Differential effects of "Advanced glycation endproducts" and beta-amyloid peptide on glucose utilization and ATP levels in the neuronal cell line SH-SY5Y.

Beta-amyloid peptide (Abeta) and "Advanced glycation endproducts" (AGEs) are components of the senile plaques in Alzheimer's disease patients. It has been proposed that both AGEs and Abeta exert many of their effects, which include the upregulation of pro-inflammatory cytokines, through RAGE ("receptor for advanced glycation endproducts"). To investigate whether Abeta and AGEs cause similar or identical effects on cell survival and energy metabolism, we have compared the effects of a model-AGE and Abeta on cell viability, ATP level, glucose consumption and lactate production in the neuroblastoma cell line SH-SY5Y. The results show that AGEs and Abeta increase glucose consumption and decrease ATP levels in a dose dependent manner. Furthermore, both compounds decrease mitochondrial activity measured by the MTT assay. However, only AGEs decrease the number of cells and significantly increase lactate production. These data indicate that both AGEs and Abeta can cause differential disturbances in neuronal metabolism, which may contribute to the pathophysiological findings in Alzheimer's disease. However, their signalling pathways are apparently quite distinct, a fact which should stimulate a more detailed investigation in this field, e.g. for the purpose of a rational design of potential "neuroprotective" RAGE antagonists.

Advanced glycation endproducts change glutathione redox status in SH-SY5Y human neuroblastoma cells by a hydrogen peroxide dependent mechanism.

The reaction of proteins with reducing sugars leads to the formation of 'advanced glycation endproducts' (AGEs). They accumulate in Alzheimer's disease brain in the vicinity of beta-amyloid plaques. AGEs are cytotoxic by a mechanism involving reactive oxygen species, which implies that they could compromise glutathione redox status. In this study, we show that AGEs (BSA-AGE and beta-amyloid-AGE) persistently increase the ratio of oxidized to reduced glutathione in a dose- and time-dependent manner in SH-SY5Y neuroblastoma cells. The level of oxidized glutathione accounted to 10-14% and persisted for up to 24 h in the presence of added AGEs. In contrast, the unmodified beta-amyloid peptides A beta (1-40) and A beta (25-35) had no significant effect on glutathione redox status. The AGE-induced increase in oxidized glutathione could be prevented by the radical scavengers N-acetylcysteine, alpha-lipoic acid and 17beta-estradiol or by application of catalase, indicating that superoxide and hydrogen peroxide production precedes the AGE-mediated depletion of reduced glutathione.

Advanced glycation endproducts: activators of cardiac remodeling in primary fibroblasts from adult rat hearts.

BACKGROUND: Cardiovascular diseases are the leading cause of death in the Western world, especially in the elderly. Myocardial fibrosis induced by activated cardiac fibroblasts is thought to play a key role in the pathogenesis of cardiovascular disease. Accumulation of advanced glycation endproducts (AGEs), products of nonenzymatic glycation of proteins, correlate with the stiffness of the heart and large vessels. To elucidate a potential role of AGEs as a trigger of fibrosis, the effects of AGEs on primary fibroblasts from hearts of adult rats were investigated. MATERIAL AND METHODS: The activation of intracellular signaling pathways was shown by Western blotting. In addition, the expression of genes of the extracellular matrix proteins, metalloproteases (MMPs), their inhibitors, and TGF-beta were analyzed by semiquantitative PCR. Activation of MMPs were controlled by Zymography. RESULTS: It was shown that treatment of cardiac fibroblasts with AGEs leads to an activation of different signaling molecules, such as the p38MAP-kinase, the extracellular regulated kinases (ERKs), the jun kinase (JNK), as well as transcription factors like ATF-2 and NF-kappaB. In addition, the expression and activation of MMP-2, MMP-9, and MMP-13 were induced, which may be responsible for tissue remodeling followed by fibrosis. CONCLUSION: Due to their effects on the expression and activation of metalloproteases, AGEs should be regarded as a potential therapeutic target for the prevention of pathologic remodeling.

Protein "AGEing"--cytotoxicity of a glycated protein increases with its degree of AGE-modification.

Non-enzymatic glycation of proteins with reducing sugars and subsequent transition metal-catalyzed oxidations leads to the formation of protein-bound "advanced glycation endproducts" (AGEs). They accumulate on long-lived proteins including on and in the vicinity of the beta-amyloid plaques in Alzheimer's disease (AD). Since the AGE modification of a protein increases with time, and such a "long-term incubation" might also occur in the AD brain, we investigated whether an increase in the cytotoxic effects of an AGE-modified model protein occurs over time. Bovine serum albumin (BSA) was modified by glucose for defined time periods, and the viability of SH-SY5Y neuroblastoma cells, incubated with the differentially AGE-modified BSA samples, was measured with the MTT assay. Cytotoxicity of the AGE-modified BSAs increased in correlation to the incubation time with glucose. Among the AGE-specific markers, browning (OD 400) correlated best with cytotoxicity, followed by AGE-specific fluorescence and the defined AGE, carboxymethyllysine. Since AGEs accumulate in AD over time, they may be one of the "age-related" factors contributing to neuronal cell death in Alzheimer's disease.

Transition metal-mediated glycoxidation accelerates cross-linking of beta-amyloid peptide.

beta-Amyloid deposits, hallmarks of Alzheimer's disease, contain both sugar-derived 'advanced glycation end products' (AGEs) and copper and iron ions. Our in vitro experiments using synthetic beta-amyloid peptide and glucose or fructose show that formation of covalently cross-linked high-molecular-mass beta-amyloid peptide oligomers is accelerated by micromolar amounts of copper (Cu+, Cu2+) and iron (Fe2+, Fe3+) ions. Formation of these covalent AGE cross-links can be inhibited by capping agents of amino groups, redox-inactive metal chelators and antioxidants, suggesting that these drugs may be able to slow down the formation of insoluble beta-amyloid deposits in vivo and possibly the progression of Alzheimer's disease.

Cytotoxicity of advanced glycation endproducts is mediated by oxidative stress.

Non-enzymatic glycation of proteins with reducing sugars and subsequent transition metal catalysed oxidations leads to the formation of protein bound "advanced glycation endproducts" (AGEs). They accumulate on long-lived proteins and are for example structural components of the beta-amyloid plaques in Alzheimer's disease. Since the oxidation of glycated proteins as well as the interaction of AGEs with cell surface receptors produces superoxide radicals, it was tested in BHK 21 hamster fibroblast cells and SH-SY5Y human neuroblastoma cells if AGEs can exert cytotoxic effects on cells. Cell viability was assessed with three independent tests: MTT-assay (activity of the mitochondrial respiratory chain), lactate dehydrogenase assay (release of cytoplasmatic enzymes, membrane integrity) and Neutral Red assay (active uptake of a hydrophilic dye). Two model AGEs, chicken egg albumin-AGE and BSA-AGE, both caused significant cell death in a dose-dependent manner. The cytotoxic effects of AGEs could be attenuated by alpha-ketoglutarate and pyruvate, by antioxidants such as thioctic acid and N-acetylcysteine, and by aminoguanidine, an inhibitor of nitric oxide synthase. This suggests that reactive oxygen species as well as reactive nitrogen species contribute to AGE mediated cytotoxicity. Since AGEs accumulate on beta-amyloid plaques in AD over time, they may additionally contribute to oxidative stress, cell damage, functional loss and even neuronal cell death in the Alzheimer's disease brain.

Alzheimer's disease--synergistic effects of glucose deficit, oxidative stress and advanced glycation endproducts.

Many approaches have been undertaken to understand Alzheimer's disease (AD) but the heterogeneity of the etiologic factors makes it difficult to define the clinically most important factor determining the onset and progression of the disease. However, there is increasing evidence that the previously so-called "secondary factors" such as a disturbed glucose metabolism, oxidative stress and formation of "advanced glycation endproducts" (AGEs) and their interaction in a vicious cycle are also important for the onset and progression of AD. AGEs are protein modifications that contribute to the formation of the histopathological and biochemical hallmarks of AD: amyloid plaques, neurofibrillary tangles and activated microglia. Oxidative modifications are formed by a complex cascade of dehydration, oxidation and cyclisation reactions, subsequent to a non-enzymatic reaction of sugars with amino groups of proteins. Accumulation of AGE-crosslinked proteins throughout life is a general phenomenon of ageing. However, AGEs are more than just markers of ageing since they can also exert adverse biologic effects on tissues and cells, including the activation of intracellular signal transduction pathways, leading to the upregulation of cytokine and free radical production (oxidative stress). Oxidative stress is involved in various divergent events leading to cell damage, including an increase in membrane rigidity, DNA strand breaks and an impairment in glucose uptake. In addition, other age-related metabolic changes such as depletion of antioxidants or decreased energy production by a disturbed glucose metabolism diminish the ability of the cell to cope with the effects of radical-induced membrane, protein and DNA damage. With our improving understanding of the molecular basis for the clinical symptoms of dementia, it is hoped that the elucidation of the etiologic causes, particularly the positive feedback loops involving radical damage and a reduced glucose metabolism, will help to develop novel "neuroprotective" treatment strategies able to interrupt this vicious cycle of oxidative stress and energy shortage in AD.