Early Glycation Products Research Articles

Oxidative damage of vascular smooth muscle cells by the glycated protein–cupric ion system

To clarify the mechanism of cellular injury through the nonenzymatic reaction of glucose with proteins, we studied the cytotoxic effect of glycated bovine serum albumin on cultured smooth muscle cells in the presence of cupric ion. Glycated proteins were prepared by incubating bovine serum albumin with 0.5 M d-glucose in 0.3 M sodium phosphate buffer at 37°C for 2, 4 and 16 weeks (g-BSA-2, g-BSA-4 and g-BSA-16, respectively). Early glycation products, such as fructosamine, were formed more than two weeks after incubation. However, the immunoreactivity of glycated proteins to anti-AGE antibody was 12-fold higher in g-BSA-16 than in g-BSA-2. Both g-BSA-2 and g-BSA-16 showed a concentration-dependent cytotoxicity in smooth muscle cells in the presence of 80 μM cupric ion by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) dye reduction assay and dye exclusion test. Flow cytometry and spectrofluorophotometry using dihydrorhodamine 123 showed that the extracellular generation of oxidants was dose-dependently enhanced with increasing concentrations of g-BSA-2 or g-BSA-16 in the presence of cupric ion. However, no difference was observed in the intracellular generation of oxidants between the presence and absence of glycated proteins by flow cytometry using 2′, 7′-dichlorofluorescein diacetate. Cytotoxicity and oxidant generation were prevented by catalase and tiron, but not by superoxide dismutase or mannitol, a hydroxyl radical scavenger. These results indicate that smooth muscle cells may be damaged by reactive oxygen species which are produced extracellularly by the interaction with the early glycation products and cupric ion, and suggest that hydrogen peroxide may be a candidate for reactive oxygen species which contribute to such oxidative damage of smooth muscle cells.

Non-enzymatic glycosylation of alkaline phosphatase alters its biological properties.

Hyperglycaemia in poorly controlled diabetic patients induces non-enzymatic glycosylation (glycation) of proteins, altering their structure and physiological bioactivity. Alkaline phosphatase (ALP) is a membrane-bound exoenzyme which faces the extracellular compartment. We have investigated the glycation of intestinal alkaline phosphatase in vitro and the consequences of such molecular modifications on certain structural and functional characteristics. The effect of glycation on alkaline phosphatase specific activity was determined after incubation of the enzyme with different sugars for various periods of time. The formation of early reversible glycation products was determined by the measurement of fructosamine levels, while the appearance of advanced glycation end products was estimated by spectrofluorometric analysis. A decrease in the specific activity of ALP was associated both with an increase in fructosamine levels and with the appearance of AGE-characteristic fluorescence. Changes in these parameters were found to depend on the incubation time, and on the concentration and glycating capability of the sugar employed. Co-incubation with aminoguanidine slowed down the appearance of protein-linked fluorescence, and additionally curbed the decrease in enzymatic specific activity. A significant correlation between the levels of ALP-fructosamine and ALP-advanced glycation end product was observed. Patterns of protein bands fractionated by SDS-PAGE were essentially identical for the nonglycated controls and the glycated samples. The electrophoretic mobility of the band of alkaline phosphatase on cellulose acetate gels increased as a function of the incubation time and the glycosylating power of the carbohydrate used. The present study provides evidence for the in vitro glycation of alkaline phosphatase, and for the consecutive alteration of its activity and structure.

Levels of fructosyllysine in collagen are low compared with the loss of lysine in diabetic rats: involvement of oxidation in increased cross-linking.

1. It is thought that early glycation products on lysine residues of collagen are re-arranged to form collagen cross-links (advanced glycation end-products), which are increased in hyperglycaemia. We have previously reported that 4 week vitamin E supplementation in diabetic rats protected against the development of increased collagen cross-linking. In the present study we analysed early glycation products as fructosyllysine and amino acid content of tail tendon collagen in rats. 2. The amount of lysine residues in early collagen glycation products in diabetic rats was estimated to be less than 0.2 mol/mol type I collagen, whereas in the diabetic rats the loss of lysine residues was estimated to be 2.5 mol/mol. However, vitamin E supplementation protected against the loss of lysine residues without affecting early glycation products. 3. Thus, it is demonstrated that the amount of early collagen glycation products is quite small compared with the loss of lysine residues in diabetic rats and we suggest that the oxidative modification of lysine residues may be involved in the increased cross-linking observed in hyperglycaemia.

Accumulation of advanced glycation endproducts in the rat nephron: link with circulating AGEs during aging.

The accumulation of advanced glycosylation end products (AGEs) is believed to be a factor in the development of aging nephropathy. We have attempted to establish a link between the formation of AGEs and the onset of renal impairment with aging, indicated by albuminuria, using a fluorescence assay and immunohistochemical detection of AGEs in the renal extracellular matrix in rats. The fluorescence of collagenase-digested Type IV collagen from GBM increased with age, from 1.65 +/- 0.05 AU/mM OHPro (3 months) and 1.58 +/- 0.04 (10 months) to 2.16 +/- 0.06 (26 months) (p < 0.001) and 2.53 +/- 0.18 (30 months) (p < 0.001). In contrast, the extent of early glycation products significantly decreased from 5.35 +/- 0.25 nmol HCHO/nmol OHPro at 3 months to 3.14 +/- 0.19 at 10 months (p < 0.001), 3.42 +/- 0.38 at 26 months, and 0.74 +/- 0.08 at 30 months (p < 0.001). The urinary fluorescence of circulating AGE rose from 2.42 +/- 0.15 AU/mg protein (3 months), 1.69 +/- 0.07 (10 months), to 4.63 +/- 0.35 (26 months) (p < 0.01) and 4.73 +/- 0.72 (30 months), while the serum fluorescence increased from 0.39 +/- 0.02 AU/mg protein at 3 months and 0.43 +/- 0.02 at 10 months to 0.59 +/- 0.04 at 26 months (p < 0.001) and 0.54 +/- 0.03 at 30 months (p < 0.04). Polyclonal antibodies raised against AGE RNase showed faint areas of AGE immunoreactivity in mesangial areas in the nephrons of young rats. The immunolabeling of Bowman's capsule, the mesangial matrices, and the peripheral loops of glomerular and tubule basement membranes increased with rat age. The increase in circulating AGE peptides parallels the accumulation of AGEs in the nephron, and this parallels the pattern of extracellular matrix deposition, suggesting a close link between AGE accumulation and renal impairment in aging rats.

Nonenzymatic glycation of peripheral and central nervous system proteins in experimental diabetes mellitus.

Nonenzymatic glycation of neural proteins could underlie diabetic peripheral neuropathy. Cytoskeletal and myelin protein fractions of central nervous system and peripheral nervous system (PNS) tissue from rats with streptozotocin-induced diabetes of 1.5 and 8 months duration were analyzed for glycation products. In sciatic nerve cytoskeletal preparations from both diabetic and control animals we found high levels of the early glycation product (measured as furosine) after 6 weeks, which had fallen markedly by 8 months. Conversely the advanced glycation end product (AGE), pentosidine, was low at 6 weeks and high by 8 months in diabetic animals. The levels of glycation products were much lower in spinal cord and spinal nerve from diabetic animals. There was increased borotritride labeling of neurofilament subunits, and of cross-linked material, in cytoskeletal fractions of diabetic sciatic nerves. These results show that the PNS cytoskeleton is vulnerable to nonenzymatic glycation, resulting in AGE formation, in diabetic rats and to a lesser extent in normals.

Pathogenesis of diabetic nephropathy, advanced glcation and new therapy

It is well understood that persistent hyperglycemia contributes to diabetic tissue and end-organ damage [1], by promoting the formation of advanced glycosylation endproducts (AGEs) [2, 3]. AGEs form principally from the rearrangement of early glycation products, i.e. Amadori products, which produce a class o f stable moieties that possess distinctive chemical cross-linking and biological properties [2, 31 . It has been generally believed that proteins with half-lives of greater than a few weeks are most susceptible to advanced glycosylation and the highest levels o f AGEs occur on proteins that comprise the long-lived structural components of connective tissue matrix and basement membrane. Of particular interest, however, has been the recent observation that AGEs can also occur on short-lived proteins, on lipid constituents and on nucleic acids. This is most apparent under conditions o£ high AGE accumulation such as diabetes and renal failure, and results from the fact that AGE formation proceeds through a succession of reactive intermediates which can bind indiscriminantly to amino groups present on diverse "bystander" proteins. 1keactive intermediates can also be released from naturally degraded tissue AGE, only to bind again on other substances, if not cleared via the kidney, forming "second" generation AGEs. Cell surface receptors that are specific for the recognition, and degradation of AGE-modified proteins also have been identified on circulating monocytes, lymphocytes, endothelial, renal mesangial cells and other cellular systems to partake in both normal tissue remodeling and tissue damage. In this synoptic review, selected recent studies are highlighted which

Advanced glycation end products enhance osteoclast-induced bone resorption in cultured mouse unfractionated bone cells and in rats implanted subcutaneously with devitalized bone particles.

Advanced glycation end products (AGE) are formed in long-lived matrix proteins by a nonenzymatic reaction with sugar. The presence of AGE in beta 2-microglobulin-amyloid fibrils of dialysis-related amyloidosis, one of the characteristic features of which is an accelerated bone resorption around amyloid deposits, was recently demonstrated. This suggested a potential link of AGE in bone resorption and initiated this investigation of whether AGE enhance bone resorption. When mouse unfractionated bone cells containing osteoclasts were cultured on dentin slices, both AGE-modified beta 2-microglobulin and BSA increased the number of resorption pits formed by osteoclasts, whereas their normal counterparts of those modified with the early glycation products did not. AGE proteins, however, did not increase the number of newly formed osteoclasts, even in the coculture of mouse bone marrow cells with osteoblastic cells isolated from mouse calvaria. Enhanced bone resorption was also observed when unfractionated bone cells were cultured on AGE-modified dentin slices. AGE-enhanced bone resorption was effectively inhibited by calcitonin and ipriflavone, both of which are inhibitors of bone resorption. AGE-enhanced bone resorption was further supported by in vivo evidence that rat bone particles-upon incubation with glucose for 60 days (AGE-bone particles)-when implanted subcutaneously in rats, were resorbed to a much greater extent than control bone particles upon parallel incubation without glucose. These findings suggest that AGE enhance osteoclast-induced bone resorption. Although the mechanism remains unknown, AGE are unlikely to promote differentiation of osteoclast progenitors into osteoclasts, suggesting that AGE activate osteoclasts or alter microenvironments favorable for bone resorption by osteoclasts. The modification of bone matrices with AGE might play a role in the remodeling of senescent bone matrix tissues, further implicating a pathological significance of AGE in dialysis-related amyloidosis or osteoporosis associated with diabetes and aging.

Growth modulation of retinal microvascular cells by early and advanced glycation products

To investigate the possible implication of non-enzymatic glycosylation in the etiopathogenesis of the diabetic retinopathy, we studied the effect of early and advanced glycation products on the growth of retinal microvascular cells. Glucose modified products were obtained by incubating bovine serum albumin or fetal bovine serum with 0.5 M glucose for 10 (early glycation products: EG-BSA and EG-FBS, respectively) or 60 days (advanced glycation end products: AGE-BSA and AGE-FBS, respectively). Cell growth was assessed by cell counting and DNA content determination. EG-BSA or AGE-BSA significantly decreased pericyte proliferation after 8 days of culture (33 and 13% inhibition, respectively). Concerning endothelial cells, EG-BSA reduced proliferation to 40% whereas AGE-BSA increased it to 156% after 4 days of culture. The glucose-treated sera didn't exhibit the same growth effects, neither the EG-FBS nor the AGE-FBS significantly affected endothelial cell proliferation. Only the AGE-FBS showed a significant inhibitory effect on pericyte proliferation (40% inhibition). We conclude that retinal microvascular cell growth in vitro could be differently modulated by early and advanced glycation products. The inhibitory effect of AGEs observed on pericyte growth, suggests that glycoxidation could be implicated in the pericyte loss observed in diabetic retinopathy.

Discordant effects of guanidines on renal structure and function and on regional vascular dysfunction and collagen changes in diabetic rats.

We examined the effects of aminoguanidine and methylguanidine on vascular dysfunction, glomerular structural changes, and indexes of early and late nonenzymatic glycation in 7-month streptozotocin-induced diabetic rats. Kidney weight, glomerular volume, fractional mesangial volume, glomerular capillary basement membrane width, and urinary albumin excretion were increased in diabetic rats. Diabetes also 1) increased vascular albumin permeation twofold in retina, sciatic nerve, aorta, skin, and kidney; 2) decreased renal collagenase-soluble collagen; 3) increased collagen-associated fluorescence in kidney and skin but not in aorta; and 4) increased glycated hemoglobin levels and aortic pentosidine levels. Aminoguanidine reduced albuminuria by 70% after 4 months, and both guanidines 1) normalized aortic pentosidine levels and renal collagenase-soluble collagen, 2) had no effect on glycated hemoglobin levels or collagen-associated fluorescence (in aorta, kidney, or skin), and 3) had little or no effect on regional albumin permeation. These discordant effects of aminoguanidine on diabetes-induced vascular changes versus parameters of nonenzymatic glycation are consistent with a multifactorial pathogenesis of diabetic complications, including roles for metabolic imbalances independent of nonenzymatic glycation. To the extent that glomerular matrix accumulation and increased regional albumin permeation in chronically diabetic rats are sequelae of nonenzymatic glycation, these findings point to an important role for early glycation reactions and products.

Rapid assessment of early glycation products by mass spectrometry

In order to detect the early glycation products, we have reacted a model peptide (t-boc-lys-ala-ala) with L-threose (a degradation product of ascorbic acid) and analyzed the reaction products by a combination of HPLC and mass spectrometry. Amino group modification, as observed by a fluorescamine assay, indicated complete modification after 3 days of incubation with a 10-fold excess of threose. As much as 60% of the adducts were acid labile and only 4% of the adducts could be observed by amino acid analysis. However, Fast atom bombardment mass spectrometry (FABMS) of the samples incubated for 6 hr showed relative molecular masses consistent with the formation of adducts corresponding to the addition of one and two molecules of L-threose to the peptide. Likewise, samples incubated for 12 hr showed peptide adducts with two and three L-threoses. The number of threose molecules added to the peptide was also confirmed from the FABMS analysis by using [1-13C]-threose as the glycating agent.

Recent progress in advanced glycation and diabetic vascular disease: role of advanced glycation end product receptors.

Advanced glycosylation end products (AGEs) form principally from the rearrangement of early glycation products, i.e., Amadori products, which produce a class of stable moieties that possess distinctive chemical crosslinking and biological properties. It has been generally believed that proteins with half-lives of longer than a few weeks are most susceptible to advanced glycosylation and that the highest levels of AGEs occur on proteins that comprise the long-lived structural components of connective tissue matrix and basement membrane.

Role of glycation in human lens protein structure change.

Protein glycation may be involved in cataract development, by altering protein structure, particularly amino acid composition, and formation of fluorophores through a Maillard reaction. This study was designed to evaluate major changes in early and advanced (fluorescent) glycation products, with special emphasis on glycation-induced changes in amino acid composition of lens proteins. We analyzed 50 human cataractous lenses (25 diabetic and 25 non-diabetic). Glycated proteins were isolated by affinity chromatography. Glycated and non-glycated proteins were separated by molecular sieve chromatography and further analyzed by RP-HPLC to establish the amino acid content. Early glycation levels were determined as furosine content and advanced glycation products were quantified by the characteristic fluorescence. Specific lens fractions (HMW and LMW) present significant differences in fluorescence levels between glycated and non-glycated proteins, specially in cataractous lenses from diabetic patients in which all proteins analyzed presented higher glycation levels than in non-diabetic patients. The amino and analysis of glycated proteins also revealed some important differences in specific basic residues (namely Lys, Arg and His) compared to the non-glycated fraction. The results suggest that protein glycation may be involved in changes in amino acid composition and fluorophore formation. This process may well account for the increased risk factor that diabetes represents for cataract development.

Possible involvement of advanced glycation end-products in bone resorption.

Advanced glycation end-products (AGEs) are formed in long-lived matrix proteins by a non-enzymatic reaction with sugar. We recently demonstrated the presence of AGEs in amyloid fibrils of dialysis-related amyloidosis, one of the characteristic features of which is an accelerated bone resorption around amyloid deposits. This suggested a potential link of AGEs in bone resorption and led us to investigate whether AGEs enhance bone resorption. An immunohistochemical study using anti-AGE antibody revealed positive immunostaining for AGEs in bone tissues from elderly subjects. AGE-modified proteins were shown to stimulate monocyte/macrophage to secrete bone-resorbing cytokines such as interleukin-1 beta, interleukin-6 and tumour necrosis factor- alpha. AGE-modified proteins enhanced net calcium efflux in cultured neonatal mouse calvariae to a much greater extent than unmodified proteins. Furthermore, when mouse unfractionated bone cells containing osteoclasts were cultured on dentin slices, AGE-modified proteins increased the number of resorption pits formed by osteoclasts, whereas their normal counterparts or those modified with the early glycation products did not. These findings suggest that AGEs enhance bone resorption by osteoclasts. The modification of bone matrices with AGEs might, therefore, play a pathophysiological role not only in the remodelling of senescent bone matrix tissues, but also in dialysis-related amyloidosis or osteoporosis associated with diabetes and ageing.

Histones from Diabetic Rats Contain Increased Levels of Advanced Glycation End Products

In a recent report we have demonstrated the in vitro formation of advanced glycation end products (AGEs) on histones in a time and sugar concentration dependent fashion. In the present work we examined histone advanced glycation in vivo. Diabetes was induced in rats by streptozotocin injection and the hyperglycemic state was maintained and surveyed for up to 24 weeks. Diabetic rats showed accumulation of early glycation products in plasma proteins and in hemoglobin. Histones from the liver of diabetic rats showed AGEs levels three-fold higher than those of their age-matched controls. Histone AGEs increased with the duration of diabetes and tended to increase with the age as well. Similar tendencies were apparent in skin collagen. Our data demonstrate that diabetes induces an increase in the accumulation of AGE products on histones. This reinforces the concept that advanced glycation occurs in intracellular proteins and suggests a possible role for intracellular glycation in the increased theratogeny associated with diabetes mellitus.

Reaction of advanced glycation endproducts with renal tissue from normal and streptozotocin-induced diabetic rats: an ultrastructural study using colloidal gold cytochemistry.

Advanced glycation endproducts (AGEs) are believed to play important roles in the molecular basis of long-term diabetes complications. In the present study we have revealed direct reactivity of AGEs towards kidney tissue structures. Bovine serum albumin, modified by advanced glycation and tagged with colloidal gold (AGE-BSA-gold) was applied to renal tissue sections from normal and long-term diabetic rats by a post-embedding procedure at the electron microscopic level. The AGE-BSA probes were characterized by fluorometry, spectrophotometry, free amines analysis, measurement of early glycation products, electrophoresis, and isoelectrofocusing. When applied to renal tissue sections, AGE-BSA-gold exhibited strong labeling on nuclei. At the extracellular level, glomerular basement membranes and mesangial matrix were labeled. Probes having a higher content of AGEs yielded more intense labeling. Control experiments demonstrated the specificity of the labeling obtained. Quantitative evaluation indicated little variation in reactivity among tissues from rats of different ages. However, a significant increase in reactivity was established for tissues from streptozotocin-induced long-term diabetic rats. This study thus provides direct evidence that soluble AGEs bind to cell nuclei and to structures implicated in the development of diabetic glomerular nephropathy, demonstrating higher AGE reactivity in tissues from diabetic animals.

Plasma protein's glycation is decreased in Sprague Dawley rats under caloric restriction

Different dietary regimens were applied to three cohorts of rats. The first was fed ad libitum every day (AL), the second was fed ad libitum every other day (EOD) and the third was fed a diet equivalent to 60% of the caloric intake (60% CI) of the AL cohort. Levels of stable early glycation products in plasma proteins were then measured according to two different methods. Glycation of plasma proteins progressively increased in AL animals belonging to the 2–12 month age interval, while it showed a less pronounced age-dependent increase in EOD and 60% CI animals. The lowest degree of glycation was detected 2–3 months after the beginning of caloric restriction. After 12 months of age a lower level of glycation was detected in 60% CI rats than in EOD animals. Body weight was lower in restricted animals than in AL animals and was lowest in 60% CI rats. During the life span, glycemia was lower in fasting 60% CI than in EOD or AL rats.

The Influence of L-Arginine on Biomechanical Parameters of Native and Nonenzymatically Glycated Rat Tail Tendons

Rat tail tendons from animals of an age range from 35 days to 900 days were glycated using different glucose concentrations. After nonenzymatic glycation the biomechanical properties of tendons from young rats approached that of old tendons. In tendons from old animals the glucose incorporation was significantly lower than in young rat tail tendons. Addition of L-arginine to the glucose solution reduced the incorporation of glucose especially in young rats. The concentration of early glycation products and of advanced glycation end products were measured via affinity chromatography on boronic acid agarose and relative fluorescence per collagen content respectively. Both parameters were significantly reduced by an addition of 10 mmol/l arginine. Biomechanical changes due to glycation were partially reversed. Incubation of rat tail tendons in L-arginine in the absence of glucose caused a dose dependent binding of the amino acid mainly to the proteoglycan matrix. High concentrations of L-arginine induced pronounced biomechanical alterations contrary to the action of glucose. The biomechanical effect of L-arginine is compared to the action of Na+ and Ca2+ and discussed on the basis of a structural model of the proteoglycan matrix. Incubation of already glycated rat tail tendons with L-arginine caused a reduction of the elastic stress component This effect was diminished by increasing the preincubation interval with glucose. The equilibrium values of the elastic fraction were achieved after approximately five days incubation with the amino acid.

Glycation of crystalline in lenses from aging and diabetic individuals

Water-soluble crystallins were obtained from clear human lenses of different age(4–81-year-olds) and lenses of individuals showing senile and diabetic cataracts. Levels of early glycation products were high in the high molecular weight material (HM) and the α-crystallin fractions, compared with β and γ-crystallins. This difference becomes more prominent upon aging. The content of total early glycation products in HM and α-crystallin increases clearly with age, whereas levels remain relatively constant in the β- and α-crystallins. There is an elevation of early products in cataractous lenses from diabetic individuals compared with those suffering from senile cataract. Specific non-tryptophan fluorescence (excitation/emission wavelengths 370/440 nm), used as an indicator for late glycation products, increased dramatically with age and was 2-fold higher in the diabetic subjects. Levels of fluorescence decreased in the order HM > α- > b > γ-crystallins. The results suggest an increase in glycation rate in α-crystallin as a result of aging and diabetes, while the rate of glycation of β- and γ-crystallins remains almost constant.

In vivo glycation of bovine lens crystallins

To investigate the possible role of glycation in the aging of lens proteins, we used bovine lenses as a model. We studied crystallins isolated from prenatal bovine lenses, calf lenses and lenses from mature animals (up to 20 years old). The experiments show an increase in glycation levels with age in all crystallin fractions. Regarding the lysine content of the different crystallins, γ-crystallin showed relatively high levels of early glycation products. The results also revealed high levels of early glycation products for the HM material (containing mainly α-crystallin). In α-crystallin, αA-subunits were glycated to a higher extent compared with the αB-subunits. There is an age-related increase in advanced glycation products, measured as specific flourescence (excitation/emission wavelengths 370/440 nm), mainly present in the HM and α-crystallin fraction.

Aminoguanidine treatment reduces the increase in collagen stability of rats with experimental diabetes mellitus.

Alterations in the biophysical properties of connective tissues in diabetes mellitus have been attributed to the nonenzymatic glycation of the collagens and the subsequent formation of browning products, cross-linking the proteins. Aminoguanidine may bind to carbonyl groups of these nonenzymatic glycation products and thereby block the process. Rats with streptozotocin-induced diabetes were treated with aminoguanidine, 25 mg.kg-1.day-1, for 120 days. The aminoguanidine treatment did not counteract the increase in blood glucose concentrations, nor did it prevent the arrest in weight gain of diabetic rats. The increased stability in 7 mol/l urea and increased tensile strength of tail tendons from the diabetic rats, however, were prevented by the aminoguanidine treatment. Aminoguanidine did not reduce the formation of early nonenzymatic glycation products (aldimine and Amadori rearrangement products), whereas the amount of browning products (fluorescent compounds) was reduced in the tail tendon collagen of the diabetic rats. Aminoguanidine treatment of intact rats did not influence these parameters. These findings indicate that the biophysical alterations of collagens induced by experimental diabetes are caused by cross-links derived from the nonenzymatic glycation, and furthermore, that aminoguanidine treatment may prevent the concomitant changes in biophysical properties of connective tissues.