The induction of surface beta-amyloid binding proteins and enhanced cytotoxicity in cultured PC-12 and IMR-32 cells by advanced glycation end products.

During aging the non-enzymatic glycation of proteins and other molecules increases significantly, leading to the accumulation of advanced glycation end-products (AGEs). These AGEs enhance inflammatory and autoimmune reactions with resultant cytotoxicity. We noted in an earlier study that individuals with Alzheimer's disease exhibit enhanced expression of the receptor for advanced glycation end-products (RAGE) on the surface of their leukocytes. In order to better understand the relationship between AGEs and the cell surface binding of amyloid-beta protein (Abeta) 42 we studied the effect of two AGEs: glycated bovine serum albumin (BSA), and epsilon-carboxymethyllysine-BSA (CML), a glycoxidation product, on the binding of Abeta42 to rat PC-12 and IMR-32 cells. We measured the expression of three potential cell surface receptors binding Abeta42: RAGE, beta-amyloid precursor protein (beta-APP), and the alpha7 subtype of the nicotinic acetylcholine receptor (alpha7nAChR) by using specific antibody probes. Incubation of PC-12 or IMR-32 cells with bovine serum albumin-advanced glycation end-product (BSA-AGE) or with CML induced small but significant concentration-dependent increases in the expression of beta-APP, RAGE, and alpha7nAChRs as measured by flow cytometry or by ELISA. Incubation of the cells with 48 microM of either of the AGEs combined with varying concentrations (138-1100 nM) of Abeta42 resulted in the enhanced binding of the Abeta42 to the cell surface as compared with cells not exposed to the AGE co-treatment. The combination of AGE and Abeta treatment also resulted in the heightened expression of all three potential Abeta binding sites as well as their gene precursors. Exposure of cells to the same regimen of AGE plus Abeta resulted in the production of reactive oxygen species and mitochondrial toxicity. These results are consistent with the ability of AGEs to enhance the cell surface expression of diverse Abeta42 binding sites, a factor that can lead to the enhanced binding of amyloid and subsequent cell death.

Autoimmunity in Alzheimer's disease as evidenced by plasma immunoreactivity against RAGE and Abeta42: complication of diabetes.

Features of autoimmunity have been associated with both Alzheimer's disease (AD) and with diabetes. In both diseases high levels of advanced glycation end products (AGEs) and their receptor (RAGE) have been detected in tissues and in the circulation. In addition high titers of antibodies directed against a RAGE-like peptide occur in the circulation. In this study we report the presence of auto-antibodies directed against RAGE and the cytotoxic amyloid peptide Abeta42 in plasma samples derived from four study groups. Anti-RAGE IgG titers were greatest in the AD-diabetic cohort. They were followed in decreasing order by the AD-non-diabetic cohort, the elderly diabetic cohort, and lastly by the control non-diabetic elderly cohort. The same profile of IgG differences was evident for the anti-Abeta42 titers. When all of the data were combined, there was a strong linear correlation between the RAGE and Abeta42 titers suggesting that the two peptides exist as a tight complex in plasma. Plasma IgG titers were not correlated with cognitive status except that AD and AD-diabetic participants were significantly cognitively impaired relative to the two non-AD groups. There also was no significant correlation between IgG titers and subject age, except that there was a trend for a negative slope for the AD participants and a positive slope for the control participants. In keeping with the human data, we also report that chemically-induced diabetes in rats was associated with high levels of AGEs, anti-RAGE-like IgGs, and anti-Abeta42-like IgGs. For non-diabetic rats, there was a clear age-dependency regarding the magnitude of the IgG levels. These data support the concept of an interrelationship between diabetes and AD. For both diseases one underlying contributing factor to cytotoxicity could be the development of an autoimmune response triggered by the presence of AGEs and amyloid peptides.

Immunolocalization and quantification of advanced glycation end products in retinal neovascular membranes and serum: a possible role in ocular neovascularization.

PURPOSE: Earlier studies have revealed the association of advanced glycation end products (AGE) with the pathogenesis of various micro and macro vascular complications. The purpose of the present study is to localize AGEs, namely carboxy methyl lysine (CML-AGE) and methyl glyoxal-derived AGEs (MG-AGE), in retinal neovascular membranes and to quantify them in serum samples. METHODS: Surgically excised retinal neovascular membranes and serum samples obtained from patients with diabetic retinopathy, Eales' disease and nondiabetics were studied. Immunolocalization of AGEs namely CML-AGE and MG-derived AGEs was done using avidin biotin complex method and quantification was done by enzyme linked immunosorbent assay (ELISA). RESULTS: CML-AGE immunoreactivity was detected in all cases of Eales' disease and 61% cases of diabetic retinopathy and none in idiopathic epiretinal membrane (ERM). MG-AGE immunoreactivity was observed in approximately 15% of diabetic retinopathy and none in Eales' disease and and idiopathic ERM. Quantification of AGEs in serum samples revealed statistically significant increased levels of MG-AGE in diabetes, in relation to nondiabetics with idiopathic ERM and CML-AGE in Eales' disease, in relation to diabetics and nondiabetics with idiopathic ERM. CONCLUSION: Results from this study suggest that AGEs formed through glycation and glycoxidation may play an important role in the development of retinal neovascularization. The immunoreactivity of CML-AGEs in neovascular membrane and its increased levels in serum suggest that inspite of the normoglycemic status, glycoxidation and lipid peroxidation due to oxidative stress may trigger retinal neovascularization in Eales' disease, while MG-AGEs in diabetic membrane and serum suggest the role of glycation. Thus the mechanism of neovascularization in different pathological conditions could be different.

Glycation decreases calmodulin binding to lens transmembrane protein, MIP.

Channels of the major intrinsic protein (MIP) of the lens transport water, thus playing an important role in lens fiber cell homeostasis. Calmodulin (CAM) interacts with MIP and possibly regulates MIP channel permeability. Protein glycation has been implicated in lens opacification. We previously identified sites of glycation of MIP, which are in close proximity to the putative CAM binding site. This study is aimed to show the effect of in vitro and in vivo glycation on CAM binding to MIP. Our results show that MIP and MP20 are the major CAM binding proteins of the lens membrane. In vitro incubation of lens membranes with 1 M glucose decreased CAM binding by 38% (P<0.001). Similarly, there was a progressive decrease in CAM binding to diabetic lens membranes compared to age-matched controls (up to 30% decrease, P<0.01). Mutation of K228 and K238 as well as a triple K mutation (K228N, K238N, K259N) of MIP resulted in a decrease in CAM binding. Thus, post-translational protein modifications of MIP influence CAM binding. Since CAM is the ubiquitous Ca(2+) receptor, decreases in CAM binding to the target protein will affect the Ca(2+)-mediated cellular processes leading to lens opacification in diabetic and aging lenses.

Evidence of a glycemic threshold for the development of cataracts in diabetic rats.

PURPOSE: This study was aimed to establish a possible correlation between the levels of plasma glucose and degree of lens opacification. Levels of glycation- and glycoxidation-products in different lens protein fractions were also estimated with an aim to determine the involvement of these products in lens opacification. METHODS: A wide range of hyperglycemia was induced by injecting different doses of streptozotocin to 1 month old rats and lenses were examined on the 75th, 90th and 150th day post-injection. Lens opacification was measured by Scheimpflug imaging and densitometry. Levels of plasma glucose and glycated hemoglobin were measured after overnight fasting. On 90th day, levels of Amadori products in lens water soluble (WS) fraction were measured by affinity chromatography. Similarly, advanced glycation end products (AGEs) in lens WS, urea soluble (US) and alkali soluble (AS) fractions were measured immunochemically using a monoclonal antibody against the major glycoxidation product, carboxymethyl lysine (CML). RESULTS: Different dosages of streptozotocin injection resulted in a broad range of plasma glucose levels in the rats which were grouped into three groups on the basis of their plasma glucose levels: mildly diabetic (< 170 mg/dl plasma glucose), moderately diabetic (190-350 mg/dl) and severely diabetic (> 400 mg/dl). On the 75th, 90th and 150th day post-injection, only the moderately and severely diabetic rats developed cataracts whereas lenses of the mildly diabetic rats remained clear. As seen on 90th day, levels of glycated hemoglobin and Amadori products in lens WS fraction increased significantly in the moderately and severely diabetic groups whereas in the mildly diabetic rats these levels remained more or less same as in the control group. Levels of CML in WS fractions remained unchanged between control rats and different diabetic groups, while US fractions showed a decrease in CML in both the moderately and severely diabetic groups compared to the controls and the mildly diabetic group. Interestingly, AS fractions contained the highest level of CML; the moderately and severely diabetic groups showed about 2-fold higher levels than the controls and the mildly diabetic group. CONCLUSIONS: This study strongly supports the existence of plasma glycemic threshold above which incidence of diabetic cataract formation increases exponentially. This threshold level seems to be at approximately 180 mg/dl or 10 mM plasma glucose. Significant increase in the levels of glycation and glycoxidation products mainly in cataract lenses suggests that glycation and glycation-mediated oxidation play an important role in the development of diabetic cataracts.

Acetyl- L -carnitine decreases glycation of lens proteins: in vitro studies.

Although the role of carnitine system in the ocular tissues is not clearly understood, earlier studies showed that lenticular levels of L -carnitine were the highest among ocular tissues and there was a dramatic depletion of lenticular L -carnitine and acetyl- L -carnitine in streptozotocin-diabetic rats. As protein glycation has been implicated in the development of several diabetic complications including cataracts, this study was initiated to show the possible effects of L -carnitine and acetyl- L -carnitine on the glycation and advanced glycation (AGEs) of lens proteins. Calf lens soluble fraction (crystallins) was incubated with 50 m m glucose (containing14C glucose) with or without 5-50 m ml -carnitine, 5-50 m m acetyl- L -carnitine and 5-50 m m acetyl salicylic acid, for 15 days. The results show that while L -carnitine did not have any effect on in vitro glycation of lens crystallins, acetyl- L -carnitine and acetyl salicylic acid decreased crystallin glycation by 42% and 63%, respectively-this decrease was concentration dependent. Glycated crystallins were separated on HPLC which showed that the rate of glycation is in the following order: alpha>beta>gamma. Interestingly, acetyl- L -carnitine inhibited glycation of alpha crystallin more than other crystallins. In vitro incubations with [3H-acetyl] acetyl- L -carnitine showed that acetyl- L -carnitine acetylates lens crystallins (non-enzymatically) and alpha crystallin is the major acetylated protein. Furthermore, there was a 70% reduction in anti-AGE antibody reactivity when 50 m m acetyl- L -carnitine was included in the incubation of lens crystallins and 10 m m erythrose, suggesting that inhibition of glycation by acetyl- L -carnitine also affected the generation of AGEs. This in vitro study shows, for the first time, that acetyl- L -carnitine could acetylate potential glycation sites of lens crystallins, and protect them from glycation-mediated protein damage.

Expression and characterization of lens membrane intrinsic protein, MIP, in a baculovirus expression system.

PURPOSE: Major intrinsic protein (MIP) is the transmembrane protein in the lens involved in homeostasis of the fiber cell. Although MIP has some intrinsic water and glycerol permeability and can form a gap junction like channels in the reconstituted systems, its role in the lens remained enigmatic. This study is aimed at developing a heterologous expression system for understanding the role of MIP. METHODS: The coding sequence of bovine MIP was subcloned into pBlueBacIII baculovirus transfer vector to facilitate transfer to AcMNPV genome, and the recombinant baculovirus was used to transfect Sf9 cells. Expression of MIP was confirmed by immunoblotting and purified by HPLC. Reconstituted liposomes were used to study the function of the recombinant protein. RESULTS: SDS-PAGE and immunoblots confirmed that the expressed protein was targeted to the cell membrane. MIP accounted for about 10% of total membrane proteins of the transfected Sf9 cell membranes. Molecular size of the recombinant protein is estimated to be about 30-32 kDa on SDS-PAGE, in contrast to 26 kDa of native MIP. The reconstitution studies showed that the channels formed by recombinant MIP are functionally similar to those of native MIP isolated from calf lens membranes. CONCLUSIONS: This study describes functional expression of MIP in a baculovirus expression system. This opens the way to studying the role of MIP and to understanding the effect of mutations of critical residues in the function of MIP.

Mass spectroscopic identification of in vitro glycated sites of MIP.

PURPOSE: Earlier reconstitution studies showed that glycation of the major intrinsic peptide (MIP) of the lens affected the permeability of liposomes. This study is aimed to identify in vitro glycated sites. METHODS: Urea- and alkali-washed calf lens membranes were incubated with 0 and 1 M glucose for 5 days. Following the incubation, MIP was purified by size exclusion HPLC. The C-terminus peptide of MIP was then cleaved by cyanogen bromide (CNBr) and purified by C4 reversed-phase HPLC. The CNBr peptide was analyzed, either directly or after digestion with trypsin, by electrospray ionization mass spectrometry (ESIMS) and matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS). RESULTS: The ESIMS and MALDI-MS analyses of the intact C-terminus peptide from 1 M glucose incubated samples showed a mass shift equivalent to one, two and three glucose adducts. The MALDI-MS of the tryptic digest of the same sample showed peptides with mass shift equivalent to one or more glucose adducts. Sequence assignment confirmed that these glycated peptides contained lys238 and lys259. Although the intact C-terminus peptide showed up to three glucose adducts, we could not assign any tryptic peptide that contained glycated lys228. Samples incubated with 0 M glucose did not show any protein modifications. CONCLUSIONS: The data suggest that in vitro glycation sites of calf lens MIP are lys238 and lys259, and possibly lys228.

Inhibition of cataracts in moderately diabetic rats by aminoguanidine.

The effect of aminoguanidine (AG), an inhibitor of advanced glycation, on the development of cataracts was studied in diabetic rats. Rats were made diabetic with streptozotocin, and based on the level of plasma glucose they were grouped as moderately (< 350 mg dl-1 plasma glucose) and severely (> 350 mg dl-1 plasma glucose) diabetic. One half of the animals in each group received AG (25 mg kg-1 body weight each day), intraperitoneally, starting from the day of streptozotocin injection. Progression of lens opacification was recorded using Fundus and Scheimpflug photography at regular time intervals. On the ninetieth day all the rats were killed and the levels of advanced glycation end products (AGE) was determined by measuring the non-tryptophan fluorescence of the lens soluble and insoluble fractions. Densitometric analysis of Scheimpflug images showed that in diabetic rats lens opacification progressed in a biphasic manner, an initial slow progression for the first 60 days, followed by a steep increase during next 30 days. Moderately and severely diabetic rats developed lens opacities more or less at the same time. AGE fluorescence in the lens soluble fractions increased three-fold and seven-fold in the moderately and severely diabetic rats, respectively; whereas in insoluble fractions there was a 30% and three-fold increase in the moderately and severely diabetic rats, respectively. Although AG treatment inhibited the AGE fluorescence of lens soluble and insoluble fractions by about 56% and 75% in moderately diabetic and by 19% and 52% severely diabetic rats, respectively, the development of cataracts was delayed only in the moderately diabetic rats. These results thus suggest that the effect of AG is indeed inhibition of the formation of AGEs. However, in the severely diabetic rats the beneficial effect of AG is overwhelmed by the excessive accumulation of AGEs.

Scheimpflug densitometric analysis of cataracts in diabetic rats: correlation with glycation.

Glycation has been implicated in cataract formation. Our earlier studies showed that crystallin glycation enhances oxidation and aggregation, whereas MIP26 glycation affects the membrane permeability. Scheimpflug densitometric analysis has been used to quantify the lens opacification. In this study, we measured the progressive changes in lens opacification and correlated them with protein glycation in streptozotocin-induced diabetic rats. The lens opacification progressed in a biphasic manner: an initial slow increase between 0 and 60 days, followed by a steep increase between 60 and 90 days of diabetes. There was a strong correlation between lens opacification and lens crystallin and MIP26 glycation. The correlation was relatively weak with plasma glucose. This study suggests that glycation of lens crystallin and MIP26 plays a significant role in the development of lens opacification in diabetic rats.