Biomedical subjects

Paul J Beisswenger

Publications and source records attributed to Paul J Beisswenger.

8 recordsLinked to original sources

PubMed2005

Methylglyoxal can modify GAPDH activity and structure.

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 microM 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 microM (IEP 8.0). With MALDI, GAPDH mass increased from 36.012 kDa to 37.071 after exposure to 50 microM 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.

Diabetes Mellitus↗

PubMed2005

Ketosis leads to increased methylglyoxal production on the Atkins diet.

In the popular and widely used Atkins diet, the body burns fat as its main fuel. This process produces ketosis and hence increased levels of beta-hydroxybutyrate (BOB) acetoacetate (AcAc) and its by-products acetone and acetol. These products are potential precursors of the glycotoxin methylglyoxal. Since methylglyoxal and its byproducts are recognized as a significant cause of blood vessel and tissue damage, we measured methylglyoxal, acetone, and acetol in subjects on the Atkins diet. We found that by 14-28 days, methylghyoxal levels rose 1.67-fold (P = 0.039) and acetol and acetone levels increased 2.7- and 6.12-fold, respectively (P = 0.012 and 0.028). Samples from subjects with ketosis showed even greater increases in methylglyoxal (2.12-fold), as well as acetol and acetone, which increased 4.19- and 7.9-fold, respectively; while no changes were seen in samples from noncompliant, nonketotic subjects. The increase in methylglyoxal implies that potential tissue and vascular damage can occur on the Atkins diet and should be considered when choosing a weight-loss program.

Adult↗

PubMed2005

Some clues as to the regulation, expression, function, and distribution of fructosamine-3-kinase and fructosamine-3-kinase-related protein.

Fructosamine-3-kinase (FN3K) and the more recently discovered fructosamine-3-kinase-related protein (FN3KRP) appear to protect proteins from nonenzymatic glycation. To gain a better understanding of these enzymes we performed a series of investigations including (1) in silico comparisons of their promoters; (2) real-time PCR analysis of their expression in human tissues; (3) effects of hyperglycemia, interleukin-1beta (IL-1beta), and nuclear factor kappa-B (NFkappaB) activation on their mRNA levels; (4) effects of small interfering RNA (siRNA) suppression of FN3K expression (knockdown) in cultured cells and (5) search of FN3K and FN3KRP homologs in available genomic and EST (expressed sequence tag) databases. Our results indicate that (1) both FN3K and FN3KRP promoters are TATA-less and CAAT-less and contain several homologous CpG islands and Sp1 binding sites. (2) Both genes are expressed in all human tissue examined, with FN3K showing significantly higher levels in tissues susceptible to nonenzymatic glycation and diabetic complications. (3) Treatment of fibroblasts with high glucose, IL-1beta, and activation of NFkappaB does not affect the expression of either FN3K or FN3KRP. (4) Knockdown of FN3K in cultured cells inhibits or arrests their growth. (5) FN3K-like genes are widely distributed in nature, with the notable exception of insects and yeasts. These data suggest that FN3K and FN3KRP are constitutive "housekeeping" genes and that they play an important role in cell metabolism, possibly as deglycating enzymes.

Animals↗

PubMed2005

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

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 (Schiff's bases). We postulate that the first step in this Schiff's-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.

Erythrocytes↗

PubMed2005

Susceptibility to diabetic nephropathy is related to dicarbonyl and oxidative stress.

Dicarbonyl and oxidative stress may play important roles in the development of diabetes complications, and their response to hyperglycemia could determine individual susceptibility to diabetic nephropathy. This study examines the relationship of methylglyoxal, 3-deoxyglucosone (3DG), and oxidative stress levels to diabetic nephropathy risk in three populations with diabetes. All subjects in the Overt Nephropathy Progressor/Nonprogressor (ONPN) cohort (n = 14), the Natural History of Diabetic Nephropathy study (NHS) cohort (n = 110), and the Pima Indian cohort (n = 45) were evaluated for clinical nephropathy, while renal structural measures of fractional mesangial volume [Vv(Mes/glom)] and glomerular basement membrane (GBM) width were determined by electron microscopy morphometry in the NHS and Pima Indian cohorts. Methylglyoxal and 3DG levels reflected dicarbonyl stress, while reduced glutathione (GSH) and urine 8-isoprostane (8-IP) measured oxidative stress. Cross-sectional measures of methylglyoxal production by red blood cells incubated in 30 mmol/l glucose were increased in nephropathy progressors relative to nonprogressors in the ONPN (P = 0.027) and also reflected 5-year GBM thickening in the NHS cohort (P = 0.04). As nephropathy progressed in the NHS cohort, in vivo levels of methylglyoxal (P = 0.036), 3DG (P = 0.004), and oxidative stress (8-IP, P = 0.007 and GSH, P = 0.005) were seen, while increased methylglyoxal levels occurred as nephropathy progressed (P = 0.0016) in the type 2 Pima Indian cohort. Decreased glyceraldehyde-3-phosphate dehydrogenase activity also correlated with increased methylglyoxal levels (P = 0.003) in the NHS cohort. In conclusion, progression of diabetic nephropathy is significantly related to elevated dicarbonyl stress and possibly related to oxidative stress in three separate populations, suggesting that these factors play a role in determining individual susceptibility.

Adolescent↗

PubMed2005

Glycated and oxidized protein degradation products are indicators of fasting and postprandial hyperglycemia in diabetes.

OBJECTIVE: To assess the relative importance of fasting and postprandial hyperglycemia to vascular dysfunction in diabetes, we have measured indicators of glycation, oxidative and nitrosative stress in subjects with type 1 diabetes, and different postprandial glucose patterns. RESEARCH DESIGN AND METHODS: Plasma and urinary levels of specific arginine- and lysine-derived advanced glycation end products, as well as oxidative and nitrosative products, were measured by liquid chromatography with triple quadrupole mass spectrometric detection (LC-MS/MS) after 2 months of treatment with insulin lispro or human regular insulin in 21 subjects participating in a cross-over study. Hb-bound early glycation (Amadori) products were also measured after each treatment period by high-performance liquid chromatography (fructosyl-valine Hb or HbA1c [A1C]:Diamat) and fructosyl-lysine Hb by LC-MS/MS (A1C:fructosyl-lysine). RESULTS: In diabetic patients, the concentrations of protein glycation and oxidation-free adducts increased up to 10-fold, while urinary excretion increased up to 15-fold. Decreasing postprandial hyperglycemia with lispro gave 10-20% decreases of the major free glycation adducts, hydroimidazolones derived from methylglyoxal and 3-deoxyglucosone, and glyoxal-derived Nepsilon-carboxymethyl-lysine. No differences were observed in A1C:Diamat or A1C:fructosyl-lysine with lispro or regular insulin therapy in spite of significant decreases in postprandial glycemia with lispro. CONCLUSIONS: We conclude that the profound increases in proteolytic products of proteins modified by advanced glycation endproducts in diabetic patients are responsive to changes in mean hyperglycemia and also show responses to changes in postprandial hyperglycemia.

Adult↗

PubMed2004

The expression of the genes for fructosamine-3-kinase and fructosamine-3-kinase-related protein appears to be constitutive and unaffected by environmental signals.

Fructosamine-3-kinase (FN3K) and the more recently discovered fructosamine-3-kinase related protein (FN3KRP) appear to protect proteins from nonenzymatic glycation. To elucidate the patterns of transcriptional regulation of these two genes, we performed in silico comparisons of their promoters along with real-time PCR assays of their expression in a variety of human tissues. Both promoters were TATA-less and CAAT-less, and contained several homologous CpG islands and Sp1 binding sites. The genes were expressed in all human tissues examined, with FN3K showing significantly higher levels in organs susceptible to nonenzymatic glycation and diabetic complications. Cultured fibroblasts treated with conditions mimicking the hormonal and biochemical profile of the diabetic state showed no changes in FN3K and FN3KRP expression relative to untreated cells. These data suggest that FN3K and FN3KRP act as protein repair enzymes and are expressed constitutively in human cells independently of some of the variables altered in the diabetic state.

Amino Acid Sequence↗

PubMed2003

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.

Adult↗