Immunohistochemical localization of advanced glycosylation end products in coronary atheroma and cardiac tissue in diabetes mellitus

Advanced glycosylation end products (AGEs) accumulate on long-lived extracellular matrix proteins and have been implicated in the micro- and macrovascular complications of diabetes mellitus. Within the arterial wall, AGE-modified proteins increase vascular permeability, inactivate nitric oxide activity, and induce the release of growth-promoting cytokines. Recently developed anti-AGE antibodies were used in an immunohistochemical analysis of coronary arteries obtained from type II diabetic and nondiabetic patients. High levels of AGE reactivity were observed within the atherosclerotic plaque present in vessels from selected patients with diabetes. Considered together with the pathological effects of AGEs on vascular wall homeostasis, these data support the role of advanced glycosylation in the rapidly progressive atherosclerosis associated with diabetes mellitus.     

Depletion of reactive advanced glycation endproducts from diabetic uremic sera using a lysozyme-linked matrix

Diabetic uremic sera contain excessive amounts of reactive advanced glycation endproducts (AGEs), which accelerate the vasculopathy of diabetes and end-stage renal disease. To capture in vivo-derived toxic AGEs, high affinity AGE-binding protein lysozyme (LZ) was linked to a Sepharose 4B matrix. Initial studies showed that > 80% of 125I-AGE-BSA was retained by the LZ matrix, compared with < 10% retained by a control matrix. More than 60% of AGE-lysine was captured by the LZ matrix, and the LZ-bound fraction retained immunoreactivity and cross-linking activity, but had little intrinsic fluorescence (370/440 nm). After passage through the LZ matrix, AGE levels in diabetic sera (0.37+/-0.04 U/mg) were significantly reduced to a level (0.09+/-0.01 U/mg; n = 10; P < 0. 0001) comparable with the level of normal human serum, whereas total protein absorption was < 3%. The AGE-enriched serum fraction exhibited cross-linking activity, which was completely prevented by aminoguanidine. Among numerous LZ-bound proteins in diabetic uremic sera, three major proteins "susceptible" to AGE modification were identified: the immunoglobulin G light chain, apolipoprotein J (clusterin/SP-40,40), and the complement 3b beta chain. These findings indicate that the LZ-linked AGE affinity column may serve as an efficient method for the depletion of toxic AGEs from sera, including specific AGE-modified proteins that may be linked to altered immunity, lipoprotein metabolism, and accelerated vasculopathy in renal failure patients with or without diabetes.     

Glycolaldehyde-modified low density lipoprotein leads macrophages to foam cells via the macrophage scavenger receptor

It was shown that proteins modified with advanced glycation end products (AGE) are effectively endocytosed by macrophages or macrophage-derived cells in vitro, and immunohistochemical studies involving anti-AGE antibodies demonstrated the accumulation of AGE-modified proteins (AGE-proteins) in macrophage-derived foam cells in human atherosclerotic lesions in situ, suggesting the involvement of AGE-modified LDL in the atherogenic process in vivo. To examine this suggestion, LDL was modified with glycolaldehyde, a highly reactive intermediate of the Maillard reaction. Physicochemically, glycolaldehyde-modified LDL (GA-LDL) was characterized by increases in negative charge, fluorescence intensity, and reactivity to anti-AGE antibodies, properties highly similar to those of AGE-proteins. The cellular interaction of GA-LDL with mouse peritoneal macrophages showed that GA-LDL was specifically recognized and endocytosed, followed by lysosomal degradation. The endocytic uptake of GA-LDL by these cells was competitively inhibited by acetylated LDL (acetyl-LDL), and the endocytic degradation of acetyl-LDL was also competed for by GA-LDL. Furthermore, incubation of GA-LDL with these macrophages and Chinese hamster ovary cells overexpressing the macrophage scavenger receptor (MSR), but not with peritoneal macrophages from MSR-knockout mice, led to the intracellular accumulation of cholesteryl esters (CE). These results raised the possibility that AGE-modified LDL, if available in situ, is taken up by macrophages mainly via MSR and then contributes to foam cell formation in early atherosclerotic lesions.     

Identification of potential ferric binding residues in the iron-binding protein of pathogenic Neisseria meningitidis through structure-based multiple sequence alignments

Toxic effects of hyperglycemia-induced advanced glycosylated end products (AGEs) may explain some vasculopathic complications of diabetes. Aminoguanidine, a known inhibitor of AGE formation, was administered by gavage to Sprague-Dawley streptozotocin-induced diabetic rats made azotemic by surgical reduction of renal mass. All rats became hyperglycemic. Renal ablation caused renal insufficiency, as evidenced by markedly reduced endogenous creatinine clearances at days 7 and 14. Aminoguanidine-treated rats had significantly (P < 0.04) superior survival to that of untreated azotemic diabetic rats. We infer from the extended life in a rat model of uremia in diabetic nephropathy that aminoguanidine may prove beneficial in human diabetes.     

Deposition of advanced glycation end products (AGE) and expression of the receptor for AGE in cardiovascular tissue of the diabetic rat

Advanced glycation end products (AGE) in tissues are important for the central pathological features of diabetic complication. Although AGE bind to several cell-surface sites, resulting in altered cellular functions, receptor for AGE (RAGE) appears to have a central role. We examined AGE accumulation and RAGE expression in the aorta and heart of rats with streptozotocin (STZ)-induced diabetes, 0, 4, 8, 12, 16 and 24 weeks after STZ administration. Early atherosclerotic findings in the intima and medial thinning were observed in the aorta after 16 weeks of STZ-Induced diabetes. Immunohistochemistry and microscope spectrophotometry showed that AGE deposition increased significantly in the aorta and vessels of the myocardium, depending on the period of hyperglycaemia. RAGE was expressed in the endothelial cells and vascular smooth muscle cells of all animals. The number of smooth muscle cells with RAGE immunoreactivity increased until 12 weeks after STZ injection, and then decreased in rats with diabetes between 16 and 24 weeks. On the other hand, total RAGE mRNA levels in the aorta and heart continued to increase with the duration of hyperglycaemia. Furthermore, AGE-BSA induced RAGE mRNA expression of human umbilical vein endothelial cells in vitro. Taken together, the AGE accumulation might initiate diabetic macroangiopathy through RAGE, and the increase of RAGE expression by endothelial cells could be a reason that diabetes mellitus accelerates atherosclerosis rapidly.     

Insulin enhances macrophage scavenger receptor-mediated endocytic uptake of advanced glycation end products

Hyperglycemia accelerates the formation and accumulation of advanced glycation end products (AGE) in plasma and tissue, which may cause diabetic vascular complications. We recently reported that scavenger receptors expressed by liver endothelial cells (LECs) dominantly mediate the endocytic uptake of AGE proteins from plasma, suggesting its potential role as an eliminating system for AGE proteins in vivo (Smedsrod, B., Melkko, J., Araki, N., Sano, H., and Horiuchi, S. (1997) Biochem. J. 322, 567-573). In the present study we examined the effects of insulin on macrophage scavenger receptor (MSR)-mediated endocytic uptake of AGE proteins. LECs expressing MSR showed an insulin-sensitive increase of endocytic uptake of AGE-bovine serum albumin (AGE-BSA). Next, RAW 264.7 cells expressing a high amount of MSR were overexpressed with human insulin receptor (HIR). Insulin caused a 3.7-fold increase in endocytic uptake of 125I-AGE-BSA by these cells. The effect of insulin was inhibited by wortmannin, a phosphatidylinositol-3-OH kinase (PI3 kinase) inhibitor. To examine at a molecular level the relationship between insulin signal and MSR function, Chinese hamster ovary (CHO) cells expressing a negligible level of MSR were cotransfected with both MSR and HIR. Insulin caused a 1.7-fold increase in the endocytic degradation of 125I-AGE-BSA by these cells, the effect of which was also inhibited by wortmannin and LY294002, another PI3 kinase inhibitor. Transfection of CHO cells overexpressing MSR with two HIR mutants, a kinase-deficient mutant, and another lacking the binding site for insulin receptor substrates (IRS) resulted in disappearance of the stimulatory effect of insulin on endocytic uptake of AGE proteins. The present results indicate that insulin may accelerate MSR-mediated endocytic uptake of AGE proteins through an IRS/PI3 kinase pathway.     

Lipid advanced glycosylation: pathway for lipid oxidation in vivo

To address potential mechanisms for oxidative modification of lipids in vivo, we investigated the possibility that phospholipids react directly with glucose to form advanced glycosylation end products (AGEs) that then initiate lipid oxidation. Phospholipid-linked AGEs formed readily in vitro, mimicking the absorbance, fluorescence, and immunochemical properties of AGEs that result from advanced glycosylation of proteins. Oxidation of unsaturated fatty acid residues, as assessed by reactive aldehyde formation, occurred at a rate that paralleled the rate of lipid advanced glycosylation. Aminoguanidine, an agent that prevents protein advanced glycosylation, inhibited both lipid advanced glycosylation and oxidative modification. Incubation of low density lipoprotein (LDL) with glucose produced AGE moieties that were attached to both the lipid and the apoprotein components. Oxidized LDL formed concomitantly with AGE-modified LDL. Of significance, AGE ELISA analysis of LDL specimens isolated from diabetic individuals revealed increased levels of both apoprotein- and lipid-linked AGEs when compared to specimens obtained from normal, nondiabetic controls. Circulating levels of oxidized LDL were elevated in diabetic patients and correlated significantly with lipid AGE levels. These data support the concept that AGE oxidation plays an important and perhaps primary role in initiating lipid oxidation in vivo.     

Potential benefit of inhibitors of advanced glycation end products in the progression of type II diabetes: a study with aminoguanidine in C57/BLKsJ diabetic mice

Prolonged hyperglycemia in type II diabetic patients is linked both with diabetic complications and with further impairment of glucose homeostasis, possibly due to glucose toxicity of the beta cell. While the connection between the accumulation of extracellular advanced glycation end products (AGEs) and the development of complications is well established, it has only recently been suggested that intracellular glycation may be equally adverse and could be involved in the pathogenesis of glucose toxicity in vitro. Aminoguanidine is a recognized inhibitor of the formation of both extracellular and intracellular AGEs. In this study, we show that the development of diabetes, measured by increased water intake and concomitant midday blood glucose levels in type II genetically diabetic mice, is reduced by treatment with aminoguanidine at a dosage of 500 mg/kg/d for 12 weeks in the diet. In addition, at the end of the study, aminoguanidine reduced the decline in serum and pancreatic insulin levels and the degree of pancreatic islet morphological degeneration, all of which are associated with pancreatic insufficiency following prolonged hyperglycemia in this animal model. These results suggest that AGEs may be involved in the aggravation of type II diabetes in vivo and aminoguanidine may be beneficial in its treatment.     

In vitro transcription of Myxococcus xanthus genes with RNA polymerase containing sigmaA, the major sigma factor in growing cells

Long-term incubation of proteins with glucose leads to advanced glycation end products (AGEs) with fluorescence and a brown color. We recently demonstrated immunologically the intracellular AGE accumulation in smooth muscle cell (SMC)-derived foam cells in advanced atherosclerotic lesions. To understand the mechanism of AGE accumulation in these foam cells, we have now characterized the interaction of AGE proteins with rabbit-cultured arterial SMCs. In experiments at 4 degrees C, 125I-labeled AGE-bovine serum albumin (AGE-BSA) showed a dose-dependent saturable binding to SMCs with an apparent dissociation constant (Kd) of 4.0 microg/ml. In experiments at 37 degrees C, AGE-BSA underwent receptor-mediated endocytosis and subsequent lysosomal degradation. The endocytic uptake of 125I-AGE-BSA was effectively inhibited by unlabeled AGE proteins such as AGE-BSA and AGE-hemoglobin, but not by acetylated LDL and oxidized LDL, well-known ligands for the macrophage scavenger receptor (MSR). Moreover, the binding of 125I-AGE-BSA to SMCs was affected neither by amphoterin, a ligand for one type of the AGE receptor, named RAGE, nor by 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole-hexanoic acid-BSA, a ligand for the other AGE receptors, p60 and p90. This indicates that the endocytic uptake of AGE proteins by SMCs is mediated by an AGE receptor distinct from MSR, RAGE, p60, and p90. To examine the functional role of this AGE receptor, the migratory effects of AGE-BSA on these SMCs were tested. Incubation with 1-50 microg/ml of AGE-BSA for 14 h resulted in significant dose-dependent cell migration. The AGE-BSA-induced SMC migration was chemotactic in nature and was significantly inhibited (approximately 80%) by an antibody against transforming growth factor-beta (TGF-beta), and the amount of TGF-beta secreted into the culture medium from SMC by AGE-BSA was sevenfold higher than that of control, indicating that TGF-beta is involved in the AGE-induced SMC chemotaxis. These data suggest that AGE may play a role in SMC migration in advanced atherosclerotic lesions.     

Down-regulatory effect of Mycobacterium leprae cell wall lipids on phagocytosis, oxidative respiratory burst and tumour cell killing by mouse bone marrow derived macrophages

The authors have previously demonstrated that lipids from Mycobacterium leprae cell walls inhibit macrophage functions and are endowed with anti-inflammatory properties in vivo. To investigate these observations further, the authors describe here the influence of dead M. leprae or of the lipids extracted from the cell wall of the mycobacterium, enclosed in liposomes, on the phagocytic, oxidative respiratory burst and tumouricidal ability of bone marrow derived macrophages in vitro. Dead M. leprae or its cell wall lipids abrogated the oxidative respiratory burst and phagocytic ability of mouse bone marrow derived macrophages. A dose-dependent inhibitory effect of the bacterial lipid extract on tumour cell killing by lipopolysaccharide (LPS)-activated bone marrow derived macrophages was demonstrated. However, when delipidated M. leprae was added to cultures of bone marrow derived macrophages, immune phagocytosis and superoxide production was up-regulated. Mycobacterium leprae or its lipids did not appear to be toxic to those cells assayed by the MTT (methyl thiazol tetrazolium) test. These data, added to our preceding observations, support the hypothesis that the down-regulatory activity of M. leprae wall lipids on macrophage function might be one of the evasive mechanisms of the bacterium to enable it to perpetuate itself in the host tissues.     

Advanced glycation end products in vitreous: Structural and functional implications for diabetic vitreopathy

PURPOSE: Advanced glycation end products (AGEs) form irreversible cross-links with many macromolecules and have been shown to accumulate in tissues at an accelerated rate in diabetes. In the present study, AGE formation in vitreous was examined in patients of various ages and in patients with diabetes. Ex vivo investigations were performed on bovine vitreous incubated in glucose to determine AGE formation and cross-linking of vitreous collagen. METHODS: By means of an AGE-specific enzyme-linked immunosorbent assay (ELISA), AGE formation was investigated in vitreous samples obtained after pars plana vitrectomy in patients with and without diabetes. In addition, vitreous AGEs were investigated in bovine vitreous collagen after incubation in high glucose, high glucose with aminoguanidine, or normal saline for as long as 8 weeks. AGEs and AGE cross-linking was subsequently determined by quantitative and qualitative assays. RESULTS: There was a significant correlation between AGEs and increasing age in patients without diabetes (r = 0.74). Furthermore, a comparison between age-matched diabetic and nondiabetic vitreous showed a significantly higher level of AGEs in the patients with diabetes (P < 0.005). Collagen purified from bovine vitreous incubated in 0.5 M glucose showed an increase in AGE formation when observed in dot blot analysis, immunogold labeling, and AGE ELISA. Furthermore, there was increased cross-linking of collagen in the glucose-incubated vitreous, when observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein separation. This cross-linking was effectively inhibited by coincubation with 10 mM aminoguanidine. CONCLUSIONS: This study suggests that AGEs may form in vitreous with increasing age. This process seems to be accelerated in the presence of diabetes and as a consequence of exposure to high glucose. Advanced glycation and AGE cross-linking of the vitreous collagen network may help to explain the vitreous abnormalities characteristic of diabetes.     

Specific fluorescence assay for advanced glycation end products in blood and urine of diabetic patients

Late rearrangement products that accumulate by glycation of proteins, known as advanced glycation end products (AGEs), have been implicated in the pathogenesis of complications related to diabetes. Circulating AGEs, especially in the form of a small peptide (AGE-peptide) of less than 10 kd, increase in the blood of diabetic patients with end-stage renal disease (ESRD). The aim of the study was to evaluate AGE-peptide levels by measuring AGE-specific fluorescence (excitation at 370 nm and emission at 440 nm) and to examine the relationship between AGE-peptide and diabetic nephropathy. AGE-specific fluorescence in serum and urine were examined in diabetic subjects with various levels of renal complications of varying severity: normoalbuminuria (N), microalbuminuria (Mi), macroalbuminuria (Ma), chronic renal failure (C), and hemodialysis (HD). We also assessed correlations among the AGE-peptide level and age, duration of diabetes, hemoglobin A1c (HbA1c), serum creatinine, and creatinine clearance. Serum and urine AGE-peptide levels in C and HD were significantly higher than in N, Mi, and Ma. Serum AGE-peptide levels were significantly correlated with serum creatinine (r=.866, P < .0001) and creatinine clearance (r=-.720, P < .0001) but not with duration of diabetes or age. There was a significant correlation between AGE-peptide levels measured by enzyme-linked immunosorbent assay (ELISA) and levels determined from the specific fluorescence intensity (r=.688, P < .0001). These findings suggest that renal function may play a greater role in the accumulation of AGEs than persistent hyperglycemia in diabetic patients. Measurement of AGE-specific fluorescence (ie, AGE-peptide) may serve as a simple and useful test to assess circulating AGE levels and monitor AGE excretion.     

Effect of glycated collagen on proliferation of human smooth muscle cells in vitro

While non-enzymatic glycation of long-lived tissue proteins such as collagen has been implicated in chronic complications of diabetes mellitus, its role in the aetiology of diabetic macroangiopathy has not been elucidated. To test the hypothesis that glycation of collagen abolishes the inhibitory effect of native collagen on the proliferation of human smooth muscle cells, we obtained smooth muscle cells from human gastric arteries and cultured them on dishes coated with glycated or non-glycated collagen. The proliferation of human smooth muscle cells in the presence of 10% fetal calf serum or platelet derived growth factor-BB (10 ng/ml) was inhibited by type 1 collagen coated on the dishes. Glycation of collagen with glucose 6-phosphate for 7 days abolished the growth-inhibitory effect of native collagen. Succinylation of collagen, which like glycation blocked the lysyl residues in collagen, also abolished the growth-inhibitory effect. Adhesion of human smooth muscle cells to collagen-coated dishes was not affected by glycation of collagen. Addition of glycated albumin to the medium did not affect the growth of human smooth muscle cells on plastic dishes. The inhibition of human smooth muscle cell proliferation by collagen was not reversed by the glycation of collagen in the presence of aminoguanidine. Results suggest that early glycation abolishes the inhibitory effect of collagen on human smooth muscle cell proliferation and may thus participate in the progression of macro-angiopathy in diabetes.     

GA strategy for variable selection in QSAR studies: GA-based PLS analysis of calcium channel antagonists

To clarify a possible role of advanced glycation end-products (AGE) on photoaging of human skin, the interaction between AGE and ultraviolet A light (UVA) was examined from both a biological and chemical perspective. Human dermal fibroblasts that were exposed to UVA in the presence of AGE bound with bovine serum albumin (AGE-BSA) exhibited a significant decrease of cell viability as compared to control cells, which were exposed to UVA in the absence of AGE-BSA. Further, UVA-irradiated AGE-BSA reduced nitroblue tetrazolium to its formazan, but the reaction was inhibited by addition of superoxide dismutase in the system. UVA dose-dependent formation of H2O2 in AGE-BSA was also observed. An ESR spin-trapping study revealed the generation of unstable free radicals in AGE-BSA under UVA irradiation. After addition of Fe2+ in the system, an ESR spectrum due to the formation of hydroxyl radicals was observed. On the basis of these results, the authors propose that AGE is an important factor for promoting photoaging in the skin via generation of active oxygen species involving .O2-, H2O2, and .OH.     

Effects of aminoguanidine on adhesion molecule expression of human endothelial cells

The effect of aminoguanidine (AG) on the expression of adhesion molecules on nonactivated human umbilical vein endothelial cells (HUVEC) was investigated in vitro. Nonactivated HUVEC cultivated on long-term glycated fibronectin (FN) as compared to native FN showed a significant upregulation of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1) and CD31 which could be further promoted by long-term glycated bovine serum albumin. AG, at a concentration of 0.01 mol/l, caused an upregulation of ICAM-1 of 48 +/- 17.4% in HUVEC cultivated on gelatin. In contrast, VCAM-1 and E-selectin remained unaffected. At this concentration, formation of advanced glycation end products (AGE) was inhibited by 57%, as determined immunologically, and by 50%, as verified by AGE-specific fluorescence. A hypothesis concerning the upregulation of ICAM-1 by AG as compared to VCAM-1 is proposed relating to its relative redox insensitivity. Our results demonstrate that the beneficial effect of AG in reducing the risk of accelerated development of atherosclerosis in diabetic patients by inhibiting formation of AGE on matrix proteins such as FN might be hampered by its tendency to upregulate ICAM-1 on endothelial cells.     

Moderate amplifications of the c-myc gene correlate with molecular and clinicopathological parameters in colorectal cancer

Advanced glycation end product (AGE) formation is related to hyperglycemia in diabetes but not in uremia, because plasma AGE levels do not differ between diabetic and nondiabetic hemodialysis patients. The mechanism of this phenomenon remains elusive. Previously, it was suggested that elevation of AGE levels in uremia might result from the accumulation of unknown AGE precursors. The present study evaluates the in vitro generation of pentosidine, a well identified AGE structure. Plasma samples from healthy subjects and nondiabetic hemodialysis patients were incubated under air for several weeks. Pentosidine levels were determined at intervals by HPLC assay. Pentosidine rose to a much larger extent in uremic than in control plasma. Pentosidine yield, i.e., the change in pentosidine level between 0 and 4 wk divided by 28 d, averaged 0.172 nmol/ml per d in uremic versus 0.072 nmol/ml per d in control plasma (P < 0.01). The difference in pentosidine yield between uremic and control plasma was maintained in samples ultrafiltrated through a filter with a 5000-Da cutoff value and fortified with human serum albumin (0.099 versus 0.064 nmol/ml per d; P < 0.05). Pentosidine yield was higher in pre- than in postdialysis plasma samples (0.223 versus 0.153 nmol/ml per d; P < 0.05). These results suggest that a large fraction of the pentosidine precursors accumulated in uremic plasma have a lower than 5000 Da molecular weight. Addition of aminoguanidine and OPB-9195, which inhibit the Maillard reaction, lowered pentosidine yield in both uremic and control plasma. When ultrafiltrated plasma was exposed to 2,4-dinitrophenylhydrazine, the yield of hydrazones, formed by interaction with carbonyl groups, was markedly higher in uremic than in control plasma. These observations strongly suggest that the pentosidine precursors accumulated in uremic plasma are carbonyl compounds. These precursors are unrelated to glucose or ascorbic acid, whose concentration is either normal or lowered in uremic plasma. They are also unrelated to 3-deoxyglucosone, a glucose-derived dicarbonyl compound whose level is raised in uremic plasma: Its addition to normal plasma fails to increase pentosidine yield. This study reports an elevated level of reactive carbonyl compounds ("carbonyl stress") in uremic plasma. Most have a lower than 5000 Da molecular weight and are thus partly removed by hemodialysis. Their effect on pentosidine generation can be inhibited by aminoguanidine or OPB-9195. Carbonyl stress might contribute to AGE modification of proteins and thus to clinically relevant complications of uremia.     

Advanced glycated albumin impairs protein degradation in the kidney proximal tubules cell line LLC-PK1

Advanced glycation end-products (AGEs) are assumed to play a major role in the genesis of diabetic nephropathy and other diabetic complications. We studied the potential effect of AGEs on protein turnover and lysosomal proteinase activities in LLC-PK1 cells, a pig kidney proximal tubules cell line. Advanced glycated bovine serum albumin (AGE-BSA) was used as a model of AGEs and its action was compared to that of nonglycated BSA. AGE-BSA but not BSA (50 micromol/l) induced a significant increase in cell volume (BSA: 4870.6 +/- 74.8 fl, AGE-BSA: 5718.0 +/- 20.7 fl, p<0.01). Protein degradation rate was decreased by 13.8% after 48 hrs. incubation with AGE-BSA (p<0.01) while protein synthesis increased by 19,1%, (p<0.01). After incubation with AGE-BSA but not BSA activities of lysosomal cathepsins (B, L+B and H) decreased in a time- and dose-dependent fashion. This decline was neither caused by a shift in lysosomal pH outside the optimal range for cathepsins, nor by a direct inhibitory effect of AGEs modified proteins or peptides but most probably by inhibition of cathepsin B expression as measured by RT-PCR. It is supposed that impaired protease activities participated in decreased protein breakdown and cell enlargement. For the first time our data provide the evidence that AGEs induce hypertrophy of LLC-PK1 cells due to decreased protein breakdown resulting from reduced lysosomal proteinase activities with a concomitant stimulation of protein synthesis.     

Plasma levels of pentosidine in diabetic patients: an advanced glycation end product

Nonenzymatic reactions between glucose and proteins yield advanced glycation end products (AGE) such as pentosidine. AGE accumulate in diabetic patients, alter the structure and function of tissue proteins, stimulate cellular response, and have thus been implicated in diabetic tissue damage. The present study was undertaken to assess the factors determining plasma total pentosidine level in diabetic patients and the possible relation between plasma pentosidine level and diabetic complications. In diabetic patients, including patients with renal failure, plasma pentosidine levels, assessed by HPLC assay, were correlated with serum creatinine (P < 0.0001). In patients with normal renal function, pentosidine levels were correlated with blood glucose control (hemoglobin Alc: P = 0.0028; fructoselysine: P = 0.0133), serum creatinine (P = 0.029), patient age (P = 0.0416), duration of diabetes (P = 0.0431), and total cholesterol (P = 0.0056) and LDL-cholesterol (P = 0.0208). Multiple regression analysis revealed an independent influence of hemoglobin Alc and serum creatinine on pentosidine levels (r2 = 0.216, P = 0.0026). Pentosidine levels were higher in patients with than in those without hypertension (P = 0.043) or ischemic heart diseases (P = 0.0061). No such differences were observed between patients with and without albuminuria or retinopathy. Multiple regression analysis revealed an independent influence of plasma pentosidine on the presence of hypertension (r2 = 0.129, P = 0.0382) and of plasma pentosidine and HDL-cholesterol on the presence of ischemic heart disease (r2 = 0.326, P = 0.0012). The present study demonstrated that plasma pentosidine level was significantly influenced by the quality of glycemic control and renal function. Pentosidine level was also correlated with hypertension and ischemic heart disease, and might be taken as a biomarker of diabetic cardiovascular risk.     

Tobacco smoke is a source of toxic reactive glycation products

Smokers have a significantly higher risk for developing coronary and cerebrovascular disease than nonsmokers. Advanced glycation end products (AGEs) are reactive, cross-linking moieties that form from the reaction of reducing sugars and the amino groups of proteins, lipids, and nucleic acids. AGEs circulate in high concentrations in the plasma of patients with diabetes or renal insufficiency and have been linked to the accelerated vasculopathy seen in patients with these diseases. Because the curing of tobacco takes place under conditions that could lead to the formation of glycation products, we examined whether tobacco and tobacco smoke could generate these reactive species that would increase AGE formation in vivo. Our findings show that reactive glycation products are present in aqueous extracts of tobacco and in tobacco smoke in a form that can rapidly react with proteins to form AGEs. This reaction can be inhibited by aminoguanidine, a known inhibitor of AGE formation. We have named these glycation products "glycotoxins." Like other known reducing sugars and reactive glycation products, glycotoxins form smoke, react with protein, exhibit a specific fluorescence when cross-linked to proteins, and are mutagenic. Glycotoxins are transferred to the serum proteins of human smokers. AGE-apolipoprotein B and serum AGE levels in cigarette smokers were significantly higher than those in nonsmokers. These results suggest that increased glycotoxin exposure may contribute to the increased incidence of atherosclerosis and high prevalence of cancer in smokers.