Formation Of Advanced Glycosylation End Products Research Articles

Diabetic nephropathy. Its relationship to hypertension and means of pharmacological intervention.

Hypertension and diabetes mellitus are common chronic conditions which frequently coexist. Diabetic nephropathy is a major cause of elevated blood pressure in patients with insulin-dependent diabetes mellitus (IDDM). Diabetic nephropathy, arterial sclerosis, obesity and association of essential hypertension can be the causes of hypertension in patients with non-insulin-dependent diabetes mellitus (NIDDM). Ambulatory blood pressure monitoring has revealed that the nocturnal fall of blood pressure is blunted in patients with diabetic nephropathy. A blunted diurnal blood pressure variation is seen in microalbuminuric diabetic patients and even in some normoalbuminuric patients. Accumulating data suggest that normalisation of blood pressure in hypertensive IDDM patients is most important to minimise the loss of kidney function. Angiotensin converting enzyme (ACE) inhibitors have been reported to be effective in postponing the development of nephropathy and in slowing its progression. Whether only ACE inhibitors have such beneficial renal effects on diabetic nephropathy is under discussion. While many studies have suggested that insulin resistance and hyperinsulinaemia are related to an elevated blood pressure in hypertensive patients, there does not seem to be enough evidence to prove that insulin per se can raise blood pressure in humans. Neither an insulin infusion within a physiological range nor sustained hyperinsulinaemia and insulin resistance (e.g. patients with insulinoma, cystic ovary syndrome) have been associated with an elevated blood pressure. Insulin resistance in some hypertensive patients may be a consequence of a decreased blood flow due to an increased peripheral resistance. Preliminary evidence suggests that low birth weight or impaired fetal growth is related to hypertension and NIDDM. Familial clustering of diabetic nephropathy suggests the contribution of genetic susceptibility and/or environmental inheritance. The frequent association of nephropathy with hypertension has led to research on the genes related to hypertension (ACE, angiotensinogen). Nevertheless, to date no reliable and clinically useful genetic marker has been found. Attempts to correct the metabolic abnormalities derived from diabetes are a new topic in the treatment of diabetic nephropathy. The effects of HMG CoA reductase inhibitors (antihypercholesterolaemic drugs), aldose reductase inhibitors (inhibitors of the polyol pathway) and glycation inhibitors (inhibitors of formation of advanced glycosylation end-products) on diabetic nephropathy have been evaluated in animal studies and in some clinical trials. Thus far, results with HMG CoA reductase and aldose reductase inhibitors have been somewhat conflicting. The potential therapeutic role of glycation inhibition in the treatment of diabetes deserves further study.

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

Chronic dosing with aminoguanidine and novel advanced glycosylation end product-formation inhibitors ameliorates cross-linking of tail tendon collagen in STZ-induced diabetic rats

Chronic dosing with aminoguanidine and novel advanced glycosylation end product-formation inhibitors ameliorates cross-linking of tail tendon collagen in STZ-induced diabetic rats

Alterations in Venous Endothelial Cell and Smooth Muscle Cell Relaxation Induced by High Glucose Concentrations Can Be Prevented by Aminoguanidine

High glucose concentrations lead to the formation of advanced glycosylation end-products (AGE). Increased glucose concentrations are found during the systemic inflammatory response syndrome and during coronary artery bypass surgery. This study examines the pharmacological effect of AGE on venous endothelial-dependent and -independent relaxation and seeks to determine if aminoguanidine, a known AGE inhibitor, can prevent changes induced by the presence of high glucose concentrations. Standard isometric tension studies on rings from endothelialized and deendothelialized rabbit external jugular veins were performed after incubation for 6 hr in 5.5 mMglucose (control) or 44 mMglucose. The effects of preincubation with either indomethacin (10 μM) to inhibit cyclo-oxygenase activity or aminoguanidine (10 μM) to inhibit protein glycosylation were also studied. In the presence of 44 mMglucose, there was a significant reduction in acetylcholine (endothelial cell based)- and forskolin-induced (smooth muscle cell based) relaxation without associated alterations in serotonin-, calcium ionophore-, and sodium nitroprusside-mediated relaxations. The alterations in acetylcholine-mediated relaxation were inhibited by the addition of indomethacin; co-incubation with aminoguanidine prevented the decrease in acetylcholine-mediated and forskolin-mediated responses. This study shows thatin vitroelevated glucose concentrations lead to a reduction in both endothelial cell and smooth muscle cell relaxation, which may be due to AGE-mediated generation of endothelial cell cyclo-oxygenase products and AGE-induced changes in cAMP-mediated relaxation. Therefore, AGE production in the vessel wall cells produces alterations in receptor-dependent and receptor-independent cyclo-oxygenase production and these changes result in altered endothelial- and nonendothelial-mediated relaxation. Alterations in the endothelial and smooth muscle cell responses can be inhibited by aminoguanidine, suggesting that means to reduce or prevent glycosylation are beneficial in ameliorating the acute changes induced by short-term exposure to elevated glucose concentrations.

Aminoguanidine has both pro-oxidant and antioxidant activity toward LDL.

We previously demonstrated that aminoguanidine (AMGN) was able to prevent oxidative modification of LDL. Initially, we thought that this occurred solely because AMGN trapped reactive breakdown products of lipid peroxidation and prevented apoB modification, similar to AMGN's proposed ability to trap reactive glucose intermediates and prevent advanced glycosylation end-product formation. We now demonstrate that AMGN also displays dose-dependent pro-oxidant and antioxidant activity toward LDL. Moderate doses of AMGN (0.05 to 1.0 mmol/L) prevented lipid peroxidation in LDL exposed to copper. AMGN prevented the loss of polyunsaturated fatty acids and delayed or prevented conjugated-diene formation, both of which are sensitive indicators of lipid peroxidation. The same doses of AMGN also prevented apoB modification, a step distal to lipid peroxidation, as evidenced by the ability to (1) prevent fluorescence at 420 nm, (2) block enhanced electrophoretic mobility, and (3) prevent changes leading to enhanced macrophage uptake. Thus, AMGN inhibits LDL modification both by inhibiting lipid peroxidation as well as by trapping reactive breakdown products of lipid peroxidation. It was also demonstrated that for every LDL, there was also a very low dose of AMGN (about 0.01 mmol/L) that actually promoted lipid oxidation and subsequent protein modification. This activity of AMGN could be enhanced by increasing the content of lipid hydroperoxide in the LDL, eg, by aging or radioiodinating the LDL. Conversely, the pro-oxidant activity could be reduced by pretreatment of LDL with ebselen or vitamin E. We propose a mechanism by which AMGN effects pro-oxidant activity toward LDL at very low concentrations and antioxidant activity at higher concentrations and discuss the practical implications of these observations.

'Toxicity of glucose: is AGE the answer?'

Despite a growing knowledge of the pathogenesis of diabetic nephropathy, the increased incidence of end-stage renal disease in diabetic patients continue to pose problems of enormous public health and economic importance. Recently, a growing body of evidence has linked the accumulation of the late products of glucose-protein interaction to a variety of chronic complications, including diabetic nephropathy. The formation of irreversible 'advanced glycosylation endproducts' resulting from the spontaneous reaction between glucose and proteins occurs most noticeably on long-lived structural proteins. In this article, I review recent studies suggesting that the pathogenesis of diabetic nephropathy leading to end-stage renal disease is caused by the hyperglycaemia-accelerated formation of advanced glycosylation endproducts. Recent studies suggest that reactive AGE peptides in the circulation potentially play a role as a new version of so called 'middle molecules toxic substances'. The evidence is opening a new window for our understanding of the pathogenesis of diabetic end-stage renal disease and the benefits of various of treatment modalities.

The cognition-enhancing drug tenilsetam is an inhibitor of protein crosslinking by advanced glycosylation.

Non-enzymatic glycosylation of proteins, also called Maillard reaction, which occurs at an accelerated rate in diabetes, can lead to the formation of advanced glycosylation endproducts (AGEs). Tenilsetam (CAS 997: (+/-)-3-(2-thienyl)-2-piperazinone), a cognition-enhancing drug successfully used for treatment of patients suffering from Alzheimer's disease, when included in the Maillard reaction apparently inhibits protein crosslinking by AGEs in vitro. According to the mechanism proposed, Tenilsetam acts via covalent attachment to glycated proteins, thus blocking the reactive sites for further polymerisation reactions. A beneficial effect of Tenilsetam in Alzheimer's disease could come from the interference with AGE-derived crosslinking of amyloid plaques and a decreased inflammatory response by diminished activation of phagocytosing microglia.

Diabetic nephropathy: Are we making progress in delaying renal failure?

Patients suffering from the severe complications associated with both insulin- (IDDM) and noninsulin-dependent diabetes mellitus (NIDDM): nephropathy, retinopathy, neuropathy, and atherosclerosis are still largely left without a prospect of an efficient treatment. This is the case even if it has been assumed for decades and now finally proved by the results from the Diabetes Control and Complications Trial (DCCT) that hyperglycemia is the single main cause of these complications. Improved glycemic control as a result of intensive insulin treatment has the potential to reduce the incidence and progression of complications, but implementation and monitoring of improved glycemic control in all groups of IDDM and NIDDM patients in different communities will be difficult and expensive. Results from the recently terminated DCCT have shown that even with intensive insulin treatment, there will still be a significant burden of complications on the diabetic population. It will, therefore, still be of immense importance for the long-term quality of life for the diabetic patient that additional possibilities are developed for prevention and intervention against diabetic complications. Almost two decades of research, animal model testing, and clinical trials have been conducted on various efficient aldose reductase inhibitors. Now the concept of inhibition of formation of advanced glycosylation endproducts on proteins and lipids resulting from extra- and intracellular hyperglycemia is entering the scene as an alternative or perhaps supplementary approach to reduce the occurrence of diabetic complications. An overview of the results from these two fields of research and associated drug-development programs will be presented along with thoughts on possible future developments.

Diabetic Nephropathy in Insulin-Dependent Patients

Diabetic nephropathy is a serious complication of insulin-dependent diabetes mellitus (IDDM) that affects 30% to 40% of IDDM patients with a predictable time of onset. Epidemiologic data suggest that either a genetic susceptibility, perhaps for hypertension (HTN), or an environmental exposure selects out that subset of IDDM patients and destines them to develop diabetic nephropathy. Hopefully, assessing glomerular hyperfiltration, urinary albumin excretion rate (AER), glycemic control, mean arterial pressure (MAP), and perhaps early morphologic changes will allow early identification of this high-risk group of IDDM patients before overt nephropathy is present. Once nephropathy appears, renal function inexorably declines, although the natural history of this progression may be changing with earlier therapeutic intervention. IDDM patients with nephropathy suffer a high mortality rate compared with IDDM patients without nephropathy or with nondiabetic end-stage renal disease patients. This is primarily due to malignant atherosclerotic disease manifested as coronary, peripheral, and cerebral arterial disease. Therapeutic interventions of demonstrated benefit in slowing the rate of decline of glomerular filtration rate (GFR) include blood pressure control and low-protein diets. Strict blood sugar control or treatment with aldose reductase inhibitors, converting enzyme inhibitors (CEIs), or inhibitors of advanced glycosylation end-product formation are of possible benefit, but are awaiting clinical trial results.

Mechanisms of glucose-induced extracellular matrix overproduction by cultured vascular cells

Mesangial expansion, the characteristic anatomical lesion of diabetic glomerulosclerosis, is linked predominantly to accumulation of extracellular matrix, due to either increased production or reduced degradation. Although increased matrix synthesis and/or transcription have been demonstrated in vitro in vascular cells (endothelial cells, pericytes and mesangial cells) exposed to elevated glucose concentrations, the mechanisms of high glucose-induced matrix overproduction are still unclear. Several metabolic and biochemical alterations associated with hyperglycaemia have been implicated in the pathogenesis of these changes. A growing body of experimental evidence suggests that non-enzymatic glycosylation of circulating and matrix proteins with formation of advanced glycosylation end-products, diacylglycerol accumulation and associated protein kinase C activation, or glucose per se, possibly via increased osmolarity, may mediate glucose-induced extracellular matrix overproduction.

Glycosylation Products as Toxic Mediators of Diabetic Complications

Hyperglycemia causes excessive amounts of irreversible advanced glycosylation end products (AGEPs) to accumulate on long-lived extracellular matrix proteins and probably also on DNA in tissues that develop diabetic complications. AGEPs induce permanent abnormalities in extracellular protein cross-linking, cell-matrix interactions, and DNA structure in vitro. Pharmacologically inhibiting AGEP formation in long-term diabetic animals prevents both retinal capillary pathology and thickening of the glomerular basement membrane.

Non-enzymatic glycosylation.

Experience with the use of glycosylated haemoglobin throughout the 1980s has confirmed its uniqueness and usefulness as an objective index of long-term glycaemia in diabetes mellitus, and has enabled the definition of realistic and achievable targets for outpatient management. Measurement of glycosylated serum proteins yields information over a much shorter time-scale which may be particularly useful in diabetic pregnancy. The formation of advanced glycosylation end-products may provide a link between hyperglycaemia and chronic diabetic complications. Therapeutic inhibition or the promotion of alternative metabolic pathways, to yield inert glycosylated products, represents an innovative approach to the problem of preventing these complications.