Distribution Of Advanced Glycation End Products Research Articles

Effects of exercise on the nephron of Goto-Kakizaki rats: morphological, and advanced glycation end-products and inducible nitric oxide synthase immunohistochemical analyses

The current study aimed to examine how exercise affects morphology of the nephron, and localization of advanced glycation end-products (AGEs) and inducible nitric oxide synthase (iNOS) immunoreactivity in diabetic Goto-Kakizaki rats. Four groups of male rats were studied. WIS SED (Wistar rats; sedentary) group served as a control. Other groups were WIS EX (Wistar rats; exercise), GK SED (Goto-Kakizaki diabetic rats; sedentary) and GK EX (Goto-Kakizaki diabetic rats; exercise) groups. The rats in EX groups were subjected to 15weeks of treadmill running at a speed of 15m/min for a total of 30minutes, three times a week. Changes in the structure of renal corpuscles and in the distribution of AGEs- and iNOS-immunoreactive cells of the uriniferous tubules were evaluated. Every parameter of GK EX was significantly different from that of GK SED (area of Bowman's capsules: p<0.001, area of glomeruli: p<0.05 and the occupancy of a glomerulus: p<0.05). These findings suggest that exercise may ameliorate glomerular filtration rate (GFR). The localizations of AGEs and iNOS immunostaining in the uriniferous tubules were similar in each group. Immunohistochemical assays revealed that the number of the AGEs and iNOS immunopositive cells of the proximal tubule of cortico-deep layer in EX groups were markedly greater than those in SED groups and that iNOS expression in GK EX was significantly higher than GK SED (p<0.05). Exercise seems to normalize the GFR and glomerular filtrate absorption from the uriniferous tubules in Goto-Kakizaki diabetic rats with the recovered shape of renal corpuscles and may be involved in the absorption and catabolization of AGEs with iNOS-related reactions for reabsorption.

Up-Regulated Expression of Advanced Glycation End-Products and Their Receptor in the Small Intestine and Colon of Diabetic Rats

Gastrointestinal disorders and symptoms are common in diabetic patients. Advanced glycation end-products (AGEs) and their receptor (RAGE) have been proposed as an important pathological mechanism underlying diabetic complications, such as diabetic cardiopathy, retinopathy, nephropathy, etc. The aims were to study the distribution of AGE and RAGE in the normal and diabetic small intestine and colon in rats and the possible relationship between AGEs/RAGE and diabetes-induced intestinal structural remodeling. Diabetic and age-matched normal rats survived for 56 days. The body weight and blood glucose were measured regularly until day 56. Jejunal, ileal, and colonic segments were excised. The wet weight per unit length and the layer thickness were measured. AGE and RAGE were detected by immunohistochemical staining. The wet weight per unit length in the three segments and the layer thickness in jejunum and ileum increased in the diabetic rats. The staining density of AGE in diabetic rats was higher in the villi of jejunum and ileum, and in the crypt and circumferential muscle layer of ileum compared to normal rats. The staining intensity of RAGE increased in ganglia, crypt, and brush border of diabetic jejunum and ileum as well as in ganglia of diabetic colon. Positive association was found between the accumulation of AGE and RAGE and the thickness of the different layers. The expression of AGE and RAGE is up-regulated in the small intestine and colon of diabetic rats. The increased AGE and RAGE levels may contribute to diabetic GI dysfunction.

In situ accumulation of advanced glycation endproducts (AGEs) in bone matrix and its correlation with osteoclastic bone resorption

Advanced glycation end products (AGEs) have been observed to accumulate in bone with increasing age and may impose effects on bone resorption activities. However, the underlying mechanism of AGEs accumulation in bone is still poorly understood. In this study, human cortical bone specimens from young (31±6years old), middle-aged (51±3years old) and elderly (76±4years old) groups were examined to determine the spatial-temporal distribution of AGEs in bone matrix and its effect on bone resorption activities by directly culturing osteoclastic cells on bone slices. The results of this study indicated that the fluorescence intensity (excitation wave length 360nm and emission wave length 470±40nm) could be used to estimate the relative distribution of AGEs in bone (pentosidine as its marker) under an epifluorescence microscope. Using the fluorescence intensity as the relative measure of AGEs concentration, it was found that the concentration of AGEs varied with biological tissue ages, showing the greatest amount in the interstitial tissue, followed by the old osteons, and the least amount in newly formed osteons. In addition, AGEs accumulation was found to be dependent on donor ages, suggesting that the younger the donor the less AGEs were accumulated in the tissue. Most interestingly, AGEs accumulation appeared to initiate from the region of cement lines, and spread diffusively to the other parts as the tissue aged. Finally, it was observed that the bone resorption activities of osteoclasts were positively correlated with the in situ concentration of AGEs and such an effect was enhanced with increasing donor age. These findings may help elucidate the mechanism of AGEs accumulation in bone and its association with bone remodeling process.

Immunohistochemical localization of advanced glycation end-products (AGEs) and their receptor (RAGE) in polycystic and normal ovaries

The aim of the present study was to investigate the localization/immunohistochemical distribution of AGEs and RAGE, as well as their putative signalling mediator NF-kappaB in ovaries of women with polycystic ovary syndrome (PCOS) compared to normal. Archival ovarian-tissue samples from biopsies of six women with PCOS and from six healthy of similar age women, were examined immunohistochemically with monoclonal anti-AGEs, anti-RAGE and anti-NF-kappaB(p50/p65) specific antibodies. In healthy women, AGE immunoreactivity was observed in follicular cell layers (granulosa and theca) and luteinized cells, but not in endothelial cells. PCOS specimens displayed AGE immunoexpression in theca interna and granulosa cells as well as in endothelial cells, but staining of granulosa cells was stronger than in that of normal ovaries. RAGE was highly expressed in normal and PCOS tissues. Normal tissue exhibited no staining differences between granulosa cell layer and theca interna. However, in PCOS ovaries, granulosa cells displayed stronger RAGE expression compared to theca interna cells in comparison to controls. NF-kappaB(p50/p65) was expressed in the cytoplasm of theca interna and granulosa cells of both normal and PCOS ovaries; whereas the NF-kappaB p65 subunit was only observed in granulosa cells nuclei in PCOS tissue. In conclusion, these findings demonstrate for the first time that RAGE and AGE-modified proteins with activated NF-kappaB are expressed in human ovarian tissue. Furthermore, a differential qualitative distribution of AGE, RAGE and NF-kappaB p65 subunit was observed in women with PCOS compared to healthy controls, where a stronger localization of both AGE and RAGE was observed in the granulosa cell layer of PCOS ovaries.

Age- and stage-dependent accumulation of advanced glycation end products in intracellular deposits in normal and Alzheimer's disease brains.

In this immunohistochemical study, the age- and stage-dependent accumulation of advanced glycation end-products (AGEs) in Alzheimer's disease (AD) and their relation to the formation of neurofibrillary tangles and neuronal cell death was investigated. For this purpose, the distribution of AGEs in neurons and glia was analyzed in the auditory association area of superior temporal gyrus (Brodmann area 22) of young and old non-demented controls and compared with early- and late-stage AD. A possible co-localization of AGEs with typical hallmarks of AD, such as hyperphosphorylated tau (as a marker for disturbed kinase/phosphatase activity), nNOS (as a marker for nitroxidative stress) and caspase-3 (as a marker of apoptotic cell death), was also investigated. Our results show that the percentage of AGE-positive neurons (and astroglia) increase both with age and, in AD patients, with the progression of the disease (Braak stages). Interestingly, nearly all if those neurons which show diffuse cytosolic AGE immunoreactivity also contain hyperphosphoryated tau, suggesting a link between AGE accumulation and the formation of early neurofibrillary tangles. Many, but not all, neurons show a co-localization of AGEs with other markers of neurodegeneration, such as nNOS and caspase-3.

Advanced glycation endproducts and pro-inflammatory cytokines in transgenic Tg2576 mice with amyloid plaque pathology.

Increased expression and altered processing of the amyloid precursor protein (APP) and generation of beta-amyloid peptides is important in the pathogenesis of amyloid plaques in Alzheimer's disease (AD). Transgenic Tg2576 mice overexpressing the Swedish mutation of human APP exhibit beta-amyloid deposition in the neocortex and limbic areas, accompanied by gliosis and dystrophic neurites. However, murine plaques appear to be less cross-linked and the mice show a lower degree of inflammation and neurodegeneration than AD patients. 'Advanced glycation endproducts (AGEs)', formed by reaction of proteins with reactive sugars or dicarbonyl compounds, are able to cross-link proteins and to activate glial cells, and are thus contributing to plaque stability and plaque-induced inflammation in AD. In this study, we analyze the tissue distribution of AGEs and the pro-inflammatory cytokines IL-1beta and TNF-alpha in 24-month-old Tg2576 mice, and compare the AGE distribution in these mice with a younger age group (13 months old) and a typical Alzheimer's disease patient. Around 70% of the amyloid plaque cores in the 24-month-old mice are devoid of AGEs, which might explain their solubility in physiological buffers. Plaque associated glia, which express IL-1beta and TNF-alpha, contain a significant amount of AGEs, suggesting that plaques, i.e. Abeta as its major component, can induce intracellular AGE formation and the expression of the cytokines on its own. In the 13-month-old transgenic mice, AGEs staining can neither be detected in plaques nor in glial cells. In contrast, AGEs are present in high amounts in both plaques and glia in the human AD patient. The data obtained in this show interesting differences between the transgenic mouse model and AD patients, which should be considered using the transgenic approach to test therapeutical strategies to eliminate plaques or to attenuate the inflammatory response in AD.

Localization in human diabetic peripheral nerve of N(epsilon)-carboxymethyllysine-protein adducts, an advanced glycation endproduct.

The present study was designed to elucidate in situ distribution of advanced glycation endproducts (AGE) in human peripheral nerve and whether the reaction products were excessive in the diabetic condition. For the detection of AGE, immunoperoxidase staining was undertaken on peripheral nerve samples obtained from 5 non-insulin-dependent diabetic patients and 5 non-diabetic control subjects. The anti-AGE antibody used in this study contained an epitope against N(epsilon)-carboxymethyllysine. Light microscopically, AGE localized in the perineurium, endothelial cells and pericytes of endoneurial microvessels as well as myelinated and unmyelinated fibers. At the submicroscopic level, AGE deposition appeared focally as irregular aggregates in the cytoplasm of endothelial cells, pericytes, axoplasm and Schwann cells of both myelinated and unmyelinated fibers. Interstitial collagens, basement membranes of the perineurium also reacted with this antibody. The AGE depositions were detected in both control and diabetic nerves, but were more intense in the latter. The excessive AGE deposition correlated with a reduction in myelinated fiber density. However, the localization of AGE was not directly associated with degeneration of nerve fibers and the link between AGE deposition and nerve fiber degeneration is yet to be determined. The present study thus demonstrated the excessive deposition of intra- and extracellular AGE in human diabetic peripheral nerve and strengthened the contention that the enhanced glycation may play a role in the development of diabetic neuropathy.

Elevated AGE-Modified ApoB in Sera of Euglycemic, Normolipidemic Patients with Atherosclerosis: Relationship to Tissue AGEs

Advanced glycation endproducts (AGEs) are implicated in the pathogenesis of atherosclerotic vascular disease of diabetic and nondiabetic etiology. Recent research suggests that advanced glycation of ApoB contributes to the development of hyperlipidemia. AGE-specific receptors, expressed on vascular endothelium and mononuclear cells, may be involved in both the clearance of, and the inflammatory responses to AGEs. The aim of this study was to examine whether there is a relationship between serum AGE-ApoB and AGEs in arterial tissue of older normolipidemic nondiabetic patients with occlusive atherosclerotic disease, compared with age-matched and younger asymptomatic persons. Serum AGE-ApoB was measured by ELISA in 21 cardiac bypass patients. Furthermore, an AGE-specific monoclonal antibody, and polyclonal antibodies against anti-AGE-receptor (anti-AGE-R) 1 and 2 were used to explore the localization and distribution of AGEs and AGE-R immunoreactivity (IR) in arterial segments excised from these patients. Serum AGE-ApoB levels were significantly elevated in the asymptomatic, older population, compared with those in young healthy persons (259 +/- 24 versus 180 +/- 21 AGE U/mg of ApoB, p < 0.01). Higher AGE-ApoB levels were observed in those patients with atherosclerosis (329 +/- 23 versus 259 +/- 24 AGE U/mg ApoB, p < 0.05). Comparisons of tissue AGE-collagen with serum AGE-ApoB levels showed a significant correlation (r = 0.707, p < 0.01). In early lesions, AGE-IR occurred mostly extracellularly. In fatty streaks and dense, cellular atheromatous lesions, AGE-IR was visible within lipid-containing smooth muscle cells and macrophages, while in late-stage, acellular plaques, AGE-IR occurred mostly extracellularly. AGE-R1 and -R2 were observed on vascular endothelial and smooth-muscle cells and on infiltrating mononuclear cells in the early-stage lesions, whereas in dense, late-stage plaques, they colocalized mostly with lipid-laden macrophages. On tissue sections, scoring of AGE-immunofluorescence correlated with tissue AGE and plasma AGE-ApoB. (1) The correlation between arterial tissue AGEs and circulating AGE-ApoB suggests a causal link between AGE modification of lipoproteins and atherosclerosis. AGE-specific receptors may contribute to this process. (2) Serum AGE-ApoB may serve to predict atherosclerosis in asymptomatic patients.

Determination of advanced glycation end products in serum by fluorescence spectroscopy and competitive ELISA.

Recent studies suggest that advanced glycation endproducts play an important role in cardiovascular complications of ageing, diabetes and end-stage renal failure. Since highly elevated levels of advanced glycation endproducts are present in serum of patients on maintenance haemodialysis, an accurate and rapid assay for their determination would be useful. This would be particularly valuable for monitoring the removal of advanced glycation endproducts by novel dialysis membranes, as well as the effect of new drugs for the inhibition of their formation. Measurement of advanced glycation endproducts in serum was performed by two competitive ELISAs, using a monoclonal antibody directed against imidazolone, an advanced glycation endproduct formed by the reaction of arginine with 3-deoxyglucosone, and a polyclonal antibody directed against keyhole limpet haemocyanin-advanced glycation endproduct, as well as by quantitative fluorescence spectroscopy. Each of the assays showed significant differences between the controls and the maintenance haemodialysis patients. Advanced glycation endproduct levels determined by each of the ELISAs correlated with total and protein-bound fluorescence, but not with each other, suggesting a variable distribution of advanced glycation endproducts on serum proteins among the maintenance haemodialysis patients.