Scott K. Howell

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.

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.

Transglycation - a potential new mechanism for deglycation of Schiff's bases.

Abstract: Nonenzymatic glycation is believed to play a major role in the development of diabetic complications. Over the past several years we and others have shown that in cells this nonenzymatic process can be reversed by an ATP‐dependent reaction catalyzed by fructosamine‐3‐kinase (FN3K) and possibly by its isozyme, fructosamine‐3‐kinase‐related protein (FN3KRP). In this study we provide the first evidence that this FN3K‐dependent deglycation, acting on the Amadori products, is complemented by another deglycation process operating on the very first product of nonenzymatic glycation, glucosylamines (Schiffs bases). We postulate that the first step in this Schiffs‐base deglycation process occurs by transfer of the sugar moiety from macromolecule‐bound glucosylamine to one of the low‐molecular weight intracellular nucleophiles—in particular, glutathione. We term this reaction transglycation, and in this study we demonstrate that it occurs readily and spontaneously in vitro. We further propose that one of the spontaneously formed glucose‐glutathione adduct(s) is subsequently removed from cells by a multidrug‐resistance pump (MRP, MDR‐protein, ATP‐binding‐cassette protein), metabolized, and excreted in urine. In support of this latter contention, we show that at least one transglycation product, glucose‐cysteine, is found in human urine and that its concentrations are increased in diabetes.

Protein depletion and replenishment in mice: different roles of muscle and liver

Fully grown male CD-1 mice, fed a protein-free diet for 3 days, received 1 g of starch with or without 300 mg casein by intragastric intubation. We surveyed the acute effects of these nutrients on protein synthesis in all tissues (by extrapolating to infinity the incorporation of radioactive leucine after its injection in massive doses) and protein degradation in skeletal muscle and liver (by the accumulation of bestatin-induced peptide intermediates). Muscle proteolysis was the major source of N during depletion. Compared with postabsorptive animals, starch suppressed muscle protein loss (synthesis +21%, degradation -24%, P < 0.01) and stimulated hepatic proteolysis (degradation +28%, P < 0.01). Addition of casein to the starch was anabolic in liver (synthesis +41%, degradation -33%, P < 0.01), gastrointestinal tract, pancreas, and skin (synthesis +38, +69 and +38%, respectively, P < 0.01) but had no effect on muscle. Protein turnover proved uniquely sensitive to the dietary supply of carbohydrates in muscle and to the endogenous or exogenous supply of amino acids in liver.

Nonenzymatic glycation/enzymatic deglycation: a novel hypothesis on the etiology of diabetic complications

Abstract Nonenzymatic glycation appears to play a major role in the development of diabetic complications. Key early intermediates in the nonenzymatic glycation cascade are glucoselysines (GL) and fructoselysines (FL). In 1997, we proposed that intracellular nonenzymatic glycation is controlled by an enzymatic deglycation process catalyzed by fructosamine-3-kinase (FN3K). FN3K phosphorylates FL to fructoselysine-3-phosphate (FL3P), which then decomposes regenerating an unmodified lysine residue. With the recent purification, sequencing and cloning of FN3K [Diabetes 50 (2001) 2139; Diabetes 49 (2000) 1627] the concept of enzymatic deglycation has received considerable experimental support. In this paper, we provide evidence of enzymatic deglycation activity in vivo involving both FN3K-dependent and FN3K-independent mechanisms. Based on these data, we propose a new theory on the development of diabetic complications: the nonenzymatic glycation/enzymatic deglycation hypothesis. We postulate that enzymatic deglycation is an essential defense system in mammalian cells. In diabetes, this system is stressed and often overwhelmed by episodes of extreme hyperglycemia during which nonenzymatic glycation proceeds unchecked. This results in cumulative damage to essential proteins and leads ultimately to cellular dysfunction. Susceptibility to diabetic complications is thus a consequence of two factors: severity of the hyperglycemic stress and ability of the deglycating system to cope with that stress.

Advanced Glycation End Products, Oxidation Products, and the Extent of Atherosclerosis During the VA Diabetes Trial and Follow-up Study*

OBJECTIVE To determine whether plasma levels of advanced glycation end products and oxidation products play a role in the development of atherosclerosis in patients with type 2 diabetes (T2D) over nearly 10 years of the VA Diabetes Trial and Follow-up Study. RESEARCH DESIGN AND METHODS Baseline plasma levels of methylglyoxal hydroimidazolone, Nε-carboxymethyl lysine, Nε-carboxyethyl lysine (CEL), 3-deoxyglucosone hydroimidazolone and glyoxal hydroimidazolone (G-H1), 2-aminoadipic acid (2-AAA), and methionine sulfoxide were measured in a total of 411 participants, who underwent ultrasound assessment of carotid intima-media thickness (CIMT), and computed tomography scanning of coronary artery calcification (CAC) and abdominal aortic artery calcification (AAC) after an average of 10 years of follow-up. RESULTS In risk factor–adjusted multivariable regression models, G-H1 was associated with the extent of CIMT and CAC. In addition, 2-AAA was strongly associated with the extent of CAC, and CEL was strongly associated with the extent of AAC. The combination of specific advanced glycation end products and oxidation products (G-H1 and 2-AAA) was strongly associated with all measures of subclinical atherosclerosis. CONCLUSIONS Specific advanced glycation end products and metabolic oxidation products are associated with the severity of subclinical atherosclerosis over the long term and may play an important role in the “negative metabolic memory” of macrovascular complications in people with long-standing T2D.

Advanced Glycation End Products Predict Loss of Renal Function and Correlate With Lesions of Diabetic Kidney Disease in American Indians With Type 2 Diabetes

We examined associations of advanced glycation end products (AGEs) with renal function loss (RFL) and its structural determinants in American Indians with type 2 diabetes. Data were from a 6-year clinical trial that assessed renoprotective efficacy of losartan. Participants remained under observation after the trial concluded. Glomerular filtration rate (GFR) was measured annually. Kidney biopsies were performed at the end of the trial. Five AGEs were measured in serum collected at enrollment and at kidney biopsy. RFL was defined as ≥40% decline of measured GFR from baseline. Of 168 participants (mean baseline age 41 years, HbA1c 9.2%, GFR 164 mL/min, and albumin-to-creatinine ratio 31 mg/g), 104 reached the RFL end point during median follow-up of 8.0 years. After multivariable adjustment, each doubling of carboxyethyl lysine (hazard ratio [HR] 1.60 [95% CI 1.08–2.37]) or methylglyoxal hydroimidazolone (HR 1.30 [95% CI 1.02–1.65]) concentration was associated with RFL. Carboxyethyl lysine, carboxymethyl lysine, and methylglyoxal hydroimidazolone correlated positively with cortical interstitial fractional volume (partial r = 0.23, P = 0.03; partial r = 0.25, P = 0.02; and partial r = 0.31, P = 0.003, respectively). Glyoxyl hydroimidazolone and methylglyoxal hydroimidazolone correlated negatively with total filtration surface per glomerulus (partial r = −0.26, P = 0.01; and partial r = −0.21, P = 0.046, respectively). AGEs improve prediction of RFL and its major structural correlates.

Advanced Glycation End Products, Oxidation Products, and Incident Cardiovascular Events in Patients With Type 2 Diabetes

OBJECTIVE The goal of this study was to determine whether plasma levels of advanced glycation end products (AGE) and oxidation products (OP) predict the incidence of cardiovascular disease (CVD) in type 2 diabetes. RESEARCH DESIGN AND METHODS Five specific AGE (methylglyoxal hydroimidazolone, carboxymethyl lysine, carboxyethyl lysine, 3-deoxyglucosone hydroimidazolone, and glyoxal hydroimidazolone) and two OP (2-aminoadipic acid and methionine sulfoxide [MetSO]) were measured at baseline in two intensive glucose-lowering studies: 1) a subcohort of the Veterans Affairs Diabetes Trial (VADT) (n = 445) and 2) a nested case-control subgroup from the Action to Control Cardiovascular Risk in Diabetes (ACCORD) study (n = 271). RESULTS Increased levels of several AGE and OP were associated with older age, decreased kidney function, previous CVD, and longer diabetes duration, but not with hemoglobin A1c. In the VADT, increased risk of incident CVD events (n = 107) was associated with lower MetSO after adjusting for age, race/ethnicity, sex, prior CVD event, kidney function, treatment assignment, and diabetes duration (hazard ratio [HR] 0.53; 95% CI 0.28–0.99; P = 0.047). Individuals with both low MetSO and high 3-deoxyglucosone hydroimidazolone concentrations were at highest risk for CVD (HR 1.70; P = 0.01). In the ACCORD study, those with incident CVD events (n = 136) had lower MetSO (by 14%; P = 0.007) and higher glyoxal hydroimidazolone and carboxymethyl lysine (by 18% and 15%, respectively; P = 0.04 for both); however, only the difference in MetSO remained significant after adjustment for prior CVD event (P = 0.002). CONCLUSIONS Lower levels of MetSO and higher levels of select AGE are associated with increased incident CVD and may help account for the limited benefit of intensive glucose lowering in type 2 diabetes.