The succinated proteome.

The post-translational modifications (PTMs) of cysteine residues include oxidation, S-glutathionylation, S-nitrosylation, and succination, all of which modify protein function or turnover in response to a changing intracellular redox environment. Succination is a chemical modification of cysteine in proteins by the Krebs cycle intermediate, fumarate, yielding S-(2-succino)cysteine (2SC). Intracellular fumarate concentration and succination of proteins are increased by hyperpolarization of the inner mitochondrial membrane, in concert with mitochondrial, endoplasmic reticulum (ER) and oxidative stress in 3T3 adipocytes grown in high glucose medium and in adipose tissue in obesity and diabetes in mice. Increased succination of proteins is also detected in the kidney of a fumarase deficient conditional knock-out mouse which develops renal cysts. A wide range of proteins are subject to succination, including enzymes, adipokines, cytoskeletal proteins, and ER chaperones with functional cysteine residues. There is also some overlap between succinated and glutathionylated proteins, suggesting that the same low pKa thiols are targeted by both. Succination of adipocyte proteins in diabetes increases as a result of nutrient excess derived mitochondrial stress and this is inhibited by uncouplers, which discharge the mitochondrial membrane potential (ΔΨm) and relieve the electron transport chain. 2SC therefore serves as a biomarker of mitochondrial stress or dysfunction in chronic diseases, such as obesity, diabetes, and cancer, and recent studies suggest that succination is a mechanistic link between mitochondrial dysfunction, oxidative and ER stress, and cellular progression toward apoptosis. In this article, we review the history of the succinated proteome and the challenges associated with measuring this non-enzymatic PTM of proteins by proteomics approaches.

Chelation therapy for the management of diabetic complications: a hypothesis and a proposal for clinical laboratory assessment of metal ion homeostasis in plasma.

In a recent article, we presented the hypothesis that decompartmentalized metal ions are a major contributor to the development of diabetic complications and supported the use of chelation therapy for the treatment of diabetic complications [Nagai R, Murray DB, Metz TO, Baynes JW. Chelation: a fundamental mechanism of action of AGE inhibitors, AGE breakers, and other inhibitors of diabetes complications. Diabetes 2012;61:549-59]. Evidence in support of this hypothesis included the observation that many drugs used in the treatment of diabetes are chelators, that advanced glycation end product (AGE) inhibitors and AGE breakers lack carbonyl-trapping or AGE-breaker activity but are potent chelators, and that simple copper chelators inhibit vascular pathology in diabetes and aging. In the present article, we extend this hypothesis, proposing the interplay between copper and iron in the development of pathology in diabetes and other chronic age-related diseases, including atherosclerosis and neurodegenerative diseases. We also discuss the need and provide a framework for the development of a clinical laboratory test to assess plasma autoxidative catalytic activity and transition metal homeostasis in vivo.

Mitochondrial stress causes increased succination of proteins in adipocytes in response to glucotoxicity.

2SC [S-(2-succino)-cysteine] is a chemical modification formed by a Michael addition reaction of fumarate with cysteine residues in proteins. Formation of 2SC, termed 'succination' of proteins, increases in adipocytes grown in high-glucose medium and in adipose tissues of Type 2 diabetic mice. However, the metabolic mechanisms leading to increased fumarate and succination of protein in the adipocyte are unknown. Treatment of 3T3 cells with high glucose (30 mM compared with 5 mM) caused a significant increase in cellular ATP/ADP, NADH/NAD+ and Δψm (mitochondrial membrane potential). There was also a significant increase in the cellular fumarate concentration and succination of proteins, which may be attributed to the increase in NADH/NAD+ and subsequent inhibition of tricarboxylic acid cycle NAD+-dependent dehydrogenases. Chemical uncouplers, which dissipated Δψm and reduced the NADH/NAD+ ratio, also decreased the fumarate concentration and protein succination. High glucose plus metformin, an inhibitor of complex I in the electron transport chain, caused an increase in fumarate and succination of protein. Thus excess fuel supply (glucotoxicity) appears to create a pseudohypoxic environment (high NADH/NAD+ without hypoxia), which drives the increase in succination of protein. We propose that increased succination of proteins is an early marker of glucotoxicity and mitochondrial stress in adipose tissue in diabetes.

Gut barrier dysfunction in the Apc(Min/+) mouse model of colon cancer cachexia.

BACKGROUND: The Apc(Min/+) mouse, an animal model of colorectal cancer and cachexia, has a heterologous mutation in the Apc tumor suppressor gene, predisposing the mouse to intestinal and colon tumor development. This mouse develops intestinal polyps by ~4 weeks of age, and loses body weight gradually between ~14 and ~20 weeks of age. The strengths of this cachexia model derive from several features that mimic human cancer, including a gradual increase in tumor burden, chronic inflammation, and anemia. Little is known about the role of gut barrier dysfunction and endotoxemia in the development of cancer cachexia. We sought to determine how gut permeability and resultant endotoxemia change with the progression of cachexia. METHODS: Intestinal gut barrier integrity was assessed by permeability to FITC-dextran (MW(av)=4000kDa; FD4). Plasma glucose and triglycerides were measured by enzymatic assays, IL-6 by enzyme-linked immunosorbent assay, and endotoxin by the limulus amoebocyte assay. Body temperature was measured using a rectal probe. RESULTS: Progression of cachexia was accompanied by development of gut barrier dysfunction (permeability to FD4), hypertrophy of mesenteric lymph nodes, and an increase in plasma endotoxin concentration. Changes in blood glucose and glucose tolerance, plasma IL-6, triglycerides, and body temperature were characteristic of endotoxemia. CONCLUSION: We propose a role for gut barrier dysfunction (GBD) and subsequent endotoxemia in the development of inflammation and progression of cachexia in the Apc(Min/+) mouse.

Aberrant succination of proteins in fumarate hydratase-deficient mice and HLRCC patients is a robust biomarker of mutation status.

Germline mutations in the FH gene encoding the Krebs cycle enzyme fumarate hydratase predispose to hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. FH-deficient cells and tissues accumulate high levels of fumarate, which may act as an oncometabolite and contribute to tumourigenesis. A recently proposed role for fumarate in the covalent modification of cysteine residues to S-(2-succinyl) cysteine (2SC) (termed protein succination) prompted us to assess 2SC levels in our existing models of HLRCC. Herein, using a previously characterized antibody against 2SC, we show that genetic ablation of FH causes high levels of protein succination. We next hypothesized that immunohistochemistry for 2SC would serve as a metabolic biomarker for the in situ detection of FH-deficient tissues. Robust detection of 2SC was observed in Fh1 (murine FH)-deficient renal cysts and in a retrospective series of HLRCC tumours (n = 16) with established FH mutations. Importantly, 2SC was undetectable in normal tissues (n = 200) and tumour types not associated with HLRCC (n = 1342). In a prospective evaluation of cases referred for genetic testing for HLRCC, the presence of 2SC-modified proteins (2SCP) correctly predicted genetic alterations in FH in every case. In two series of unselected type II papillary renal cancer (PRCC), prospectively analysed by 2SCP staining followed by genetic analysis, the biomarker accurately identified previously unsuspected FH mutations (2/33 and 1/36). The investigation of whether metabolites in other tumour types produce protein modification signature(s) that can be assayed using similar strategies will be of interest in future studies of cancer.

Muscle oxidative capacity during IL-6-dependent cancer cachexia.

Many diseases are associated with catabolic conditions that induce skeletal muscle wasting. These various catabolic states may have similar and distinct mechanisms for inducing muscle protein loss. Mechanisms related to muscle wasting may also be related to muscle metabolism since glycolytic muscle fibers have greater wasting susceptibility with several diseases. The purpose of this study was to determine the relationship between muscle oxidative capacity and muscle mass loss in red and white hindlimb muscles during cancer cachexia development in the Apc(Min/+) mouse. Gastrocnemius and soleus muscles were excised from Apc(Min/+) mice at 20 wk of age. The gastrocnemius muscle was partitioned into red and white portions. Body mass (-20%), gastrocnemius muscle mass (-41%), soleus muscle mass (-34%), and epididymal fat pad (-100%) were significantly reduced in severely cachectic mice (n = 8) compared with mildly cachectic mice (n = 6). Circulating IL-6 was fivefold higher in severely cachectic mice. Cachexia significantly reduced the mitochondrial DNA-to-nuclear DNA ratio in both red and white portions of the gastrocnemius. Cytochrome c and cytochrome-c oxidase complex subunit IV (Cox IV) protein were reduced in all three muscles with severe cachexia. Changes in muscle oxidative capacity were not associated with altered myosin heavy chain expression. PGC-1α expression was suppressed by cachexia in the red and white gastrocnemius and soleus muscles. Cachexia reduced Mfn1 and Mfn2 mRNA expression and markers of oxidative stress, while Fis1 mRNA was increased by cachexia in all muscle types. Muscle oxidative capacity, mitochondria dynamics, and markers of oxidative stress are reduced in both oxidative and glycolytic muscle with severe wasting that is associated with increased circulating IL-6 levels.

Succination of thiol groups in adipose tissue proteins in diabetes: succination inhibits polymerization and secretion of adiponectin.

S-(2-Succinyl)cysteine (2SC) is formed by reaction of the Krebs cycle intermediate fumarate with cysteine residues in protein, a process termed succination of protein. Both fumarate and succination of proteins are increased in adipocytes cultured in high glucose medium (Nagai, R., Brock, J. W., Blatnik, M., Baatz, J. E., Bethard, J., Walla, M. D., Thorpe, S. R., Baynes, J. W., and Frizzell, N. (2007) J. Biol. Chem. 282, 34219-34228). We show here that succination of protein is also increased in epididymal, mesenteric, and subcutaneous adipose tissue of diabetic (db/db) mice and that adiponectin is a major target for succination in both adipocytes and adipose tissue. Cys-39, which is involved in cross-linking of adiponectin monomers to form trimers, was identified as a key site of succination of adiponectin in adipocytes. 2SC was detected on two of seven monomeric forms of adiponectin immunoprecipitated from adipocytes and epididymal adipose tissue. Based on densitometry, 2SC-adiponectin accounted for approximately 7 and 8% of total intracellular adiponectin in cells and tissue, respectively. 2SC was found only in the intracellular, monomeric forms of adiponectin and was not detectable in polymeric forms of adiponectin in cell culture medium or plasma. We conclude that succination of adiponectin blocks its incorporation into trimeric and higher molecular weight, secreted forms of adiponectin. We propose that succination of proteins is a biomarker of mitochondrial stress and accumulation of Krebs cycle intermediates in adipose tissue in diabetes and that succination of adiponectin may contribute to the decrease in plasma adiponectin in diabetes.

Citric acid inhibits development of cataracts, proteinuria and ketosis in streptozotocin (type 1) diabetic rats.

Although many fruits such as lemon and orange contain citric acid, little is known about beneficial effects of citric acid on health. Here we measured the effect of citric acid on the pathogenesis of diabetic complications in streptozotocin-induced diabetic rats. Although oral administration of citric acid to diabetic rats did not affect blood glucose concentration, it delayed the development of cataracts, inhibited accumulation of advanced glycation end-products (AGEs) such as N(epsilon)-(carboxyethyl)lysine (CEL) and N(epsilon)-(carboxymethyl)lysine (CML) in lens proteins, and protected against albuminuria and ketosis. We also show that incubation of protein with acetol, a metabolite formed from acetone by acetone monooxygenase, generate CEL, suggesting that inhibition of ketosis by citric acid may lead to the decrease in CEL in lens proteins. These results demonstrate that the oral administration of citric acid ameliorates ketosis and protects against the development of diabetic complications in an animal model of type 1 diabetes.

Glycation of wood frog (Rana sylvatica) hemoglobin and blood proteins: in vivo and in vitro studies.

The effects of in vivo freezing and glucose cryoprotectant on protein glycation were investigated in the wood frog, Rana sylvatica. Our studies revealed no difference in the fructoselysine content of blood plasma sampled from control, 27 h frozen and 18 h thawed wood frogs. Glycated hemoglobin (GHb) decreased slightly with 48 h freezing exposure and was below control levels after 7d recovery, while glycated serum albumin was unchanged by 48 h freezing but did increase after 7d of recovery. In vitro exposure of blood lysates to glucose revealed that the GHb production in wood frogs was similar to that of the rat but was lower than in leopard frogs. We conclude that wood frog hemoglobin was glycated in vitro; however, GHb production was not apparent during freezing and recovery when in vivo glucose is highly elevated. It is possible that wood frog blood proteins have different in vivo susceptibilities to glycation.

Improved methods for the enrichment and analysis of glycated peptides.

Nonenzymatic glycation of tissue proteins has important implications in the development of complications of diabetes mellitus. Herein we report improved methods for the enrichment and analysis of glycated peptides using boronate affinity chromatography and electron-transfer dissociation mass spectrometry, respectively. The enrichment of glycated peptides was improved by replacing an off-line desalting step with an online wash of column-bound glycated peptides using 50 mM ammonium acetate, followed by elution with 100 mM acetic acid. The analysis of glycated peptides by MS/MS was improved by considering only higher charged (> or = 3) precursor ions during data-dependent acquisition, which increased the number of glycated peptide identifications. Similarly, the use of supplemental collisional activation after electron transfer (ETcaD) resulted in more glycated peptide identifications when the MS survey scan was acquired with enhanced resolution. Acquiring ETD-MS/MS data at a normal MS survey scan rate, in conjunction with the rejection of both 1+ and 2+ precursor ions, increased the number of identified glycated peptides relative to ETcaD or the enhanced MS survey scan rate. Finally, an evaluation of trypsin, Arg-C, and Lys-C showed that tryptic digestion of glycated proteins was comparable to digestion with Lys-C and that both were better than Arg-C in terms of the number of glycated peptides and corresponding glycated proteins identified by LC-MS/MS.

Glutaraldehyde is an effective cross-linker for production of antibodies against advanced glycation end-products.

Immunohistochemical approaches have been widely used in the localization and quantification of advanced glycation end-products (AGEs). Traditional approaches for production of anti-AGE antibodies use cross-linkers such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) to conjugate the AGE antigen to the carrier protein. However, these approaches often fail to produce antibodies that are specific to the particular AGE of interest. In the present study, Nepsilon-(carboxymethyl)lysine (CML), a major antigenic AGE structure, was conjugated to human serum albumin (HSA) using various cross-linkers, including EDC, bis(sulfosuccinimidyl)suberate (BS3) and glutaraldehyde, to compare their efficiency for the production of epitope-specific antibodies. All of the cross-linkers tested were capable of conjugating CML to HSA, and each CML-conjugated HSA was recognized by previously characterized anti-CML antibody. However, only the use of glutaraldehyde as the cross-linker resulted in the production of a CML-specific monoclonal antibody, termed 2G11. 2G11 significantly recognized CML-modified HSA and peptide, whereas it did not recognize Nepsilon-(carboxyethyl)lysine (CEL)-modified HSA and peptide, indicating that 2G11 is highly specific to CML, and can distinguish the difference of a single methyl group between the two epitopes. To further demonstrate the use of glutaraldehyde, anti-AGE antibodies against CEL, S-(2-succinyl)cysteine and S-(carboxymethyl)cysteine were obtained by conjugation with glutaraldehyde. These studies demonstrate the efficacy of glutaraldehyde as a cross-linker for the production of antibodies against small molecules.

Succination of proteins by fumarate: mechanism of inactivation of glyceraldehyde-3-phosphate dehydrogenase in diabetes.

S-(2-succinyl)cysteine (2SC) is a chemical modification of proteins formed by a Michael addition reaction between the Krebs cycle intermediate, fumarate, and thiol groups in protein--a process known as succination of protein. Succination causes irreversible inactivation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in vitro. GAPDH was immunoprecipitated from muscle of diabetic rats, then analyzed by ultra-performance liquid chromatography-electrospray ionization-mass spectroscopy. Succination of GAPDH was increased in muscle of diabetic rats, and the extent of succination correlated strongly with the decrease in specific activity of the enzyme. We propose that 2SC is a biomarker of mitochondrial and oxidative stress in diabetes and that succination of GAPDH and other thiol proteins may provide the chemical link between glucotoxicity and the pathogenesis of diabetic complications.

Skin autofluorescence is a strong predictor of cardiac mortality in diabetes.

OBJECTIVE: Advanced glycation end products (AGEs) are biomarkers of metabolic stress and are thought to contribute to the increase of coronary heart disease (CHD) in diabetes. Tissue autofluorescence is related to the accumulation of AGEs. The aim of the present study was to evaluate the relationship between skin autofluorescence and metabolic burden (hyperglycemia and hyperlipidemia) and its relationship with CHD and mortality. RESEARCH DESIGN AND METHODS: Skin autofluorescence was measured noninvasively with an autofluorescence reader in 48 type 1 and 69 type 2 diabetic patients and 43 control subjects. The presence of CHD was observed at baseline and mortality during a follow-up period of 5 years. RESULTS: Autofluorescence correlated with mean A1C, triglycerides, and LDL. Autofluorescence values further increased with age, microalbuminuria, dialysis treatment, and diabetes duration. Autofluorescence was strongly related to the presence of CHD (odds ratio 7.9) and predicted mortality (3.0). Multivariate analysis showed that autofluorescence was more strongly associated with CHD and mortality compared with A1C, triglycerides, and LDL. CONCLUSIONS: Skin autofluorescence is strongly related to cumulative metabolic burden. Skin autofluorescence seems strongly associated with cardiac mortality and may provide important clinical information for risk assessment.

The anti-cancer drug, doxorubicin, causes oxidant stress-induced endothelial dysfunction.

The anticancer drug doxorubicin (DOX) is toxic to target cells, but also causes endothelial dysfunction and edema, secondary to oxidative stress in the vascular wall. Thus, the mechanism of action of this drug may involve chemotoxicity to both cancer cells and to the endothelium. Indeed, we found that the permeability of monolayers of bovine pulmonary artery endothelial cells (BPAEC) to albumin was increased by approximately 10-fold above control, following 24-h exposure to clinically relevant concentrations of DOX (up to 1 microM). DOX also caused >4-fold increases in lactate dehydrogenase leakage and large decreases in ATP and reduced glutathione (GSH) in BPAECs, which paralleled the increases in endothelial permeability. A large part of the ATP loss could be attributed to DOX-induced hydrogen peroxide production which inhibited key thiol-enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glucose-6-phosphate dehydrogenase (G6PDH). Depletion of reduced nicotinamide adenine dinucleotide phosphate (NADPH) appeared to be a major factor leading to DOX-induced GSH depletion. At low concentrations, the sulfhydryl reagent, iodoacetate (IA), inhibited GAPDH, caused a decrease in ATP and increased permeability, without inhibiting G6PDH or decreasing GSH. These results, coupled with those of previous work on a related quinone, menadione, suggest that depletion of either GSH or ATP may lead independently to endothelial dysfunction during chemotherapy, contributing to the cardiotoxicity and other systemic side-effects of the drug.

Detection and identification of arginine modifications on methylglyoxal-modified ribonuclease by mass spectrometric analysis.

Analysis of the broad range of trace chemical modifications of proteins in biological samples is a significant challenge for modern mass spectrometry. Modification at lysine and arginine residues, in particular, causes resistance to digestion by trypsin, producing large tryptic peptides that are not readily sequenced by mass spectrometry. In this work, we describe the analysis of ribonuclease (RNase) modified by methylglyoxal (MGO) under physiological conditions. For detection of modifications, we use comparative analysis of the single combined spectra extracted from the full-scan MS data of the tryptic digests from native and modified proteins. This approach revealed 11 ions unique to MGO-modified RNase, including a 32-amino acid peptide containing a modified Arg-85 residue. Sequential digestion of MGO-modified RNase by endoproteinase Glu-C and trypsin was required to obtain peptides that were amenable to sequencing analysis. Arg-39 was identified as the main site of modification (35% modification) on MGO-modified Rnase, and the dihydroxyimidazolidine and hydroimidazolone derivatives were the main adducts formed, with minor amounts of the tetrahydropyrimidine and argpyrimidine derivatives. For identification of these products, we used variations in source voltage and collision energy to obtain the dehydration and decarboxylation products of the tetrahydropyrimidine-containing peptides and dehydration of the dihydroxyimidazoline-containing peptides. The resultant spectra were dependent on the cone voltage and collision energy, and analysis of spectra at various settings permitted structural assignments. These studies illustrate the usefulness of single combined mass spectra extracted from full-scan data and variations in source and collision cell voltages for detection and structural characterization of chemical adducts on proteins.

Dietary ALEs are a risk to human health--NOT!

Advanced lipoxidation end-products (ALEs) are formed by reaction of protein with lipid-derived reactive peroxyl and carbonyl compounds produced during food processing and cooking. There is concern that ALEs may induce damage in the gastrointestinal tract, affecting gut health, or enter the body and promote vascular inflammation and tissue damage. However, there is no direct evidence that ALE-proteins are a source of damage in the intestines or that they are transported into the circulation and cause pathology. Modification of proteins by ALEs impedes their digestion, and reactive ALEs released by gastrointestinal proteases would react with proteins or peptides in the gut, limiting their absorption. There are also potent enzymatic mechanisms for detoxifying ALEs or their precursors prior to their entry into the circulation. If ALEs gain access to the circulation, a battery of protective enzymes in tissue provides a second level of defense. These enzymes may be induced in intestinal epithelia and liver by low doses of ALEs, and adaptive responses would provide enhanced protection against future exposure to ALEs. Overall, except in persons with compromised organ function, e. g., vascular, hepatic, or renal diseases, there is little evidence that food ALEs will have any significant pathological effects.

What is the future of diabetic wound care?

With diabetes affecting 5% to 10% of the US population, development of a more effective treatment for chronic diabetic wounds is imperative. Clinically, the current treatment in topical wound management includes debridement, topical antibiotics, and a state-of-the-art topical dressing. State-of-the-art dressings are a multi-layer system that can include a collagen cellulose substrate, neonatal foreskin fibroblasts, growth factor containing cream, and a silicone sheet covering for moisture control. Wound healing time can be up to 20 weeks. The future of diabetic wound healing lies in the development of more effective artificial "smart" matrix skin substitutes. This review article will highlight the need for novel smart matrix therapies. These smart matrices will release a multitude of growth factors, cytokines, and bioactive peptide fragments in a temporally and spatially specific, event-driven manner. This timed and focal release of cytokines, enzymes, and pharmacological agents should promote optimal tissue regeneration and repair of full-thickness wounds. Development of these kinds of therapies will require multidisciplinary translational research teams. This review article outlines how current advances in proteomics and genomics can be incorporated into a multidisciplinary translational research approach for developing novel smart matrix dressings for ulcer treatment. With the recognition that the research approach will require both time and money, the best treatment approach is the prevention of diabetic ulcers through better foot care, education, and glycemic control.

Menadione causes endothelial barrier failure by a direct effect on intracellular thiols, independent of reactive oxidant production.

Menadione (MQ), a quinone used with cancer chemotherapeutic agents, causes cytotoxicity to endothelial cells (EC). Previous studies have suggested that MQ induces an oxidative stress and dysfunction in EC by either increasing hydrogen peroxide (H(2)O(2)) production or depleting intracellular glutathione (GSH), the main intracellular antioxidant. Since a primary function of EC is to form a barrier to fluid movement into tissues, protecting organs from edema formation and dysfunction, our aim was to see if MQ would cause a barrier dysfunction and to ascertain the mechanism. Using diffusional permeability to fluorescein isothiocyanate-labeled bovine serum albumin (FITC-BSA) as a measure of barrier function, we found that 15 micro M MQ incubated with a bovine pulmonary artery EC (BPAEC) monolayer for 4 h produced a profound barrier failure ( approximately 7-fold increase in permeability) with a parallel fall in glutathione, almost to depletion. These two events were highly correlated. Immunofluorescent imaging showed formation of paracellular holes consistent with a loss or rearrangement of cell-cell and cell-matrix adhesion molecules. H(2)O(2) (100 micro M), a concentration which gave about the same increase in permeability as MQ, only slightly decreased GSH concentration. Antioxidants, such as catalase (CAT) and dimethylthiourea (DMTU), which were able to block the H(2)O(2)-induced changes, had no effect on the MQ-induced permeability and GSH changes, suggesting that H(2)O(2) was not involved in MQ-induced effects. MQ caused a severe EC cytotoxicity as judged by lactate dehydrogenase (LDH) leakage from the EC, whereas H(2)O(2) caused only a minor increase. Also, MQ profoundly inhibited the activities of glucose-6-phosphate dehydrogenase (G6PDH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), key thiol enzymes involved in glutathione and ATP metabolism, whereas H(2)O(2) produced only a slight decrease in these activities. We conclude that the cytotoxicity of MQ and resulting barrier dysfunction correlate with GSH depletion and inactivation of key metabolic enzymes, compromising antioxidant defenses, rather than being consistent with H(2)O(2)-mediated oxidative stress.

The AGE inhibitor pyridoxamine inhibits development of retinopathy in experimental diabetes.

We examined the ability of pyridoxamine (PM), an inhibitor of formation of advanced glycation end products (AGEs) and lipoxidation end products (ALEs), to protect against diabetes-induced retinal vascular lesions. The effects of PM were compared with the antioxidants vitamin E (VE) and R-alpha-lipoic acid (LA) in streptozotocin-induced diabetic rats. Animals were given either PM (1 g/l drinking water), VE (2,000 IU/kg diet), or LA (0.05%/kg diet). After 29 weeks of diabetes, retinas were examined for pathogenic changes, alterations in extracellular matrix (ECM) gene expression, and accumulation of the immunoreactive AGE/ALE N( epsilon )-(carboxymethyl)lysine (CML). Acellular capillaries were increased more than threefold, accompanied by significant upregulation of laminin immunoreactivity in the retinal microvasculature. Diabetes also increased mRNA expression for fibronectin (2-fold), collagen IV (1.6-fold), and laminin beta chain (2.6-fold) in untreated diabetic rats compared with nondiabetic rats. PM treatment protected against capillary drop-out and limited laminin protein upregulation and ECM mRNA expression and the increase in CML in the retinal vasculature. VE and LA failed to protect against retinal capillary closure and had inconsistent effects on diabetes-related upregulation of ECM mRNAs. These results indicate that the AGE/ALE inhibitor PM protected against a range of pathological changes in the diabetic retina and may be useful for treating diabetic retinopathy.