Paul J. Beisswenger

Overexpression of glyoxalase-I in bovine endothelial cells inhibits intracellular advanced glycation endproduct formation and prevents hyperglycemia-induced increases in macromolecular endocytosis.

Methylglyoxal (MG), a dicarbonyl compound produced by the fragmentation of triose phosphates, forms advanced glycation endproducts (AGEs) in vitro. Glyoxalase-I catalyzes the conversion of MG to S-D-lactoylglutathione, which in turn is converted to D-lactate by glyoxalase-II. To evaluate directly the effect of glyoxalase-I activity on intracellular AGE formation, GM7373 endothelial cells that stably express human glyoxalase-I were generated. Glyoxalase-I activity in these cells was increased 28-fold compared to neo-transfected control cells (21.80+/-0.1 vs. 0. 76+/-0.02 micromol/min/mg protein, n = 3, P < 0.001). In neo-transfected cells, 30 mM glucose incubation increased MG and D-lactate concentration approximately twofold above 5 MM (35.5+/-5.8 vs. 19.6+/-1.6, P < 0.02, n = 3, and 21.0+/-1.3 vs. 10.0+/-1.2 pmol/ 10(6) cells, n = 3, P < 0.001, respectively). In contrast, in glyoxalase-I-transfected cells, 30 mM glucose incubation did not increase MG concentration at all, while increasing the enzymatic product D-lactate by > 10-fold (18.9+/-3.2 vs. 18.4+/- 5.8, n = 3, P = NS, and 107.1+/-9.0 vs. 9.4+/-0 pmol/10(6) cells, n = 3, P < 0.001, respectively). After exposure to 30 mM glucose, intracellular AGE formation in neo cells was increased 13.6-fold (2.58+/-0.15 vs. 0.19+/-0.03 total absorbance units, n = 3, P < 0.001). Concomitant with increased intracellular AGEs, macromolecular endocytosis by these cells was increased 2.2-fold. Overexpression of glyoxalase-I completely prevented both hyperglycemia-induced AGE formation and increased macromolecular endocytosis.

Formation of Immunochemical Advanced Glycosylation End Products Precedes and Correlates With Early Manifestations of Renal and Retinal Disease in Diabetes

Elevated levels of advanced glycosylation end products (AGEs) have been found in multiple tissues in association with diabetic vascular complications and during the microalbuminuric phase of diabetic nephropathy. In this study, we have used an AGE-specific enzyme-linked immunosorbent assay (ELISA) to measure skin AGEs to determine whether elevated levels can be detected before the onset of overt microangiopathy. Subjects with type I diabetes (n = 48) were graded for the degree of nephropathy (normal [23], microalbuminuria [12], or macroalbuminuria [12]) and retinopathy (none [13], background [20], or proliferative [15]). Subgroups with a premicroalbuminuric phase of albumin excretion (≤28 mg/24 h, n = 27) or with the earliest stages of retinopathy (n = 27) were identified. A significant increase in tissue AGEs was found as urinary albumin increased during the premicroalbuminuric phase of nephropathy even when the data were adjusted for age and duration of diabetes (P = 0.005). Immunoreactive AGEs also increased as normal renal status advanced to microalbuminuria and macroalbuminuria (P = 0.0001 across groups). Significant elevation of AGEs was also found in association with the earliest stages of clinically evident retinopathy (early background versus minimal grades). In addition, higher AGE levels were found in subjects with proliferative retinopathy when compared with those with less severe retinopathy (P < 0.004 across groups). In contrast, no significant differences were found in tissue AGE levels between groups with or without early retinopathy based on pentosidine or fluorescent AGE measurements, although fluorescent AGEs correlated with albumin excretion. In conclusion, levels of collagen-linked AGEs, when measured by an AGE-specific ELISA, reveal a correlation with preclinical stages of diabetic nephropathy and early retinopathy not indicated by other methods and may prove useful as early markers of microangiopathy in type I diabetes.

Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes

Aims/hypothesisHyperglycaemia in diabetes is associated with increased glycation, oxidative stress and nitrosative stress. Proteins modified consequently contain glycation, oxidation and nitration adduct residues, and undergo cellular proteolysis with release of corresponding free adducts. These free adducts leak into blood plasma for eventual renal excretion. The aim of this study was to perform a comprehensive quantitative analysis of protein glycation, oxidation and nitration adduct residues in plasma protein and haemoglobin as well as of free adducts in plasma and urine to quantify increased protein damage and flux of proteolytic degradation products in diabetes.MethodsType 1 diabetic patients (n=21) and normal healthy control subjects (n=12) were studied. Venous blood samples, with heparin anticoagulant, and 24-h urine samples were taken. Samples were analysed for protein glycation, oxidation and nitration adducts by a quantitative comprehensive screening method using liquid chromatography with triple quadrupole mass spectrometric detection.ResultsIn type 1 diabetic patients, the concentrations of protein glycation, oxidation and nitration adduct residues increased up to three-fold in plasma protein and up to one-fold in haemoglobin, except for decreases in pentosidine and 3-nitrotyrosine residues in haemoglobin when compared with normal control subjects. In contrast, the concentrations of protein glycation and oxidation free adducts increased up to ten-fold in blood plasma, and urinary excretion increased up to 15-fold in diabetic patients.Conclusions/interpretationWe conclude that there are profound increases in proteolytic products of glycated and oxidised proteins in diabetic patients, concurrent with much lower increases in protein glycation and oxidation adduct residues.

Increased collagen-linked pentosidine levels and advanced glycosylation end products in early diabetic nephropathy.

RATIONALE Advanced glycosylation end products (AGEs) may play an important role in the development of diabetic vascular sequelae. An AGE cross-link, pentosidine, is a sensitive and specific marker for tissue levels of AGEs. OBJECTIVES To evaluate the role of AGEs in the development of diabetic nephropathy and retinopathy, we studied pentosidine levels and the clinical characteristics of 48 subjects with insulin-dependent diabetes mellitus. Diabetic nephropathy was classified as normal, microalbuminuria, or gross proteinuria, and retinopathy was graded as none, background, or proliferative. NEWLY OBSERVED FINDINGS: Significant elevation of pentosidine (P = 0.025) was found in subjects with microalbuminuria or gross proteinuria (73.03 +/- 9.47 vs 76.46 +/- 6.37 pmol/mg col) when compared with normal (56.96 +/- 3.26 pmol/mg col). Multivariate analysis to correct for age, duration of diabetes, and gender did not modify the results. Elevated pentosidine levels were also found in those with proliferative when compared with those with background retinopathy (75.86 +/- 5.66 vs 60.42 +/- 5.98 pmol/mg col) (P < 0.05). CONCLUSIONS Microalbuminuria is associated with elevated levels of pentosidine similar to those found in overt diabetic nephropathy suggesting that elevated AGE levels are already present during the earliest detectable phase of diabetic nephropathy.

Studies on the Human Glomerular Basement Membrane: Composition, Nature of the Carbohydrate Units and Chemical Changes in Diabetes Mellitus

The basement membrane has been isolated in purified form from pooled normal human glomeruli and shown by detailed analyses to be composed of glycoprotein material. The peptide portion was characterized by the presence of large amounts of glycine, as well as by the occurrence of a substantial number of hydroxyproline, hydroxylysine, and cystine residues. Approximately 7 per cent of the weight of the membrane was found to be made up of sugar residues. The membrane was extensively solubilized by digestion with bacterial collagenase, and a study of glycopeptides obtained after additional Pronase treatment indicated the occurrence of glucosylgalactose disaccharide units linked to hydroxylysine as well as heteropolysaccharides consisting of N-acetylneuraminic acid, fucose, galactose, mannose and hexosamines similar to those previously found in the bovine glomerular basement membrane. Chemical analyses of glomeruli obtained from diabetic and nondiabetic kidneys indicated that the former contained more basement membrane-like material. Furthermore basement membranes isolated from the diabetic glomeruli had a composition different from those obtained from control subjects. The diabetic membranes were found to have a significant increase (P < 0.01) in their hydroxylysine content and in the number of glucosylgalactose disaccharides linked to this amino acid. The increase in hydroxylysine was accompanied by a decrease in the lysine level so that the sum of these amino acids remained unchanged from the normal. Changes of lesser significance were observed in hydroxyproline, glycine, valine and tyrosine with the former two amino acids showing an increase and the latter two a decrease from the nondiabetic level. No change was found in the number of the heteropolysaccharide units. These results suggest an overproduction in the diabetic state of those subunits of the basement membrane that are rich in hydroxylysmejand its glycosidically linked disaccharide unit. Such alterations in the subunit composition may lead to an abnormal packing of the peptide chains and thereby form the basis for the detective filtration function observed in diabetes.

Reduction of Glycosylated Hemoglobin and Postprandial Hyperglycemia by Acarbose in Patients With NIDDM: A placebo-controlled dose-comparison study

OBJECTIVE To compare the safety and efficacy of three doses of acarbose (100, 200, and 300 mg three times daily) with placebo for the treatment of non-insulin-dependent diabetes mellitus (NIDDM) in patients maintained on dietary therapy alone. RESEARCH DESIGN AND METHODS This multicenter double-blind placebo-controlled trial was 22 weeks in duration. The trial consisted of a 2-week screening period, a 4-week placebo run-in period, and a 16-week double-blind treatment period. The primary measure of drug efficacy was the mean change from baseline in HbA1c levels. Additional efficacy variables included the mean change from baseline in fasting and postprandial plasma glucose and serum insulin levels. RESULTS After 16 weeks of treatment, acarbose-treated patients had statistically significant reductions in mean HbA1c levels of 0.78, 0.73, and 1.10% (relative to placebo) in the 100-, 200-, and 300-mg t.i.d. groups, respectively. Significant reductions in fasting and postprandial plasma glucose levels, glucose area under the time-concentration curve, and maximum glucose concentration were also observed in acarbose-treated patients. Although there were no statistically significant differences among the 100-, 200-, and 300-mg treatment groups, there was a trend toward a dose-response relationship for most plasma glucose variables that were measured. Gastrointestinal side effects (e.g., abdominal pain, flatulence, and diarrhea) and serum transaminase elevations (e.g., aspartate aminotransferase [AST] and alanine aminotransferase [ALT] were more frequently reported in the acarbose-treated patients than in the placebo-treated control patients. Transaminase elevations occurred only at the 200-, and 300-mg dosages and were readily reversible on discontinuation of treatment. CONCLUSIONS Acarbose at doses of 100, 200, and 300 mg administered three times daily for 16 weeks significantly reduced HbA1c levels and postprandial hyperglycemia. Treatment with acarbose is a safe and effective adjunct to dietary therapy for the treatment of NIDDM.

Metformin inhibition of glycation processes

A number of studies have shown that metformin is beneficial in reducing diabetes associated vascular risk beyond the benefits expected from its antihyperglycaemic effect. One of the main pathogenic mechanisms leading to chronic complications of diabetes is non-enzymatic glycation where damage is mediated through increased production of highly chemically reactive glucose and alpha-dicarbonyl compounds which lead to production of advanced glycation products (AGEs). We present laboratory and clinical data supporting the hypothesis that one important explanation of metformins effect on diabetic complications could be its ability to reduce toxic dicarbonyls and AGEs. This effect could be related either to the binding of the alpha-dicarbonyls, methylglyoxal (MG) or 3-deoxyglucosone, or to an increase in enzymatic detoxification. Our studies presented in this manuscript document extracellular binding of MG by metformin to form a specific product (triazepinone) in vivo. This condensation product appears to be only one of several inactive end products resulting from this chemical reaction and we discuss the possibility that these or other condensation products (hydroimidazolones) could be indicative of inactivation of MG by metformin. Additional studies of other possible condensation products, as well as other potential cellular effects of metformin on MG production, will help to clarify this potentially important effect of metformin and provide a further rationale for using metformin to prevent long-term complications.

Glyceraldehyde-3-phosphate dehydrogenase activity as an independent modifier of methylglyoxal levels in diabetes

Methylglyoxal (MG) may be an important cause of diabetic complications. Its primary source is dihydroxyacetone phosphate (DHAP) whose levels are partially controlled by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using a human red blood cell (RBC) culture, we examined the effect of modifying GAPDH activity on MG production. With the inhibitor koningic acid (KA), we showed a linear, concentration-dependent GAPDH inhibition, with 5 microM KA leading to a 79% reduction of GAPDH activity and a sixfold increase in MG. Changes in redox state produced by elevated pH also resulted in a 2.4-fold increase in MG production at pH 7.5 and a 13.4-fold increase at pH 7.8. We found substantial inter-individual variation in DHAP and MG levels and an inverse relationship between GAPDH activity and MG production (R=0.57, P=0.005) in type 2 diabetes. A similar relationship between GAPDH activity and MG was observed in vivo in type 1 diabetes (R=0.29, P=0.0018). Widely varying rates of progression of diabetic complications are seen among individuals. We postulate that modification of GAPDH by environmental factors or genetic dysregulation and the resultant differences in MG production could at least partially account for this observation.

Methylglyoxal Can Modify GAPDH Activity and Structure

Abstract: The activity of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) can play an important role in regulating multiple upstream pathways relating to the development of diabetic complications. GAPDH can be modified by a number of metabolic factors, including oxidative and glycation products. To study the effect of glycation on GAPDH we have measured GAPDH structure and activity after exposure of the enzyme to the potent alpha dicarbonyl sugar methylglyoxal (MG). Rabbit GAPDH was incubated with 10‐1000 μM MG for 96 hours, and enzyme activity was measured at intervals by a spectrophotometric assay. Isoelectric focusing of purified and cellular GAPDH was performed with a PROTEAN IEF system and the bands visualized by Western blotting. The mass of glycated and native GAPDH was determined by MALDI with a Applied Biosystems Voyager System 6235. GAPDH activity (at 96 h) was decreased by 20% with 1.0 micromolar MG and showed progressively greater suppression of activity with increasing concentrations up to 1 mM, where activity was decreased by 97%. Reduction in GAPDH activity was rapidly decreasing by 69.2% by two hours with 1 mM MG. IEF showed an isoelectric point (IEP) of 8.5 for native GAPDH, while measurable changes were seen with modification by MG levels of 1 mM (IEP 7.5) and 50 μM (IEP 8.0). With MALDI, GAPDH mass increased from 36.012 kDa to 37.071 after exposure to 50 μM MG and to 40.625 following 1 mM MG. This indicates addition of 12.75 and 55.6 MG residues, respectively, to GAPDH. GAPDH can be modified by methylglyoxal intracellular concentrations close to those previously observed in vivo, with measurable changes in isoelectric point and mass. These modifications can lead to decreased enzyme activity, suggesting that conditions associated with elevated intracellular MG could modify GAPDH activity in vivo.

Early Progression of Diabetic Nephropathy Correlates With Methylglyoxal-Derived Advanced Glycation End Products

OBJECTIVE Increased advanced glycation end products (AGEs) and oxidation products (OPs) are proposed to lead to progression of diabetic nephropathy (DN). We investigated the relationship between AGEs, OPs, and progression of DN in 103 subjects with type 1 diabetes participating in the Natural History of Diabetic Nephropathy Study. RESEARCH DESIGN AND METHODS Mean age of subjects was 17.6 ± 7.4 years, and mean duration of diabetes was 8.3 ± 4.9 years. All patients were normoalbuminuric. Change in glomerular basement membrane (GBM) width from baseline to 5 years, measured using electron micrographs of renal biopsies, was our primary end point, and mesangial fractional volume was a secondary end point. Fast progressors (FPs) were defined as those in the upper quartile of GBM change, and the remaining patients were classified as slow progressors (SPs). AGEs (3-deoxyglucosone and methylglyoxal hydroimidazolones [MGHI]), carboxymethyl lysine (CML), carboxyethyl lysine (CEL), and OPs (methionine sulfoxide and 2-aminoadipic acid) were measured at year 5 by liquid chromatography/triple-quadruple mass spectroscopy on 10-K plasma filtrates. RESULTS We found that MGHI, CEL, and CML levels were significantly higher in FPs relative to SPs. No product predicted mesangial expansion. A model containing only HbA1c accounted for 4.7% of GBM width variation, with the total variability explained by the model increasing to 11.6% when MGHI, CEL, and CML were added to the regression model (7.9% increase). MGHI was a significant independent predictor of FP. Using a logistic regression model to relate each biomarker to the probability of a subject’s classification as an FP, CML, CEL, and MGHI, but not HbA1c, showed a significant relationship to the probability of FP. CONCLUSIONS The results suggest that these three major AGEs may be early indicators of progression of important DN lesions.