Importance of measuring products of non-enzymatic glycation of proteins

Abstract

Non-enzymatic glycation products are a complex and heterogeneous group of compounds which accumulate in plasma and tissues in diabetes and renal failure. There is emerging evidence that these compounds may play a role in the pathogenesis of chronic complications associated with diabetes and renal failure. So measurement of the products of non-enzymatic glycation has a twofold meaning: on one hand, measurement of early glycation products can estimate the extent of exposure to glucose and the subject's previous metabolic control; on the other hand, measurement of intermediate and late products of the glycation reaction is a precious instrument in verifying the relationship between glycation products and tissue modifications. This review summarizes current knowledge about the diagnostic utility of measuring non-enzymatic glycation products.

Introduction

The non-enzymatic glycation of proteins, or Maillard reaction, is a process which links chronic hyperglycemia to a series of physiopathological alterations considered important in the development of the chronic complications of diabetes [1]. The Maillard reaction is subdivided into three main stages: early, intermediate, and late (Fig. 1). In the early stage, glucose (or other reducing sugars as fructose, pentoses, galactose, mannose, ascorbate, xylulose) react with a free amino group of several molecules, including proteins, nucleic acids, and lipids, to form an unstable aldimine compound, the Schiff base. Through rearrangement, this base gives rise to a stable ketoamine, the Amadori product. Since this reaction does not require the participation of enzymes, the variables which regulate it in vivo are the concentrations of glucose and protein, the half-life of the protein, its reactivity in terms of free amino groups, and cellular permeability to glucose. In in vivo conditions, the Amadori product reaches equilibrium after approximately 15–20 days and, through irreversible links, accumulates on both short-lived and long-lived proteins.

In the intermediate stage, through oxidation and dehydration reactions, the Amadori product degrades into a variety of carbonyl compounds (glyoxal, methylglyoxal, deoxyglucosones) which, being much more reactive than the sugars from which they are derived, act as propagators of the reaction, again reacting with the free amino groups of proteins. In particular, methyl glyoxal is a very reactive alpha-oxaldehyde which may be formed both from reactions which depend on the levels of glucose (non-enzymatic glycation, the polyol pathway) and from intermediate products of glycolysis, metabolism of ketone bodies, and catabolism of threonine. The high reactivity and elevated plasma concentrations of methyl glyoxal indicate that this compound is one of the most important ones in vivo [2].

In the late stage, these propagators again react with free amino groups and, through oxidation, dehydration and cyclization reactions, form yellow-brown, often fluorescent, insoluble, irreversible compounds, usually called Advanced Glycation End-Products (AGEs), which accumulate on long-lived proteins and cause damage. Although the chemical nature of these compounds is not yet well-defined, recent investigations indicate that they include post-Amadori products derived from oxidation and further structural rearrangements, so that compounds which are neither cross-linked nor fluorescent have been considered to belong to the AGE group. In this context, it should be emphasized that oxidation processes are important in the formation of many AGEs [3]. There are two mechanisms through which these processes take place, both catalyzed by metals such as copper and iron. The first involves auto-oxidation of free sugar in the presence of oxygen and free metals, leading to more reactive dicarbonyl compounds, which react with proteins to form highly reactive ketoamines. The second mechanism involves protein-bound products of the Amadori pattern which, in the presence of oxygen and free metals, are oxidized and give origin to highly reactive protein-enediols and protein-dicarbonyls which can generate AGEs (Fig. 2).

The three main mechanisms by means of which AGEs cause tissue damage are: cross-link formation, interaction with specific cellular receptors, and intracellular glycation [4] (Fig. 3). In this context, one of the most important is the formation of abnormal and stable cross-links on collagen, as demonstrated both in in vitro incubation with glucose and in vivo in the collagen of diabetic patients. Glyco-oxidation causes several chemical and physical modifications of collagen, leading to high levels of absorbance (280 nm), fluorescence, increased resistance to enzymatic digestion and denaturants, increased thermal stability, and reduced solubility, all of which clearly explain some of the structural tissue modifications typical of the chronic complications of diabetes, such as abnormal vascular rigidity, arterial stiffness, and basement membrane thickness. Moreover, non-enzymatic glycation of collagen can inhibit the release of nitric oxide of endothelial derivation, with consequent vasoconstriction, reduced plasma flow, and tissue ischemia. Lastly, AGE compounds in collagen may trap multiple macromolecules such as lipoproteins, immunoglobulin, fibrin, and albumin. Immunoglobulins bound to collagen retain their ability to form antigen–antibody complexes which may be deposited on vessels. Increased glycated LDLs also bind covalently to glycated collagen, thus contributing to vessel occlusion. In this context, there are also other mechanisms which are involved in the formation of atherosclerotic plaques, which may cause vessel occlusion. First, glycated LDLs are not identified by their receptor, but are preferentially recognized by high-capacity, low-affinity scavenger receptors on monocyte/macrophages, enhancing uptake by them, with consequent stimulation of cholesterol synthesis and “foam cell” formation. Second, glycated LDLs are capable of stimulating thromboxane β₂ release and inducing platelet aggregation. Third, glycated lipoproteins may generate free radicals, with consequent increased vessel oxidative damage. Lastly, glycated LDLs, due to their altered structure, are immunogenic and thus able to stimulate the production of antibodies: the resulting immune complexes may be deposited on vessel walls and stimulate “foam cell” formation.

Recent studies have shown the existence of specific cellular receptors which bind AGE proteins in a saturable manner. The first receptor for AGE, the “scavenger”, was purified from cell membranes and reported to have different molecular weights of 60 and 90 kDa. This receptor, expressed on the cell membrane of T lymphocytes and monocytes/macrophages, has the function of degrading senescent molecules. More recently, a 35-kDa protein, “RAGE”, has been identified as a new member of the Ig superfamily of cell surface molecules, codified from a gene on chromosome 6. Subsequently, this receptor was identified in various cell systems: monocytes/macrophages, T lymphocytes, fibroblasts, smooth muscle cells, neurons, red cells, and mesangial cells. The binding of AGE on RAGE T lymphocytes stimulates production of γ-interferon, with consequent tissue damage; binding of AGE to monocytes/macrophages induces production of cytokines (interleukin 1B, TNF-α, IGF-1, PDGF) and growth factors, with consequent increased synthesis of type IV collagen, increased proliferation of vessel smooth muscle cells, and stimulation of macrophage chemotaxis. In physiological conditions, cytokines are important regulators of tissue remodeling: on one hand, they stimulate the mesenchymal cells to produce hydrolase in order to degrade tissue proteins; on the other hand, they stimulate proliferation of endothelial and mesenchymal cells through reduction of tissue-derived growth factor. In diabetes, binding of AGE to RAGE reduces tissue protein degradation and increases production of growth factors, with a consequent increase in the synthesis of the extracellular matrix and impairment of the mechanisms of tissue remodeling.

The damage induced by AGE–RAGE binding varies and depends essentially on the type of cells involved, since the AGE–RAGE interaction in fibroblasts and smooth muscle cells determines an increase in growth factors (EGF, PDGF), with consequent cell proliferation. AGE–RAGE binding on mesangial cells causes a series of alterations such as increased production of collagen IV, laminin, and fibronectin, and activation of PDGF, all of which explain some structural modifications characteristic of diabetic nephropathy. Lastly, through a mechanism of oxidative stress, AGE–RAGE binding on endothelial cells induces the transcription factor NF-kB, which in turn increases expression of the vascular cellular adhesion molecule (VCAM-1). VCAM-1 overexpression then increases the adhesivity of monocytes to endothelial cells and vascular permeability, leading to accelerated transendothelial passage of AGE-modified proteins. Other alterations observed as a consequence of this AGE–RAGE interaction are an increase in the procoagulant response to TNF-α, a reduction in thrombomodulin expression, and an increase in endothelin-1 levels (Fig. 4).

Another protein with high binding affinity for AGE is galectin 3, later known as Mac-2 or 35 protein-binding carbohydrates, which is expressed on macrophages. The binding of AGE occurs at the COOH terminal peptide and promotes the formation of complexes of high molecular weight.

The third mechanism of AGE damage is dependent upon intracellular accumulation and has been demonstrated in macrophages, endothelial and smooth muscle cells, and atherosclerotic plaques. Two possible pathways of action are rapid intracellular formation of AGE induced by hyperglycemia, with consequent alteration of cytoplasmic and nuclear structures, and endocytosis, which follows binding to specific receptors.

An example of these three mechanisms of damage involved in the development of the chronic complications in diabetes is shown in Table 1, which highlights the role of AGE in diabetic atherosclerosis.

Measurement of products arising from the non-enzymatic glycation reaction therefore has a twofold meaning: on one hand, measurement of early glycation product, depending on the half-life of the glycated protein which is measured, yields an estimate of the extent of exposure to glucose and, therefore, the subject's previous metabolic control; on the other hand, measurement of the intermediate and late products of this reaction is a precious instrument in verifying the relationship between glycation products and tissue modifications, to clarify the pathogenesis of chronic complications and the association between exposure to glucose and its development (Table 2).