Formation of the molten globule-like state during prolonged glycation of human serum albumin

The interaction of reducing carbohydrates with proteins leads to a cascade of reactions that are known as glycation or Maillard reaction. We studied the impact of incubation of human serum albumin (HSA) with glucose, at various concentrations and incubation times, on the extent of HSA glycation and structural changes using circular dichroism (CD), fluorescence, and microviscometer techniques. The number of moles of glucose bound per mole of HSA (r), the number of reacted lysine and arginine residues, and the Amadori product formation during glycation were determined using 3-(dansylamino) phenyl boronic acid, fluorescamine, 9, 10 phenanthrenequinone, and p-nitroblue tetrazoliumchloride, respectively. The formation of advanced glycation end products (AGE) was detected using the autofluorescence characteristic of samples. We identified three stages of Maillard reaction for HSA upon incubation with the physiological level of glucose (0-630 mg/dl): the early, intermediate and late stages, which occurred after 7-14, 21, and >28 days of incubation, respectively. Structural information, Stokes radius, and 1-anilinonaphthalene-8-sulfonate (ANS) binding data indicated the formation of a molten globule-like state of HSA after 21 days of incubation with 35 mM (630 mg/dl) glucose. Thus, the extent of the Maillard reaction was influenced by the concentration of glucose and incubation time, such that longer exposure of HSA to glucose may have a more deleterious effect on its structure and especially on its half-life and turnover in the circulation. Our results suggest that in acute diabetes mellitus patients, HSA, after 21 days of glycation, passes through a molten globule-like state and may contribute to the pathogenesis of diabetes, and perhaps other diseases.     

Protective effect of Withania somnifera (Solanaceae) on collagen glycation and cross-linking

Modification of collagen such as non-enzymatic glycation and cross-linking plays an important role in diabetic complications and age-related diseases. We evaluate the effect of Withania somnifera on glucose-mediated collagen glycation and cross-linking in vitro. Extent of glycation, viscosity, collagen-linked fluorescence and pepsin solubility were assessed in different experimental procedures to investigate the effect of W. somnifera. Tail tendons obtained from rats (Rattus norvegicus) weighing 250-275 g were incubated with 50 mM glucose and 100 mg of metformin or Withania root powder or ethanolic extract of Withania under physiological conditions of temperature and pH for 30 days. Formation of advanced glycation end products (AGE) was measured by fluorescent method whereas the cross-linking of collagen was assessed by pepsin digestion and viscosity measurements. Tendon collagen incubated with glucose showed an increase in glycation, AGE and cross-linking of collagen. The collagen incubated with W. somnifera and metformin ameliorates these modifications. The ethanolic extract of Withania showed more prominent effect than Withania root powder. The activity of ethanolic extract of Withania is comparable to metformin, a known antiglycating agent. In conclusion, Withania could have therapeutic role in the prevention of glycation induced pathogenesis in diabetes mellitus and aging.     

Blockade of receptor for advanced glycation endproducts: a new target for therapeutic intervention in diabetic complications and inflammatory disorders

The glycation and oxidation of proteins/lipids leads to the generation of a new class of biologically active moieties, the advanced glycation endproducts (AGEs). Recent studies have elucidated that carboxymethyllysine (CML) adducts of proteins/lipids are a highly prevalent AGE in vivo. CML-modified adducts are signal transduction ligands of the receptor for AGE (RAGE), a member of the immunoglobulin superfamily. Importantly, CML-modified adducts accumulate in diverse settings. In addition to enhanced formation in settings of high glucose, these adducts form in inflammatory milieu. Studies performed both in vitro and in vivo have suggested that the proinflammatory/tissue destructive consequences of RAGE activation in the diabetic/inflamed environment may be markedly attenuated by blockade of the ligand-RAGE axis. Here, we will summarize the known consequences of RAGE activation in the tissues and highlight novel areas for therapeutic intervention in these disease states.     

Immunological detection of a novel advanced glycation end-product.

BACKGROUND: The advanced stage of the Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it has been proposed that the intermediates contributing to AGE formation include dicarbonyl intermediates such as glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). In the present study, we developed a novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with 3-DG or D-glucose. After immunization of rabbits, anti-AGE antisera were subjected to affinity chromatography on a Sepharose 4B column coupled with CML-BSA, or AGE-BSA created by incubation with 3-DG (AGE-6) or D-glucose (AGE-1). The AGE-Ab-6 and AGE-Ab-1 thus obtained was used to investigate AGEs in serum from diabetic patients on hemodialysis. RESULTS: Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis. This antibody specifically cross-reacted with proteins modified by 3-DG. AGE-6 was detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD, while AGE-1 was detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. CONCLUSION: This study provides new data on the pathways of AGE formation from 3-DG and methods for the immunochemical detection of AGEs. We also provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis.     

Advanced glycation end products upregulate angiogenic and pro-inflammatory cytokine production in human monocyte/macrophages

Glucose can react non-enzymatically with amino groups of, for example, proteins, to yield derivatives termed advanced glycation end products (AGE), which contribute to many chronic progressive diseases associated with microvascular complications. The study aimed to determine the effect of AGE-modified albumin on THP-1 cells and human monocyte-derived macrophages. Bovine serum albumin (BSA) or human serum albumin (HSA), modified by glucose-derived AGE, was prepared by incubation with glucose for differing periods of time. Alternatively, BSA was incubated with sodium cyanoborohydride and glyoxylic acid to produce N(epsilon)-(carboxymethyl)lysine-modified BSA (CML-BSA). Stimulation for 24h of THP-1 cells with BSA, incubated for 6-8 weeks with glucose, induced significant VEGF release. Human monocyte-derived macrophages stimulated with extensively glycated HSA also showed significant VEGF release, as well as upregulation of IL-8 production, incubation for 6h with extensively glycated HSA increased release of TNFalpha and expression of tissue factor. Finally, addition of CML-BSA resulted in significant induction of TNFalpha and VEGF release. We demonstrate that a range of different methods of glycation of BSA and HSA, including CML-BSA, resulted in the induction of VEGF, TNFalpha, IL-8 and expression of tissue factor, according to length of stimulation and different glycation products used, suggesting that AGE-induced activation of macrophages may contribute to vascular complications by regulation of angiogenic, inflammatory and pro-coagulant processes.     

Advanced glycation end products as a source of artifacts in immunoenzymatic methods

The most abundant proteins in the arteries are those of extracellular matrix, ie. collagen and elastin. Due to their long half-lifes these proteins have an increased chance to undergo glycation. The aim of this study was to determine relationship between the content of the main extracellular matrix proteins and the advanced glycation end products (AGEs) in arteries. In this study 103 fragments of aorta were analyzed by ELISA and immunobloting for the content of collagens type I, III and IV and elastin and the content of advanced glycation end-products (AGE). A negative correlation between the content of collagens type III and IV and AGE (r = ?0,258, p = 0,0122, and a weak negative correlation between collagen type III and age of the sample donor (r = 0,218, p = 0,0262) were demonstrated. This result comes as a surprise and it contradicts an intuitive assumption that with more glycation substrate, i.e. matrix proteins, more AGE products are expected. We have concluded that the results of the ELISA tests must have been influenced by the glycation. As a consequence, either modified protein molecules were not being recognized by the antibodies, or the glycation, and formation of crosslinks have blocked access of the antibodies to the antigen. It will conceal the effect of the linear dependence between the result (absorbance/densitometry) from the quantity of protein to which the antibody is directed.     

Age-dependent accumulation of N epsilon-(carboxymethyl)lysine and N epsilon-(carboxymethyl)hydroxylysine in human skin collagen

N epsilon-(Carboxymethyl)lysine (CML) is formed on oxidative cleavage of carbohydrate adducts to lysine residues in glycated proteins in vitro [Ahmed et al. (1988) J. Biol. Chem. 263, 8816-8821; Dunn et al. (1990) Biochemistry 29, 10964-10970]. We have shown that, in human lens proteins in vivo, the concentration of fructose-lysine (FL), the Amadori adduct of glucose to lysine, is constant with age, while the concentration of the oxidation product, CML, increases significantly with age [Dunn et al. (1989) Biochemistry 28, 9464-9468]. In this work we extend our studies to the analysis of human skin collagen. The extent of glycation of insoluble skin collagen was greater than that of lens proteins (4-6 mmol of FL/mol of lysine in collagen versus 1-2 mmol of FL/mol of lysine in lens proteins), consistent with the lower concentration of glucose in lens, compared to plasma. In contrast to lens, there was a slight but significant age-dependent increase in glycation of skin collagen, 33% between ages 20 and 80. As in lens protein, CML, present at only trace levels in neonatal collagen, increased significantly with age, although the amount of CML in collagen at 80 years of age, approximately 1.5 mmol of CML/mol of lysine, was less than that found in lens protein, approximately 7 mmol of CML/mol of lysine. The concentration of N epsilon-(carboxymethyl)hydroxylysine (CMhL), the product of oxidation of glycated hydroxylysine, also increased with age in collagen, in parallel with the increase in CML, from trace levels at infancy to approximately 5 mmol of CMhL/mol of hydroxylysine at age 80.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycation-induced inactivation of aspartate aminotransferase, effect of uric acid

Glycation is common posttranslational modification of proteins impairing their function, which occurs during diabetes mellitus and aging. Beside extracellular glycation of long-lived proteins, intracellular modifications of short-lived proteins by more reactive sugars like fructose are possible. The process includes free oxygen radicals (glycoxidation). In an attempt to reduce glycoxidation and formation of advanced glycation products (AGE), influence of 0.2–1.2 mM uric acid as endogenous antioxidant on glycoxidation of purified pig heart aspartate aminotransferase (AST) by 50 mM and 500 mM D-fructose in vitro was studied. Uric acid at 1.2 mM concentration reduced AST activity decrease and formation of total AGE products caused by incubation in vitro of the enzyme with sugar up to 25 days at 37 °C. The results thus support the hypothesis that uric acid has beneficial effects in controlling protein glycoxidation. The in vitro system AST-fructose proved to be a useful tool for investigation of glycation process. (Mol Cell Biochem 278: 85–92, 2005)     

Cd36, a member of the class b scavenger receptor family, as a receptor for advanced glycation end products

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. Five AGE receptors identified so far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger receptor class A types I and II). Since SRA is known to belong to the class A scavenger receptor family, and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36, although belonging to the class B scavenger receptor family, can recognize AGE proteins as ligands. This was tested at the cellular level in this study using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells). Cellular expression of CD36 was confirmed by immunoblotting and immunofluorescent microscopy using anti-CD36 antibody. Upon incubation at 37 degrees C, (125)I-AGE-bovine serum albumin (AGE-BSA) and (125)I-oxidized low density lipoprotein (LDL), an authentic ligand for CD36, were endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CD36-CHO cells, but not wild-type CHO cells. In binding experiments at 4 degrees C, (125)I-AGE-BSA exhibited specific and saturable binding to CD36-CHO cells (K(d) = 5.6 microg/ml). The endocytic uptake of (125)I-AGE-BSA by these cells was inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of oxidized LDL. Our results indicate that CD36 expressed by these cells mediates the endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major oxidized LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, these results suggest that, like oxidized LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.     

Papaverine increases human serum albumin glycation

Glycation is a non-enzymatic reaction that is initiated by the primary addition of sugars to amino groups of proteins. In the early phase of glycation, the synthesis of intermediates leads to formation of Amadori compounds. In the last phase, advanced glycation end products (AGE) are irreversibly formed following a complex cascade of reactions. It has recently been shown that glycation also affects diabetes-related complications and Alzheimer’s disease. In this study, human serum albumin at a concentration of 10 mg/ml was incubated in PBS with 40 mM of glucose and in different concentrations of papaverine (25, 100, 250, 500 μM) for 42 days at 37 °C. HSA with no additives as well as with glucose 40 mM were incubated as a control and as a glycated sample, respectively. Following the incubation, the samples were prepared for circular dichroism, fluorescence and absorbance techniques. The results showed that in presence of papaverine and glucose, the glycation of HSA increased notably compared with the glycated sample. In conclusion, in this work, we showed that papaverine affects HSA and increases its glycation level.     

Chemical modification of muscle protein in diabetes

Levels of glycation (fructose-lysine, FL) and advanced glycoxidation and lipoxidation end-products (AGE/ALEs) were measured in total skeletal (gastrocnemius) muscle and myofibril protein and compared to levels of the same compounds in insoluble skin collagen of control and diabetic rats. Levels of FL in total muscle and myofibril protein were 3-5% the level of FL in skin collagen. The AGE/ALEs, N(epsilon)-(carboxymethyl)lysine (CML) and N(epsilon)-(carboxyethyl)lysine, were also significantly lower in total muscle and myofibril protein, approximately 25% of levels in skin collagen. The newly described sulfhydryl AGE/ALE, S-(carboxymethyl)cysteine (CMC), was also measured in muscle; levels of CMC were comparable to those of CML and increased similarly in response to diabetes. Although FL and AGE/ALEs increased in muscle protein in diabetes, the relative increase was less than that seen in skin collagen. These data indicate that muscle protein is partially protected against the increase in both glycation and AGE/ALE formation in diabetes.     

Lysozyme enhances renal excretion of advanced glycation endproducts in vivo and suppresses adverse age-mediated cellular effects in vitro: a potential AGE sequestration therapy for diabetic nephropathy?

BACKGROUND: Lysozyme (LZ), a host-defense protein, contains an 18 amino-acid domain with high affinity binding for sugar-derived proteins or lipids, called advanced glycation endproducts (AGE), that are implicated in diabetes- and age-dependent complications (DC). MATERIALS AND METHODS: A) The effects of LZ on AGE- removal were tested in vivo. LZ was injected (200 ug/day, i.p., X2 weeks) in non-obese diabetic (NOD), db/db (+/+) mice, and non-diabetic, AGE-infused Sprague-Dawley rats. B) LZ: AGE interactions with macrophage-like T1B-183 cells (Mf) and mesangial cells (MC) were tested in vitro. RESULTS: A) In NOD mice, LZ reduced the elevated basal serum AGE (sAGE) (p < 0.05), enhanced urinary AGE (uAGE) excretion by approximately 2-fold (p < 0.01), while it reduced albuminuria (UA), p < 0.005. In db/db mice, LZ infusion also reduced the elevated sAGE (p < 0.05), doubled uAGE excretion (p < 0.05), and decreased UA (p < 0.01). In addition, LZ maintained normal sAGE in normal rats infused with AGE-BSA, as it doubled the urinary AGE (uAGE) clearance (p < 0.01). B) LZ stimulated the uptake and degradation of (125) I-labeled AGE-BSA and (25) I-human serum AGE by Mf, while suppressing AGE-induced TNFalpha and IGF-I production. In MC, LZ suppressed the AGE-promoted PDGF-B, alpha1 type IV collagen, and tenascin mRNA levels, and restored the AGE-suppressed expression and activity of MMP-9, but not MMP-2. CONCLUSION: LZ may act to: a) accelerate renal in-vivo AGE clearance, b) suppress macrophage and mesangial cell- specific gene activation in vitro, and c) improve albuminuria due to diabetes. These data suggest that LZ by sequestering AGEs may protect against diabetic renal damage.     

Leptin and connective tissue growth factor in advanced glycation end-product-induced effects in NRK-49F cells

Previously, we showed that Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Leptin is a JAK2-activating cytokine via the long form leptin receptor (Ob-Rb). Leptin and connective tissue growth factor (CTGF) may be involved in renal fibrosis. However, the relationship between leptin and CTGF in terms of AGE-induced effects remains unknown. Thus, the effects of AGE (150 microg/ml) and leptin on mitogenesis, CTGF and collagen expression in NRK-49F cells were determined. We found that leptin and AGE increased mitogenesis and type I collagen protein expression at 3 and 7 days, respectively. AGE increased leptin mRNA and protein expression at 2-3 days. AGE increased CTGF mRNA and protein expression at 3-5 days. AG-490 (JAK2 inhibitor) abrogated AGE-induced leptin mRNA and protein expression at 2-3 days. AG-490 and Ob-Rb anti-sense oligodeoxynucleotides (ODN) abrogated AGE-induced CTGF mRNA and protein expression at 3-5 days. AG-490 and CTGF anti-sense ODN abrogated AGE-induced mitogenesis and collagen protein expression at 7 days. Additionally, leptin dose (0.2-1 microg/ml) and time (1-2 days)-dependently increased CTGF protein expression. AG-490 abrogated leptin (1 microg/ml)-induced CTGF protein expression at 2 days. AG-490 and CTGF anti-sense ODN abrogated leptin-induced mitogenesis and collagen protein expression at 3 days. We concluded that AGE induced JAK2 to increase leptin while leptin induced JAK2 to increase CTGF-induced mitogenesis and type I collagen protein expression in NRK-49F cells. Additionally, AGE-induced mitogenesis and type I collagen protein expression were dependent on leptin-induced CTGF.     

Prevention and reversible solubilization of advanced glycation and products (AGE) by organic germanium compounds as derivatives of amino acids

Summary The amino-carbonyl reaction (The Maillard reaction) of bovine lens crystallin, serum albumin or skin collagen with glucose was investigated to find effective means to prevent the formation of Advanced Glycation End Products (AGE) and induce the reversible solubilization of polymerized glycated proteins. The organic germanium compounds (Ge-132, 373, 385), derivatives of amino acids containing germanium as the linker of framework, were combined by the box titration method to determine the dose that would be most effective, compared with Aminoguanidine-HCl (AMG),-tocopherol (VE), and pirenoxine (Catalin-K, CK). Although AMG suppressed the formation of AGE, effective concentrations were higher than 20 mM. Ge-385, when administered by itself at a low dose, induced the reversible solubilization of AGE made from crystallin, and albumin. The addition of any two reagents such as AMG, VE, CK and Ge-132 or 385 together to proteins lessened the effective range, and the peaks of smaller molecules in the profiles of HPLC and PAGE were quite remarkable. Examination was made of the effects of Ge-132 on the eyes of SAM mice, which show senescence accelerated cataracts at a relatively young age. The prevention of cataract-genesis and induction of reversible transparency of turbid lenses became evident following the administration of Ge-132 to the eyes 4 times a day. The mode of action of organic germanium compounds was demonstrated quite capable of disconnecting the sugar-parts from AGE by decarbonylation, resulting in the formation of glucosone and amino residues, and further leading subsequently to fewer AGE.Abbreviations used in this paper: BLC bovine lens crystallin; BSA bovine serum albumin; AsCol acid soluble bovine skin collagen type III; AGE advanced glycation end products; Ge-132 2-Carboxyethylgermanium sesquioxide, Ge-373 2-Carboxy-2-amino-6-phenyl germanium sesquioxide; Ge-385 2-Carboxy-ethyl-2-aminogermanium sesquioxide; AMG or AG aminoguanidine-HCl; V. E. vitamin E or-tocopherol; CK 1-Hydroxy-5-oxo-5H-pyrido [3, 2-a] phenoxazine-3-carboxylic acid or catalin-K or pirenoxine; PACE polyacrylamide gel electrophoresis; SAM senescence accelerated mouse; HPLC high pressure liquid chromatography; SDS sodium laurylsulfate; FT fructose-p-toluidine.     

Glucose Modification of Human Serum Albumin: A Structural Study

Structural changes associated with the exposure of human serum albumin (HSA) to glucose with or without the presence of Cu (II) have been characterized using a bank of methods for structural analysis including circular dichroism (CD), amino acid analysis (AAA), fluorescence measurements, SDS-PAGE, and boronate binding (which is a measure of Amadori product formation). We show that in the short-term (10 d) incubation mixtures, HSA is resistant to Cu (II)-mediated oxidative damage and that the early products of glycation of HSA had minimal effects on the folded structure. Amino acid analysis showed that there was no formation of advanced glycation endproducts (AGE), which can be measured by loss of lysine. This remained the case in longer term incubation of HSA (56 d) in the hyperglycemic concentration range (5–25 mM glucose) despite increased levels of Amadori product (60% boronate binding) and the formation of glycophore (Excitation 350, Emission 425). At high, nonphysiological concentrations (100 mM and 500 mM) of glucose, glycophore formation increased and 3 and 11 mol Lysine-glucose adduct/mol HSA were converted to AGE, respectively. This was accompanied by increased damage to tryptophan and protein-protein crosslinking but only minor tertiary structural change. In the presence of Cu (II), however, AGE formation was accompanied by extensive damage to histidine and tryptophan side chains, main chain fragmentation, and loss of both secondary and tertiary structure. Thus, changes in structure appear to be the result of oxidation as opposed to glycation, per se. © 1997 Elsevier Science Inc.     

Fructose-mediated damage to lens alpha-crystallin: prevention by pyruvate

Post-translational modifications in lens crystallins due to glycation and oxidation have been suggested to play a significant role in the development of cataracts associated with aging and diabetes. We have previously shown that alpha-keto acids, like pyruvate, can protect the lens against oxidation. We hypothesize that they can also prevent the glycation of proteins competitively by forming a Schiff base between their free keto groups and the free -NH(2) groups of protein as well as subsequently inhibit the oxidative conversion of the initial glycation product to advanced glycation end products (AGE). The purpose of this study was to investigate these possibilities using purified crystallins. The crystallins isolated from bovine lenses were incubated with fructose in the absence and presence of pyruvate. The post-incubation mixtures were analyzed for fructose binding to the crystallins, AGE formation, and the generation of high molecular weight (HMW) proteins. In parallel experiments, the keto acid was replaced by catalase, superoxide dismutase (SOD), or diethylene triaminepentaacetic acid (DTPA). This was done to ascertain oxidative mode of pyruvate effects. Interestingly, the glycation and consequent formation of AGE from alpha-crystallin was more pronounced than from beta-, and gamma-crystallins. The changes in the crystallins brought about by incubation with fructose were prevented by pyruvate. Catalase, SOD, and DTPA were also effective. The results suggest that pyruvate prevents against fructose-mediated changes by inhibiting the initial glycation reaction as well as the conversion of the initial glycated product to AGE. Hence it is effective in early as well as late phases of the reactions associated with the formation of HMW crystallin aggregates.     

Advanced glycation end-product-induced mitogenesis and collagen production are dependent on angiotensin II and connective tissue growth factor in NRK-49F cells

Diabetic nephropathy (DN) is characterized by glomerulopathy and tubulointerstitial expansion followed by renal fibrosis. Angiotensin II (Ang II) and connective tissue growth factor (CTGF) are involved in the pathogenesis of DN, while Janus kinase 2 (JAK2) is important in advanced glycation end-product (AGE)-induced effects in renal interstitial (NRK-49F) fibroblasts. Thus, we studied the role of Ang II, CTGF, and JAK2 in AGE-induced effects in NRK-49F cells. We found that AGE (150 microg/ml) increased mitogenesis and type I collagen production at 7 days while Ang II (10(-7)M) increased mitogenesis and type I collagen production at 3 days. We also found that AGE (150 microg/ml) increased angiotensinogen protein at 2 days, which was attenuated by AG-490 (a JAK2 inhibitor). AGE (150 microg/ml) increased CTGF mRNA and protein expression at 3 and 5 days, respectively. Ang II (10(-7)M) increased CTGF mRNA and protein expression at 1 and 2 days, respectively, which were attenuated by AG-490. Moreover, losartan (a type I angiotensin receptor blocker) and captopril (an angiotensin converting enzyme inhibitor) attenuated AGE-induced CTGF mRNA/protein expression while attenuating AGE-induced mitogenesis and type I collagen production. AG-490 and CTGF antisense (but not sense) oligodeoxynucleotide (ODN) attenuated Ang II (10(-7)M) and AGE-induced mitogenesis and type I collagen production at 3 and 7 days, respectively. We concluded that AGE (150 microg/ml)-induced mitogenesis and type I collagen production are dependent on the Ang II-JAK2-CTGF pathway in NRK-49F cells. Moreover, Ang II-induced mitogenesis and type I collagen production are dependent on the JAK2-CTGF pathway.     

Regulation of the Werner helicase through a direct interaction with a subunit of protein kinase A

Advanced glycation end products (AGE) are known to serve as ligands for the scavenger receptors such as SR-A, CD36 and SR-BI. In the current study, we examined whether AGE is recognized by lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Cellular binding experiments revealed that AGE-bovine serum albumin (AGE-BSA) showed the specific binding to CHO cells overexpressing bovine LOX-1 (BLOX-1), which was effectively suppressed by an anti-BLOX-1 antibody. Cultured bovine aortic endothelial cells also showed the specific binding for AGE-BSA, which was suppressed by 67% by the anti-BLOX-1 antibody. Thus, LOX-1 is identified as a novel endothelial receptor for AGE.     

Scavenger receptor class B type I-mediated reverse cholesterol transport is inhibited by advanced glycation end products

Cellular interactions of advanced glycation end products (AGE) are mediated by AGE receptors. We demonstrated previously that class A scavenger receptor types I and II (SR-A) and CD36, a member of class B scavenger receptor family, serve as the AGE receptors. In this study, we investigated whether scavenger receptor class B type I (SR-BI), another receptor belonging to class B scavenger receptor family, was also an AGE receptor. We used Chinese hamster ovary (CHO) cells overexpressed hamster SR-BI (CHO-SR-BI cells). (125)I-AGE-bovine serum albumin (AGE-BSA) was endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CHO-SR-BI cells. (125)I-AGE-BSA exhibited saturable binding to CHO-SR-BI cells (K(d) = 8.3 microg/ml). Endocytic uptake of (125)I-AGE-BSA by CHO-SR-BI cells was completely inhibited by oxidized low density lipoprotein (LDL) and acetylated LDL, whereas LDL exerted only a weak inhibitory effect (<20%). Cross-competition experiments showed that AGE-BSA had no effect on HDL binding to these cells and vice versa. Interestingly, however, SR-BI-mediated selective uptake of HDL-CE was completely inhibited by AGE-BSA in a dose-dependent manner (IC(50) <10 microg/ml). Furthermore, AGE-BSA partially inhibited (by <30%) the selective uptake of HDL-CE in human hepatocarcinoma HepG2 cells (IC(50) <30 microg/ml). In addition, [(3)H]cholesterol efflux from CHO-SR-BI cells to HDL was significantly inhibited by AGE-BSA in a dose-dependent manner (IC(50) <30 microg/ml). Our results indicate that AGE proteins, as ligands for SR-BI, effectively inhibit both SR-BI-mediated selective uptake of HDL-CE and cholesterol efflux from peripheral cells to HDL, suggesting that AGE proteins might modulate SR-BI-mediated cholesterol metabolism in vivo.