control fails to provide any benefit to the covalent modifications of the enzyme. Furthermore, good control did not prevent translocation of the retinal enzyme from cytosol to the nucleus, suggesting that GAPDH remains proapoptotic even after good control is reestablished. Because of tissue availability, apoptosis of retinal capillary cells was not measured in this study; however, in support of continued apoptosis, our previous studies have shown that the apoptosis execution enzyme caspase-3 remains active in the retina even after reversal of hyperglycemia in rats. The process of caspase-3 activation that starts before the appearance of retinal histopathology resists reversal by reinstitution of good control (25), and nuclear accumulation of covalently modified GAPDH could be one of the important factors associated with increased apoptosis and histopathology of diabetic retinopathy.

Inhibition of GAPDH increases the levels of glycolytic metabolite glyceraldehyde 3- phosphate, and this activates the major pathways implicated in diabetes complications, including AGEs formation, PKC activation, and hexosamine pathway (10). Here, we show that the reversal of hyperglycemia in rats, in addition to failing to provide any benefit to the inhibition of retinal GAPDH and its nuclear translocation, has no significant effect on GAPDH-mediated pathways. Thus, both the downstream and upstream consequences of GAPDH inhibition persist for at least 6 months after hyperglycemia is terminated, further strengthening the role of GAPDH in the development/progression of diabetic retinopathy.

Data presented here clearly show that the rats that presented no effect of reversal of hyperglycemia on GAPDH and its translocation to the nucleus also showed no effect on the development of retinopathy; the number of acellular capillaries remains comparable in the rats in the poor-control and poor control–good control groups. This confirms that GAPDH-mediated pathways and the process of apoptosis of retinal capillary cells that begins before histopathology and can be detected in the retinal vasculature (25) continue to progress even after hyperglycemia is terminated. The analyses of GAPDH, its translocation and covalent modification, and the consequences of its inhibition on the pathways were performed in the whole-retina samples, and this approach did not allow us to identify the specific cell type. The failure to reverse the development of retinal vascular histopathology by reinstitution of good control, however, strongly suggests that GAPDH has a significant role in the development and progression of diabetic retinopathy.

In conclusion, we have provided strong evidence demonstrating that GAPDH is inhibited and its downstream and upstream signaling pathway activated in the retina in diabetes at a duration when histopathology characteristic of retinopathy can be observed in rats. The covalent modification of the enzyme is increased. Diabetes accelerated translocation of retinal GAPDH into the nucleus, suggesting that its proapoptotic nature possibly contributes to the development of diabetic retinopathy. In addition to its role in the pathogenesis of diabetic retinopathy, GAPDH is also important in the metabolic memory phenomenon. Sustained nitration and ribosylation are possible mechanisms for resistance of GAPDH to reverse inhibition after reversal of hyperglycemia. Therapies targeted toward preventing GAPDH inhibition by blocking its covalent modification should help in the development and also in arresting the progression of diabetic retinopathy, a sight-threatening complication of diabetes.

Published ahead of print at http://diabetes.diabetesjournals.org on 13 October 2008.

Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

This study was supported in part by grants from the National Institutes of Health, the Juvenile Diabetes Research Foundation, The Thomas Foundation, and Research to Prevent Blindness.

No potential conflicts of interest relevant to this article were reported.

We thank Dr. Bindu Menon for technical assistance and Yakov Shamailov and Divyesh Sarman for their help in maintaining the rats.

1.
Engerman RL, Kern TS: Hyperglycemia as a cause of diabetic retinopathy.
Metabolism
35
:
20
–23,
1986
2.
Baynes JW: Role of oxidative stress in development of complications in diabetes.
Diabetes
40
:
405
–412,
1991
3.
Xia P, Inoguchi T, Kern TS, Engerman RL, Oates PJ, King GL: Characterization of the mechanism for the chronic activation of DAG-PKC pathway in diabetes and hypergalactosemia.
Diabetes
43
:
1122
–1129,
1994
4.
Aiello LP: Vascular endothelial growth factor and the eye: biochemical mechanisms of action and implications for novel therapies.
Ophthalmic Res
29
:
354
–362,
1997
5.
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
50
:
1938
–1942,
2001
6.
Kowluru RA, Atasi L, Ho YS: Role of mitochondrial superoxide dismutase in the development of diabetic retinopathy.
Invest Ophthalmol Vis Sci
47
:
1594
–1599,
2006
7.
Kowluru RA, Kowluru V, Ho YS, Xiong Y: Overexpression of mitochondrial superoxide dismutase in mice protects the retina from diabetes-induced oxidative stress.
Free Rad Biol Med
41
:
1191
–1196,
2006
8.
Kanwar M, Chan PS, Kern TS, Kowluru RA: Oxidative damage in the retinal mtochondria of diabetic mice: possible protection by superoxide dismutase.
Invest Ophthalmol Vis Sci
48
:
3805
–3811,
2007
9.
Du X, Matsumura T, Edelstein D, Rossetti L, Zsengeller Z, Szabo C, Brownlee M: Inhibition of GAPDH activity by poly(ADP-ribose) polymerase activates three major pathways of hyperglycemic damage in endothelial cells.
J Clin Invest
112
:
1049
–1057,
2003
10.
Brownlee M: The pathobiology of diabetic complications: a unifying mechanism.
Diabetes
54
:
1615
–1625,
2005
11.
Saunders PA, Chalecka-Franaszek E, Chuang DM: Subcellular distribution of glyceraldehyde-3-phosphate dehydrogenase in cerebellar granule cells undergoing cytosine arabinoside-induced apoptosis.
J Neurochem
69
:
1820
–1828,
1997
12.
Hara MR, Cascio MB, Sawa A: GAPDH as a sensor of NO stress.
Biochim Biophys Acta
1762
:
502
–509,
2006
13.
Kusner LL, Sarthy VP, Mohr S: Nuclear translocation of glyceraldehyde-3-phosphate dehydrogenase: a role in high glucose-induced apoptosis in retinal Muller cells.
Invest Ophthalmol Vis Sci
45
:
1553
–1561,
2004
14.
Deveze-Alvarez M, Garcia-Soto J, Martinez-Cadena G: Glyceraldehyde-3-phosphate dehydrogenase is negatively regulated by ADP-ribosylation in the fungus Phycomyces blakesleeanus.
Microbiology
147
:
2579
–2584,
2001
15.
Buchczyk DP, Grune T, Sies H, Klotz LO: Modifications of glyceraldehyde-3-phosphate dehydrogenase induced by increasing concentrations of peroxynitrite: early recognition by 20S proteasome.
Biol Chem
38
:
237
–241,
2003
16.
Batthyany C, Schopfer FJ, Baker PR, Durán R, Baker LM, Huang Y, Cerveñansky C, Branchaud BP, Freeman BA: Reversible post-translational modification of proteins by nitrated fatty acids in vivo.
J Biol Chem
281
:
450
–463,
2006
17.
Diabetes Control and Complications Trial Research Group: The effect of intensive treatment of diabetes on the development of long-term complications in insulin-dependent diabetes mellitus.
N Engl J Med
329
:
977
–986,
1993
18.
Diabetes Control and Complications Trial Research Group: Early worsening of diabetic retinopathy in the diabetes control and complication trial.
Arch Ophthalm
116
:
874
–886,
1998
19.
Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications Research Group: Effect of intensive therapy on the microvascular complications of type 1 diabetes mellitus.
JAMA
287
:
2563
–2569,
2002
20.
LeRoith D, Fonseca V, Vinik A: Metabolic memory in diabetes: focus on insulin.
Diabete Metab Res Rev
21
:
85
–90,
2005
21.
Engerman RL, Kern TS: Progression of incipient diabetic retinopathy during good glycemic control.
Diabetes
36
:
808
–812,
1987
22.
Hammes H-P, Klinzing I, Wiegand S, Bretzel RG, Cohen AM, Federlin K: Islet transplantation inhibits diabetic retinopathy in the sucrose-fed diabetic Cohen diabetic rat.
Invest Ophthalmol Vis Sci
34
:
2092
–2096,
1993
23.
Kowluru RA, Koppolu P: Termination of experimental galactosemia in rats, and progression of retinal metabolic abnormalities.
Inves Ophthalmol Vis Sci
43
:
3287
–3291,
2002
24.
Kowluru RA: Effect of re-institution of good glycemic control on retinal oxidative stress and nitrative stress in diabetic rats.
Diabetes
52
:
818
–823,
2003
25.
Kowluru RA, Chakrabarti S, Chen S: Re-Institution of good metabolic control in diabetic rats on the activation of caspase-3 and nuclear transcriptional factor (NF-kB) in the retina.
Acta Diabetologica
44
:
194
–199,
2004
26.
Kowluru RA, Kanwar M, Kennedy A: Metabolic memory phenomenon and accumulation of peroxynitrite in retinal capillaries.
Exp Diabetes Res
2007
:
2196
,
2007
27.
Chan PS, Kanwar M, Kowluru RA: Resistance of retinal inflammatory mediators to suppress after re-institution of good glycemic control: novel mechanism for metabolic memory.
J Diabetes Complicat
. In press
28.
Veluthakal R, Khan I, Tannous M, Kowluru A: Functional inactivation by interleukin-1beta of glyceraldehyde-3-phosphate dehydrogenase in insulin-secreting cells.
Apoptosis
7
:
241
–246,
2000
29.
Kowluru RA, Menon B, Gierhart D: Beneficial effect of zeaxanthin on retinal metabolic abnormalities in diabetic rat.
Inves Ophthalmol Vis Sci
49
:
1645
–1651,
2008
30.
Zachara NE, Hart GW: O-GlcNAc a sensor of cellular state: the role of nucleocytoplasmic glycosylation in modulating cellular function in response to nutrition and stress.
Biochim Biophys Acta
1673
:
13
–28,
2004
31.
Mizutani M, Gerhardinger C, Lorenzi M: Muller cell changes in human diabetic retinopathy.
Diabetes
47
:
455
–459,
1998
32.
Kern TS, Tang J, Mizutani M, Kowluru R, Nagraj R, Lorenzi M: Response of capillary cell death to aminoguanidine predicts the development of retinopathy: comparison of diabetes and galactosemia.
Invest Ophthalmol Vis Sci
41
:
3972
–3978,
2000
33.
Kiss L, Szabo C: The pathogenesis of diabetic complications: the role of DNA injury and poly(ADP-ribose) polymerase activation in peroxynitrite-mediated cytotoxicity.
Mem Inst Oswaldo Cruz
100
:
29
–37,
2005
34.
Ola MS, Berkich DA, Xu Y, King MT, Gardner TW, Simpson I, LaNoue KF: Analysis of glucose metabolism in diabetic rat retinas.
Am J Physiol Endocrinol Metab
290
:
E1057
–E1067,
2006
35.
Kowluru RA, Abbas SN: Diabetes-induced mitochondrial dysfunction in the retina.
Inves Ophthal Vis Sci
44
:
5327
–5334,
2003
36.
Zheng L, Szabo C, Kern TS: Poly(ADP-ribose) polymerase is involved in the development of diabetic retinopathy via regulation of nuclear factor-κB.
Diabetes
53
:
2960
–2967,
2004
37.
Sugawara R, Hikichi T, Kitaya N, Mori F, Nagaoka T, Yoshida A, Szabo C: Peroxynitrite decomposition catalyst, FP15, and poly(ADP-ribose) polymerase inhibitor, PJ34, inhibit leukocyte entrapment in the retinal microcirculation of diabetic rats.
Curr Eye Res
29
:
11
–16,
2004
38.
Obrosova IG, Minchenko AG, Frank RN, Seigel GM, Zsengeller Z, Pacher P, Stevens MJ, Szabó C: Poly(ADP-ribose) polymerase inhibitors counteract diabetes- and hypoxia-induced retinal vascular endothelial growth factor overexpression.
Int J Mol Med
14
:
55
–64,
2004
39.
Xu B, Chiu J, Feng B, Chen S, Chakrabarti S: PARP activation and the alteration of vasoactive factors and extracellular matrix protein in retina and kidney in diabetes.
Diabete Metab Res Rev
24
:
404
–412,
2008
40.
Padgett CM, Whorton AR: S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation.
Am J Physiol
269
:
C739
–C749,
1995
41.
Du Y, Smith MA, Miller CM, Kern TS: Diabetes-induced nitrative stress in the retina, and correction by aminoguanidine.
J Neurochem
80
:
771
–779,
2002
42.
Kowluru RA, Koppolu P, Chakrabarti S, Chen S: Diabetes-induced activation of nuclear transcriptional factor in the retina, and its inhibition by antioxidants.
Free Radic Research
37
:
1169
–1180,
2003
43.
Kowluru RA, Odenbach S: Effect of long-term administration of α-lipoic acid on retinal capillary cell death and the development of retinopathy in diabetic rats.
Diabetes
53
:
3233
–3238,
2004
44.
Kanski J, Behring A, Pelling J, Schöneich C: Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging.
Am J Physiol Heart Circ Physiol
288
:
H371
–H381,
2005
45.
Joussen AM, Poulaki V, Le ML, Koizumi K, Esser C, Janicki H, Schraermeyer U, Kociok N, Fauser S, Kirchhof B, Kern TS, Adamis AP: A central role for inflammation in the pathogenesis of diabetic retinopathy.
FASEB J
18
:
1450
–1452,
2004
46.
Kowluru RA, Odenbach S: Role of interleukin-1beta in the development of retinopathy in rats: effect of antioxidants.
Invest Ophthalmol Vis Sci
45
:
4161
–4166,
2004
47.
Kern TS: Contributions of inflammatory processes to the development of the early stages of diabetic retinopathy.
Exp Diabetes Res
2007
:
95
–103,
2007
48.
Adamis AP, Berman AJ: Immunological mechanisms in the pathogenesis of diabetic retinopathy.
Semin Immunopathol
30
:
65
–84,
2008
49.
Pacher P, Szabo C: Role of poly(ADP-ribose) polymerase-1 activation in the pathogenesis of diabetic complications: endothelial dysfunction, as a common underlying theme.
Antioxid Redox Signal
11–12
:
1568
–1580,
2005
50.
Sawa A, Khan AA, Hester LD, Snyder SH: Glyceraldehyde-3-phosphate dehydrogenase: nuclear translocation participates in neuronal and non neuronal cell death.
Proc Natl Acad Sci U S A
94
:
11669
–11674,
1997