Abstract
Modifications of extant plasma proteins, structural proteins, and other macromolecules are enhanced in diabetes because of increased glycation (secondary to increased glucose concentrations) and perhaps because of increased oxidative stress. Increased glycation is present from the time of onset of diabetes, but the relation between diabetes and oxidative stress is less clear: increased oxidative stress may occur later in the course of disease, as vascular damage becomes established, or it may be a feature of uncomplicated diabetes. The combined effects of protein modification by glycation and oxidation may contribute to the development of accelerated atherosclerosis in diabetes and to the development of microvascular complications. Thus, even if not increased by diabetes, variations in oxidative stress may modulate the consequences of hyperglycemia in individual diabetic patients. In this review, the close interaction between glycation and oxidative processes is discussed, and the theme is developed that the most significant modifications of proteins are the result of interactions with reactive carbonyl groups. While glucose itself contains a carbonyl group that is involved in the initial glycation reaction, the most important and reactive carbonyls are formed by free radical–oxidation reactions damaging either carbohydrates (including glucose itself) or lipids. The resulting carbonyl-containing intermediate products then modify proteins, yielding “glycoxidation” and “lipoxidation” products, respectively. This common pathway for glucose and lipid-mediated stress, which may contribute to diabetic complications, is the basis for the carbonyl stress hypothesis for the development of diabetic complications.
The Diabetes Control and Complications Trial (DCCT) established conclusively that intensive management can delay the onset and decrease the rate of progression of the microvascular complications of diabetes (1). A significant effect on macrovascular disease was not expected: the DCCT patient population was young, and individuals at high risk for macrovascular disease had been excluded at the outset. Nevertheless, over the average of 6 years’ follow-up reported in the study, there were almost twice as many major macrovascular events in the standard management group (40 events) as in the intensive management group (23 events) (2). While this difference did not reach statistical significance (P = 0.08), it was the first suggestion of a relationship between the intensity of diabetes management and the subsequent development of macrovascular disease. In previous epidemiological studies, both diabetes and impaired glucose tolerance have been identified as independent risk factors for the development of macrovascular disease (3–7). All of these findings are consistent with, though do not prove, the hypothesis that high glucose concentrations contribute directly to the acceleration of atherosclerosis in diabetes.
One mechanism by which hyperglycemia may cause complications is through increased glycation, the spontaneous, nonenzymatic reaction of glucose with proteins. In diabetes, elevated levels of glucose are present from the time of onset of the disease, not only in plasma but also in interstitial fluid and within cells (e.g., in the kidney, retina, and nerve) where glucose entry is not insulin dependent. As a result, glycation of a wide variety of proteins is enhanced. This realization led to the development, more than 10 years ago, of the glycation hypothesis to explain the pathogenesis of diabetic complications. Reviewed at the time by Kennedy and Baynes (8), this hypothesis proposed that enhanced protein modification leads to abnormalities in, e.g., turnover rate, receptor recognition, enzyme activity, and physicochemical properties, and that over time these abnormalities are sufficient to contribute to disease development.
Although population studies such as those mentioned above demonstrate associations between glycemic control and the development of complications, individual patients often prove exceptions to the rule. Some develop severe complications early in the course of their diabetes despite reasonable glycemic control, and others seem resistant to the development of complications despite poor long-term glycemic control. Building on the basic concept of the glycation hypothesis, i.e., that postsynthetic modifications of macromolecules are accelerated in diabetes and that this contributes to complications, two newer pathogenic mechanisms have been proposed by ourselves and others.
First, evidence is accumulating that free radical–oxidation reactions are implicated. This evidence has been reviewed recently by Baynes (9), who defines oxidative stress as “the steady state level of reactive oxygen or oxygen radicals in a biologic system.” The processes of glycation and oxidation are closely linked: free radical oxidation of glycated residues in proteins is irreversible and yields advanced glycation end products (AGEs), also known as glycoxidation products to emphasize their dual origin. Alternatively, the same products may be formed if glucose is oxidized directly, and the products of glucose oxidation then bind to proteins. The combined stresses of hyperglycemia and free radical oxidation mediate much more severe protein damage than either alone, suggesting that variations in oxidative stress may modulate the consequences of hyperglycemia. We termed this concept the glycoxidation hypothesis (reviewed in 10, and in this article).
Second, it is now clear that during glycoxidation, the modification of proteins is by reactive carbonyl groups present either in glucose itself or in oxidation products of glucose. (A carbonyl group consists simply of a carbon and an oxygen atom linked by a double bond, and it is the characteristic feature of aldehydes and ketones.) This realization allows the concept of glycoxidation to be broadened to encompass protein modification by carbonyl groups from sources other than glucose. Of particular interest are the aldehyde-containing products of oxidation of polyunsaturated fatty acids in lipoproteins and cell membranes. These lipid oxidation products are now known to react with proteins, yielding lipoxidation products, the term emphasizing the analogy with the formation of glycoxidation products from glucose oxidation products. Examples of specific carbonyl-containing compounds derived from carbohydrates and lipids are illustrated in Fig. 1. Indeed, at least one specific product, derived from glyoxal, can be formed either by glycoxidation or by lipoxidation (see details below). Oxidative damage to lipids may be enhanced in diabetes for a variety of reasons, also outlined below. In this review, therefore, we will develop the hypothesis that the complications of diabetes, including accelerated atherosclerosis, may be mediated by complex combinations of interacting biochemical stresses: these include elevated glucose concentrations, qualitative and quantitative abnormalities of plasma lipoproteins and membrane lipids, and oxidation reactions that directly damage both carbohydrates and lipids. The final common pathway is damage to macro-molecules by reactive carbonyl-containing species. Of the target molecules, proteins have been most closely studied and are the subject of this review. We have termed the ideas outlined in this paragraph the carbonyl stress hypothesis for the development of diabetic complications. An outline of the various processes is provided in Fig. 2.
Figure 1.
Chemical structures of carbonyl-containing species which may modify proteins. Glyoxal, malondialdehyde, and 4-hydroxynonenal are formed by free radical oxidation of carboxyhydrates or lipids. Reprinted with permission from Lyons and Jenkins (8a).
Figure 2.
Reactive oxygen species (ROS) attack lipids, protein, glucose, and FL, yielding reactive carbonyl-containing species that mediate the formation of glycoxidation and lipoxidation products. In the process, more ROS are generated (⇢), establishing further vicious and interactive cycles of molecular damage. MetSO, methionine sulfoxide, a protein oxidation product. Reprinted with permission from Lyons TJ and Jenkins AJ (8a).
CHEMISTRY OF GLYCATION
Glycation of proteins has conventionally been considered as being composed of early and advanced stages. The chemical reactions involved are illustrated in Fig. 3, and they are described briefly below (for greater detail, see 11 for review). The early stage involves the formation of fructoselysine (FL), while the later stages involve the subsequent formation of glycoxidation products (also known as AGEs). More recently, it has been realized that this traditional concept is an oversimplification: direct oxidative damage to glucose and other sugars (auto-oxidation) may yield reactive carbonyl-containing compounds that react directly with protein, a situation analogous to that occurring in lipids during lipoxidation (as outlined above). Thus, glycoxidation products may be formed with or without FL formation (i.e., early glycation) as an intermediate stage. Both sequences of events are illustrated in Fig. 2. Recently, Baynes’s group (12) identified the simplest of all dicarbonyls, glyoxal, as the principal product of glucose auto-oxidation.
Figure 3.
Reactions of early and late glycation, shown affecting LDL ➔, processes occurring in vivo; ⇒, processes occurring in vitro. The reduction of the ketoamine product by sodium borohydride yields a mixture of glucitollysine and its stereo-isomer, mannitollysine. From Lyons (79).
Early Glycation
In the conventional initial stage of glycation (Fig. 3), covalent bonds are formed between aldehyde groups of glucose molecules and reactive amino groups of proteins (11). The latter are usually located on lysine side chains and NH2-terminal amino-acid residues. As illustrated, this reaction can only occur with glucose in its open-chain configuration, as opposed to the ring configuration: only in the open-chain form is a reactive carbonyl group exposed. As an aside, in evolutionary terms, glucose may be the metabolic fuel of choice precisely because it exists predominantly in the ring form and is therefore less reactive with proteins. For the same reason, glucose has the advantage of being less susceptible to auto-oxidation (i.e., direct oxidative damage, fragmenting the molecule) than are most other monosaccharides.
The first reaction product of early glycation is an unstable intermediate, known as a Schiff base, whose formation is readily reversible (Fig. 3). Alternatively, following an Amadori rearrangement, the first stable product, FL, is formed. This adduct is fructoselysine, not glucoselysine, because of the shift in the position of the carbonyl group (from C1 to C2) during the Amadori rearrangement. FL is relatively stable, but it too may decay, releasing its carbohydrate moiety either as glucose or as more reactive hexoses, such as 3-deoxyglucosone (13–16), which themselves may modify proteins. Alternatively, FL may partake directly in further advanced glycation reactions involving free radical oxidation, as described in the next section. The instability of FL explains the observations that it does not accumulate as a function of age in humans, even in long-lived proteins such as insoluble collagen (17, 18). Instead, FL appears to exist in a steady-state relationship with ambient glucose concentration (Fig. 4). Consequently, skin collagen FL levels are increased in diabetes, but do not correlate with either age or duration of disease. Instead, they correlate with HbA1c (19) and, to a greater extent, with mean HbA1c values obtained at intervals over several years before skin biopsy (18). Also, FL levels in collagen respond in a matter of weeks to improved glycemic control (20). In diabetes, metabolic alterations attributable to increased FL (rather than the subsequent formation of glycoxidation products) are most likely to predominate in short-lived proteins, which do not exist long enough to accumulate high levels of advanced glycation products. The consequences of FL formation, both in short-lived plasma proteins (especially lipoproteins) and in long-lived structural proteins, are discussed below.
Figure 4.
Effects of age and diabetes on glycation (FL content) of human skin collagen. The equation of the line for control subjects aged >20 years (not shown) is: mmol FL/mol Lys = [0.029 × age (years)] + 3.27, (r = 0.54, P < 0.001). There was no significant relationship between age and skin collagen glycation in the diabetic patients. Reprinted from Dyer et al. (18) with permission from the American Society for Clinical Investigation.
Advanced Glycation
Advanced glycation is a complex process that remains only partially understood. It has been the subject of several recent review articles (21–26). In the conventional view, FL formation is a prerequisite for advanced glycation to occur (Fig. 3). Advanced glycation then comprises a series of reactions involving FL residues in proteins or interactions of proteins with FL dissociation products (14, 15). These reactions lead to the formation of a large number of stable end products, and virtually all involve free radical oxidation (see below). More recently, it has become established that these end products may also be derived from direct reactions between proteins and the auto-oxidation products of glucose itself (9, 16, 27). Thus, one product, Nɛ-carboxymethyllysine (CML, described below), may be formed either by oxidative decomposition of FL (Fig. 3) or by interactions of proteins with glyoxal, the principal dicarbonyl-containing auto-oxidation product of glucose (Fig. 1) (and also a product of lipid peroxidation). Which of these two alternatives for the formation of glycoxidation products from glucose and proteins is predominant may depend on reaction conditions (12, 16), but both involve combinations of glucose-mediated and oxidative stress.
The end products formed by advanced glycation have been variously termed Maillard products (after Louis Maillard, food chemist [28]), glycoxidation products (our preferred term, emphasizing their dual origin), browning products, or AGEs. They include many species that are colored and fluorescent and which constitute cross-links. However, only two have been identified conclusively (Fig. 3): CML (29) (also the closely related species Nɛ-carboxymethylhydroxylysine, 3-(Nɛ-lysino)-lactic acid [30], and Nɛ-carboxyethyllysine [31]) and pentosidine (32). Others have been identified in model systems and by immunologic techniques in vivo; these include pyrraline (33) and crosslines (34–36). Pyrraline has, in addition, recently been identified in skin collagen in vivo by chromatographic methods (37): it is a glucoselysine adduct that may interact with other amino acids in the formation of cross-links (38).
As already stated, it has become clear in recent years that free radical-oxidation reactions are involved in the formation of both CML and pentosidine, and indeed in most, if not all, glucose-derived cross-links (9,39). Fu et al. (39) have shown that even prolonged (several weeks) exposure of collagen to high glucose concentrations leads to CML, pentosidine, and cross-link formation only if a prooxidant environment is present. Current knowledge concerning these two best-characterized glycoxidation products is summarized below.
CML, first identified by Ahmed et al. in 1986 (29), was initially recognized as a product of free radical–mediated oxidative cleavage of FL (Fig. 3). In contrast to FL, it accumulates almost linearly with age in skin collagen in normal individuals and at an accelerated rate in diabetes (Fig. 5) (18). Also in contrast to FL, the CML content of skin collagen cannot be lowered by improved glycemic control, at least in the short term (3–4 months) (20). It has been suggested that longer-term improvements in glycemic control might lower levels of CML and other AGEs in collagen (40), but this has not been proven. Even though most glycoxidation products are uncharacterized, it is clear that CML is in some ways atypical: it is not fluorescent, and, being stable and unreactive, it is not involved in crosslink formation. It can be formed in vitro in reactions between proteins and carbohydrate species other than glucose, including other sugars (pentoses) and ascorbate (41), and so its precise origin in skin collagen is currently unclear. Recently, there have been two new discoveries concerning CML. First, it has been shown that CML may also be formed by lipoxidation of proteins, because glyoxal may be generated during oxidation of fatty acids as well as of glucose (42). In both cases, the formation of CML always involves a free radical-oxidation reaction, and as stated above, in vitro studies have shown that when collagen is incubated with glucose under antioxidant conditions, no. CML is formed (39). CML may be formed under antioxidant conditions only from glyoxal, an oxidation fragment of both glucose and fatty acids (42, 43). Second, evidence is emerging that CML may be the main epitope in the formation of antibodies against AGEs (44). Such immunologic responses may be implicated in the pathogenesis of complications of diabetes, and they are discussed below.
Figure 5.
Effects of age and diabetes on the concentration of CML in human skin collagen. The equation of the line for control subjects is: mmol CML/mol Lys = [0.018 × age] − 0.022, (r = 0.89, P < 0.001); and for the diabetic patients: mmol CML/mol Lys = [0.027 × age] − 0.015, (r = 0.84, P < 0.001). Reprinted from Dyer et al. (18) with permission from the American Society for Clinical Investigation.
Pentosidine was characterized by Sell and Monnier in 1989 (32), and its structure is illustrated in Fig. 3. It is a lysine-arginine cross-link that forms between or within protein molecules. Pentosidine is entirely different in structure from CML, yet there are many similarities between the two compounds. Pentosidine accumulates in long-lived proteins in an almost linear fashion throughout life (Fig. 6). Its accumulation is accelerated by the presence of diabetes (18, 45; Fig. 6), and in diabetic patients, levels in skin collagen do not respond to short-term improvements in glycemic control (20). As its name implies, pentosidine was originally thought to be derived from pentoses, but it is now established that it can also be formed from other carbohydrates, including glucose (46, 47). Unlike CML, it is not a lipoxidation product. In diabetes, increased levels of pentosidine are probably the direct result of elevated glucose levels, but as with CML, pentosidine formation also requires the presence of an oxidative environment (39). Therefore, again like CML, pentosidine levels could be influenced by variations in oxidative stress (9, 39), and in long-lived proteins they may provide an index of combined long-term “glycative” and oxidative stresses. In contrast to CML, pentosidine is intensely fluorescent (32, 46, 47). Pentosidine levels in collagen correlate well with total fluorescence both during aging and in the presence of diabetes (45; Fig. 7A). The common origin of CML and pentosidine is supported by their concerted increases in skin collagen with advancing age and in diabetes (Fig. 7B). In the lens, in contrast to skin collagen, a glycemic threshold for increased pentosidine levels has recently been described (48). Thus, although pentosidine contributes only a small fraction of total fluorescence in long-lived proteins, it may still be considered a biomarker for the total modification of collagen by advanced glycation (9, 47).
Figure 6.
Effects of age and diabetes on the concentration of pentosidine in human skin collagen. The equation of the line for control subjects is: μmol pentosidine/mol Lys = [0.41 × age] − 0.48, (r = 0.78, P < 0.001); and for the diabetic patients: μmol pentosidine/mol Lys = [0.93 × age] −10.27, (r = 0.83, P < 0.001). Reprinted from Dyer et al. (18) with permission from the American Society for Clinical Investigation.
Figure 7.
Correlations of fluorescence (A) and CML (B) with pentosidine in skin collagen from the control (nondiabetic) and diabetic groups. Correlation coefficients and P-values are as follows: A: for the control subjects, fluorescence units/μg Hyp = [0.059 × (μmol pentosidine/mol Lys)] + 0.39, (r = 0.92, P < 0.001), and for the diabetic patients, fluorescence units/μg Hyp = [0.048 × (μmol pentosidine/mol Lys)] + 0.57, (r = 0.96, P < 0.001); B:for the control subjects, mmol CML/mol Lys = [0.039 × (μmol pentosidine/mol Lys)] + 0.10, (r = 0.94, P < 0.001), and for the diabetic patients, mmol CML/mol Lys = [0.029 × (μmol pentosidine/mol Lys)] + 0.27, (r = 0.92, P < 0.001). Reprinted from Dyer et al. (18) with permission from the American Society for Clinical Investigation.
Another Alternative Route for Advanced Glycation
Another dicarbonyl, methylglyoxal, is generated inside cells from glucose metabolism, ketone oxidation, and amino acid metabolism. Its formation, at least from the first two sources, is enhanced in diabetes. Like glyoxal, it can readily modify proteins and generate AGEs (49). Its catabolism is dependent on the presence of reduced glutathione and is therefore likely to be diminished if oxidative stress is increased. Its potential role in the development of diabetic complications has been the subject of recent reviews (50, 51).
LIPOXIDATION
Oxidative damage affects fatty acids, especially the unsaturated fatty acid constituents of triglycerides and cholesteryl esters. As with glucose, reactive aldehydes, including glyoxal, malondialdehyde (MDA), and 4-hydroxynonenal (4-HNE), are formed, and these may react with lysine residues in proteins. The resulting lysine adducts, termed lipoxidation products, in apolipoprotein B of LDL (CML, lysine-MDA, the cross-link lysine-MDA-lysine, lysine-HNE, and histidine-HNE) have recently been identified by our group (31, 42) (Fig. 1). Furthermore, we found that formation of these products in LDL during exposure to copper-mediated oxidative stress follows after the generation of lipid peroxides, as would be expected. The role of these products in the pathogenesis of atherosclerosis or diabetic complications is presently unknown, although in one study, no increase in platelet aggregability was shown following exposure to LDL modified by MDA or 4-HNE (52).
CARBONYL STRESS
As outlined above, the process of modification of macromolecules in diabetes may now be regarded in a more general way: the reactions involve species that contain reactive carbonyl groups, many of which are generated from carbohydrate or lipid substrates by free radical–oxidation reactions. These carbonyls may damage not only proteins but also phospholipids (53, 54) and nucleotide bases in DNA (55, 56), and in so doing they may contribute significantly to the development of diabetic complications. In this review, we have introduced the term carbonyl stress hypothesis to describe these mechanisms for the pathogenesis of the complications of diabetes. The review is restricted to the effect of these modifications on proteins.
OXIDATIVE STRESS IN DIABETES
The studies outlined above support the presence of an interrelation between glycation and oxidation in the browning of proteins, and they led to the introduction of the terms glycoxidation and glycoxidation products (9). Although it is clear that the glycation component of glycoxidation is enhanced by diabetes, the effect of diabetes on oxidative stress is less well defined. This is partly because oxidative stress is much more difficult to measure than glycative stress, which may be estimated simply from serial measures of glycemic control. In any attempt to measure oxidative stress in diabetes, it is important to distinguish the effects of diabetes itself from those of any vascular complications that may have developed; atheroma, and perhaps diabetic microvascular complications, may be associated, by some measures at least, with increased oxidative stress (57; reviewed in 58, 59). It may therefore be difficult to distinguish cause and effect.
Some lines of evidence suggest that oxidative stress is not increased in diabetes per se. In studies by our group, glycoxidation products were measured in long-lived structural proteins (skin collagen and lens crystallins). These proteins are likely to undergo the greatest degree of irreversible glycoxidative modification and thus provide indices of long-term combined glycation and oxidation stress. If the glycative stress is known (from long-term glycemic history), then oxidative stress may be deduced retrospectively. By this method, our work (summarized below) has not shown evidence of a general increase in oxidative stress in diabetes. This conclusion depends on several assumptions, e.g., concerning protein turnover. Other estimates may be derived from direct measures of oxidative damage to proteins (i.e., oxidation without involvement of carbohydrates or lipids). These include determination of the oxidation products of phenylalanine, tyrosine, and methionine residues (o-tyrosine, dityrosine, and methionine sulfoxide, respectively) (60, 61). Levels of these products do not differ in lens proteins between diabetic and nondiabetic subjects (J.W. Baynes, personal communication), again suggesting no overall increase in oxidative stress in diabetes.
Conversely, significant circumstantial evidence supports an increase in oxidative stress in diabetes. Hyperglycemia and glycation itself may enhance the susceptibility of proteins in general to oxidative damage, at least in vitro (27, 62–65). Furthermore, glycation of particular proteins may be especially important. Proteins containing transition metals (e.g., ferritin, ceruloplasmin, superoxide dismutase) may undergo changes in conformation, causing release of the metal ion. Once free, such ions may act as potent catalysts for oxidative reactions (66). In the case of superoxide dismutase, not only is there release of a transition metal ion, but the enzyme, whose normal function is to detoxify the superoxide radical, is simultaneously inactivated by cleavage (66). Other considerations supporting increased oxidative stress in diabetes include decreased plasma levels of certain antioxidants, increased respiratory bursts by polymorphonuclear leukocytes secondary to increased infection, and ischemia-reperfusion injury secondary to vascular disease (reviewed in 58). Binding of erythrocytes whose cell membranes contain AGEs to specific receptors on endothelial cells (see below) has been shown to generate oxidative stress (67). Studies comparing plasma peroxyl radical-trapping capacity (68) and the rate of salicylate hydroxylation (69) in relatively complication-free type 1 diabetic patients compared with nondiabetic control subjects suggest increased oxidative stress in diabetes.
Whether or not oxidative stress is increased in diabetes therefore remains controversial. However, it is important to emphasize that even if there is no general increase caused by the presence of diabetes, oxidative stress may still be an important modulator for the development of diabetic complications. First, patients who are at the upper end of the distribution of oxidative stress in the general population may, if they develop diabetes, be the most susceptible to complications. Second, oxidative stress may increase once vascular damage becomes established in diabetic patients and may be further amplified in vicious cycles of subsequent damage, compounded by abnormalities of carbohydrate and lipid metabolism, as the disease progresses. It is of interest that the two most widely held hypotheses to explain the aging process are the glucose theory of aging (70–72) and the free-radical (oxidative) theory of aging (73), both of which are relevant to the formation of glycoxidation products. Other recent studies have linked the accumulation of glycoxidation products to the aging process. The life spans of many organisms (in fact, of all species so far studied) can be prolonged by caloric restriction (74), which recently has been shown to slow the rate of accumulation of glycoxidation products (75). Consistent with this, recent evidence suggests that AGEs can be absorbed from food (76). The presence of AGEs in plasma may accelerate diabetic complications and, conceivably, aging as well, thus perhaps linking higher food intake with shorter life span. In a related finding, a study involving eight different mammalian species showed that the rate of accumulation of pentosidine was inversely related to maximum life span (77). It follows from these considerations that in a biochemical sense, diabetes may be described as a disease of accelerated aging (Figs. 5 and 6).
GLYCATION, GLYCOXIDATION, AND LIPOXIDATION OF SHORT-LIVED PROTEINS
This section concerns plasma proteins, primarily lipoproteins. These species are short-lived, and therefore it might be expected that only the early stages of glycation would affect them. However, while advanced glycoxidation and lipoxidation may have their greatest pathogenic potential in long-lived proteins, they may be equally important in some proteins that normally are short-lived but whose existence may be prolonged in diabetes. Most plasma proteins are replaced within a matter of days or, at most, a few weeks. However, in diabetes, abnormal capillary leakage may lead to increased extravasation of plasma constituents, with subsequent sequestration in vessel walls and extravascular sites by glucose-mediated cross-linking. This process may be particularly important in the case of lipoproteins, whose lipid constituents are susceptible to oxidative damage. Evidence is accumulating that glycated, oxidized, and glycoxidized lipoproteins may contribute not only to the development of macrovascular disease in diabetes, but also to microvascular complications. Other plasma proteins may also become entrapped and further modified in the extravascular space in diabetes. The consequences of glycation, glycoxidation, and lipoxidation of plasma lipoproteins as they relate to the development of diabetic complications are described below.
Lipoproteins: General Considerations
In type 1 diabetes, conventional plasma lipoprotein levels (total cholesterol, LDL, HDL, etc.) are usually normal. An abnormal lipid profile is more common in type 2 diabetes, but irrespective of the type of diabetes, the standard lipid profile combined with other well-recognized cardiovascular risk factors is insufficient to explain the increased severity of atherosclerotic disease. For this reason, diabetes itself has been described as an independent risk factor for the development of atherosclerosis (78). In recent years, it has become recognized that qualitative, as opposed to quantitative, abnormalities of lipoproteins are important contributors to the accelerated atherogenesis of diabetes. More recently still, evidence is accumulating that glycated, oxidized, and glycoxidized lipoproteins may also mediate small-vessel damage in the retina and kidney. Modified lipoproteins may therefore be implicated in both macro- and microvascular disease in diabetes.
The delicate nature and complex structure of lipoproteins have posed major challenges to researchers. For example, the measurement of small degrees of modification present in vivo demands stringent isolation techniques and prompt analysis following minimal storage. Failure in these steps leads to artifactual modification (particularly oxidation) during processing or storage. This problem must be borne in mind when interpreting data. Particularly in earlier studies, some of the effects attributed to glycation of lipoproteins may have been the result of inadvertent in vitro oxidation of lipids. The complex structure of lipoprotein particles further compounds the problem of defining the roles of various modifications occurring in diabetes. For instance, the different apoproteins on the same particle may vary in age and therefore in their extent of glycation/glycoxidation. This hampers in vitro simulation of modifications occurring in vivo. Furthermore, separate modifications affecting the various components of the particles may interact, making it difficult to discern the effects of each individual stress. For example, protein glycation may generate free radicals and damage susceptible lipids in the cores of particles, whereas oxidation of lipids may lead to apoprotein damage. Lastly, qualitative alterations of lipoproteins in diabetes may include subtle alterations in composition. It may be difficult to distinguish effects attributable to these compositional differences from those due to glycation and oxidation. LDL is undoubtedly the most studied and best characterized of all lipoproteins (reviewed in 58, 79, 80). With LDL, investigation of the consequences of glycoxidation/lipoxidation is facilitated by the fact that each particle has only one apoprotein, apoB-100.
Glycation of LDL
Glycation of human apolipoproteins following exposure to glucose, both in vitro and in vivo, was first measured by Schleicher et al. (81) in 1981. They incubated normal human LDL and HDL with radiolabeled glucose in vitro and found that glycation of all apolipoproteins (apoA1,,A2, B, C, and E) was increased in proportion to the period of incubation and to the ambient concentration of glucose. A study of LDL isolated from diabetic compared with nondiabetic human subjects revealed a twofold increase in apoB glycation. Schleicher et al. (81) were the first to suggest that increased glycation of lipoproteins occurring in vivo in diabetes might have significant pathophysiological consequences.
Glycated LDL: interactions with human fibroblasts and murine macrophages
The work of Schleicher et al. (81) was soon followed by other studies (82–84) investigating the metabolism of LDL glycated in vitro by cultured human fibroblasts and mouse peritoneal macrophages. In some experiments, in vitro glycation (5–100 mmol/1 glucose for 1–2 weeks) was performed in the presence of the reducing agent sodium cyanoborohydride; this reduces and stabilizes the Schiff base intermediate formed during early glycation (Fig. 3), preventing the reverse reaction and enhancing the protein modification. However, the resulting adduct (glucitol-lysine) differs slightly from FL and, strictly speaking, is nonphysiological. Also, the degree of modification attained may be much greater than that occurring in vivo. With these caveats, the binding and degradation of LDL by fibroblasts, which possess the classical LDL receptor, were impaired in proportion to the degree of LDL glycation. The effect was noted even with only 6–8% of lysine residues glycated; more severe modification completely abolished recognition by the LDL receptor. With LDL from diabetic patients, a threefold increase in glycation was found. In later studies using LDL glycated in vitro to simulate this degree of modification, Steinbrecher and Witztum (85) found that modification of as few as 2–5% of lysine residues, without cyanoborohydride, decreased LDL catabolism by fibroblasts by 5–25%, and again the effect was proportional to the degree of glycation. Consistent with these data, the fractional catabolic rate of glycated LDL injected into guinea pigs and rabbits was significantly lower than that of control LDL (85). Overall, these studies suggested that impaired recognition of glycated LDL may contribute to elevations in plasma LDL cholesterol levels in diabetic patients in poor glycemic control. In contrast to fibroblasts, murine peritoneal macrophages, chosen as model scavenger cells, failed to distinguish between control and glycated LDL (82, 83). Human macrophages, however, behaved differently from their murine counterparts, as detailed below.
LDL glycated in vivo: interactions with human fibroblasts and macrophages
Subsequent studies used LDL taken from diabetic patients. Glycation of LDL in vivo was found to correlate with other indices of glycemic control (mean plasma glucose, plasma protein glycation, HbA1c). Compared with control subjects, glycation of LDL from type 1 diabetic patients in fair glycemic control was increased 1.6-fold, similar to the increases in glycation for hemoglobin (1.5-fold) and total plasma proteins (2.2-fold) (86). Interactions of LDL with human fibroblasts and macrophages were studied. Recognition of LDL from type 1 diabetic patients by human fibroblasts was impaired compared with LDL from control subjects (87), as would be expected from the studies described above. Interactions of LDL with cultured human plasma-derived monocyte-macrophages were also studied (88); these cells are the main precursors of the foam cells characteristic of atherosclerotic plaques. Like murine peritoneal macrophages, these human macrophages are scavenger cells, but they exhibited a different response from the murine cells to in vivo-glycated LDL. LDL from diabetic patients stimulated more cholesteryl ester synthesis in these cells than did LDL from nondiabetic control subjects. There were no overall compositional differences between the LDLs from the two groups, and so the increased uptake of LDL from the diabetic patients was attributed to increased apoprotein glycation. Consistent with this, cholesteryl ester synthesis in macrophages correlated with LDL glycation, and concerted increases in intracellular cholesteryl ester accumulation were observed. In a later study using LDL from type 2 diabetic patients (89), no such increase in the rate of cholesteryl ester synthesis by macrophages was found. However, this may have been because the type 2 diabetic patients were in excellent glycemic control, with mean LDL glycation increased only 1.2-fold over control values.
In summary, these findings suggest that LDL from diabetic patients is not well recognized by the classic LDL receptor, perhaps contributing to hyperlipidemia in poorly controlled diabetes, and that it stimulates more cholesteryl ester synthesis in human monocyte-macrophages, potentially accelerating foam cell formation and the atherosclerotic process. Since no other compositional alterations were detected in this LDL, the effects may be attributed to the increased in vivo glycation of apoB-100. It must be noted, however, that although efforts were made to prevent inadvertent oxidation of LDL in these experiments, the degree of lipid peroxidation was not actually measured. Bucala et al. (54) recently showed that LDL modified by exposure to AGEs exhibited impaired clearance from plasma and also identified the site of modification of the apoprotein of LDL, apoB, which determines its impaired recognition by the LDL receptor (90).
Glycated and nonglycated subfractions of LDL from subjects with and without diabetes
Studies by Klein et al. (91) also support the enhanced atherogenicity of glycated LDL. Two fractions of intact LDL were isolated by boronate affinity chromatography, as described by Jack et al. (92). The affinity columns bind FL and therefore can trap intact glycated LDL, allowing the separation of “bound” and “nonbound” (i.e., more and less glycated) LDL fractions from individual patients. These two fractions were isolated from type 1 diabetic patients and control subjects. The diabetic group had a mean HbA1c of 8.6%. Plasma lipid profiles and the extent of glycation of the nonbound LDL were similar between the groups. Glycation of the bound LDL fractions was increased irrespective of its source, twofold in the control subjects and almost threefold in the diabetic patients. The greater increase in glycation per bound LDL particle in diabetic patients suggests glycation of a higher proportion of lysine residues per particle. Bound (glycated) LDL constituted a much greater percentage of the total in the diabetic patients (21%) than in the control subjects (5%).
Interactions of bound and nonbound LDL with human fibroblasts and monocyte-derived macrophages were studied. With fibroblasts, LDL-receptor–mediated degradation of the bound (glycated) fraction was impaired compared with the nonbound fraction, whether from a diabetic or control subject. These findings confirm previous studies demonstrating impaired recognition of glycated LDL by the classic LDL receptor. With human monocyte-macrophages, the opposite effect was observed. Marked increases in the accumulation and degradation of LDL in the presence of bound compared with non-bound LDL were observed. Again, these increases occurred irrespective of the source of the LDL, but they were more pronounced in the bound fraction obtained from diabetic patients. Likewise, cholesteryl ester accumulation in the macrophages was markedly increased following exposure to bound compared with nonbound LDL, again irrespective of source. Bound LDL contained a greater percentage of triglyceride than did nonbound LDL, whether obtained from diabetic or nondiabetic subjects, and this difference in composition may have had some effect on cell interactions. In conclusion, however, the data again suggest that glycated LDL is poorly recognized by the classic LDL receptor on fibroblasts (tending to increase plasma LDL), but is recognized by monocyte-macrophages (enhancing uptake and foam cell formation).
In vitro glycated LDL: interactions with human macrophages
To characterize LDL-macrophage interactions more thoroughly, human monocyte-macrophages were exposed to LDL glycated in vitro under antioxidant conditions (93). Levels of glycation were higher than those occurring in vivo in diabetes (88) (fourfold increases over control subjects), and proportionately greater effects on macrophage cholesteryl ester synthesis, accumulation, and receptor-mediated intracellular degradation were observed than those described above. Recognition of highly glycated LDL by the classic LDL receptor in macrophages, as in fibroblasts, was diminished (93), implying the existence of a different pathway for the enhanced uptake of glycated LDL. The scavenger receptor was an obvious candidate, but it was ruled out in competition studies using acetyl-LDL, which the receptor is known to recognize. Studies with glycated albumin (native albumin is nonspecifically taken up by macrophages) and thyroglobulin (whose molecular weight is similar to that of apoB) showed clearly that enhanced uptake was not a nonspecific effect of glycation, nor could the macrophage glycoprotein receptor be implicated, since yeast mannan, known to block this receptor, failed to have any effect on degradation of control or glycated LDL. These results suggested the existence of a separate low-affinity, high-capacity receptor pathway for glycated LDL. The relationship between this putative receptor and the more recently defined receptors for AGEs is unclear.
Lipoprotein glycation and oxidation
Oxidized LDL has been extensively implicated in the development of atherosclerosis (reviewed in 94), and its role in the accelerated development of atherosclerosis in diabetes has been reviewed by one of us (58) and by Chisolm et al. (95). Lipoproteins contain varying amounts of unsaturated fatty acids in their cores, and these are vulnerable to oxidative damage. Oxidized LDL is a potent stimulator of foam cell formation by macrophages (96–98), and in the vascular wall it is chemotactic to monocytes (99, 100), the cells from which macrophages are derived. It has complex effects on cytokine release by macrophages, and these may mediate the inflammation present in atheromatous lesions (101). It also inhibits endothelial cell migration into areas of endothelial injury (102) and increases their membrane viscosity (103, 104). Oxidized LDL is cytotoxic to nearby cells (105–109). The principal toxic product produced when LDL is oxidized has recently been identified by Chisolm et al. (110) as the lipid hydroperoxide 7β-hydroperoxycholest-5-en-3 (β-ol. Also, enhanced degradation by an intrinsic phospholipase of LDL phosphatidylcholine (a phospholipid) to lysophosphatidylcholine may enhance the toxicity of oxidized LDL (111). The processes of oxidation and glycation are closely linked (9). Both FL and simple monosaccharides may auto-oxidize under physiological conditions, in the presence of traces of metal ions, generating superoxide radicals (27, 112). Thus, glycation of apolipoproteins may enhance the likelihood of oxidative damage to core lipids. Despite this, there is rather indefinite evidence that oxidation of plasma lipoproteins is increased in uncomplicated diabetes, whereas glycation clearly is. Also, no studies exist to demonstrate a correlation between oxidation of plasma lipoproteins and glycemic control in diabetic patients. However, the situation may well be different for extravasated lipoproteins sequestered in vessel walls or outside capillaries, e.g., in the retina or kidney. In these, glycation, oxidation, and glycoxidation may combine to generate vicious cycles of vascular injury (58). Concomitant glycation and glycoxidation of vascular connective tissue proteins may encourage further covalent, glucose-mediated binding of lipoproteins (113), trapping them in the vascular media and permitting greater degrees of modification, and hence, greater generation of free radicals, than could otherwise occur.
LDL glycation/glycoxidation and coagulation/fibrinolysis
Increased glycation of LDL may have prothrombotic effects. In a study by Watanabe et al. (114), LDL from young type 1 diabetic patients (in good-to-fair glycemic control) was found to be a more potent stimulator of thromboxane B2 release and thrombin-induced platelet aggregation than LDL from control subjects. Glycation of LDL from the diabetic patients was increased, while LDL composition was similar in the two groups. LDL glycated in vitro also caused a marked enhancement in thrombin-, collagen-, and ADP-induced platelet aggregation, but the enhancement occurred irrespective of the degree of glycation. Subtle alterations in the composition of platelet membranes, induced by interaction with glycated LDL, were suggested to explain these effects. When bound and nonbound (glycated and nonglycated, see above) LDL fractions were isolated from type 1 diabetic patients, platelet aggregation was enhanced to a significantly greater extent by the bound (highly glycated) than by the nonbound (less glycated) fraction (115).
The effects of modified LDL on the fibrinolytic system have been studied. Jokl et al. (116) showed that LDL isolated from diabetic patients, and also normal LDL oxidized in vitro, failed to stimulate the expected release of the pro-fibrinolytic protein tissue plasminogen activator by cultured human umbilical vein endothelial cells. Furthermore, they found that glycoxidized LDL increased the release of plasminogen activator inhibitor 1, an inhibitor of fibrinolysis, by retinal capillary endothelial cells (117). All of the above observations suggest that modifications of LDL occurring in diabetes may contribute to an increased tendency towards thrombosis.
LDL glycation/glycoxidation and diabetic retinopathy
Our group is investigating the hypothesis that modifications of LDL occurring in diabetes may contribute to micro- as well as macrovascular complications. The possibility that glycoxidized LDL may mediate an increased tendency to thrombosis in retinal vessels has already been mentioned. Other studies have shown that mild in vitro glycation or oxidation renders normal LDL toxic to cultured retinal capillary endothelial cells and pericytes, and that this toxicity is further enhanced by glycoxidation of LDL (118). In these experiments, the in vitro modification conditions were very tightly controlled so that the degrees of modification was kept pathophysiologically relevant and that unintentional oxidation did not occur. The data suggest that glycated LDL in plasma could contribute to the initiation of retinopathy by injuring endothelial cells, thereby causing capillary leakage, i.e., breakdown of the blood-retinal barrier. Later, when extravasated and more severely oxidized and glycoxidized, LDL may also contribute to the propagation of retinopathy, causing further damage to both endothelial cells and pericytes, and perhaps to the lipid-rich retinal neural tissue as well. A recent study by Yamagishi et al. (119) has demonstrated that AGE-modified serum is toxic to retinal pericytes and that this toxicity is a receptor-mediated phenomenon; it is possible that a similar mechanism is operative in our experiments with modified LDL.
More recently, studies by our group have shown that the development of toxicity of LDL towards retinal capillary cells may be mitigated if aminoguanidine is present while the lipoprotein is being subjected to in vitro glycation or oxidation, i.e., during an LDL-modifying incubation, before addition to cells (120). Remarkably low aminoguanidine concentrations were effective, down to 1 μmol/1. In contrast, aminoguanidine (10 μmol/1) was ineffective if present in culture media while already-modified LDL was exposed to cells. Aminoguanidine is a hydrazine that inhibits the formation of glycoxidation and lipoxidation products. It has little or no effect on the early stage of glycation, but it is thought to act mainly by blocking reactive carbonyl-containing intermediates (121) derived from oxidation of glucose or lipids or from dissociation of FL (122). As detailed above, these intermediates are very highly reactive, and even though they are present at very low concentrations compared with glucose, they may mediate much of the formation of AGEs. The low concentrations of these products may explain the efficacy of aminoguanidine, at similarly low concentrations, in protecting LDL from cytotoxic changes, and these findings are therefore consistent with the carbonyl stress hypothesis. In other findings consistent with the carbonyl stress hypothesis, our group has also shown that 1 μmol/1 aminoguanidine can protect cultured retinal cells from the toxic effects of high glucose (25 mmol/1) (T.J.L.,AJ.J., unpublished observations). It is established that aminoguanidine can prevent the browning process both in vitro and in vivo (121, 122): studies in experimental animals have shown that it can inhibit the cross-linking of collagen in arterial walls (121) and that it retards the development of diabetic nephropathy (123), neuropathy (124, 125), and retinopathy (126). Multicenter studies on the efficacy of aminoguanidine in nephropathic diabetic patients are now in progress both in Europe and in North America. It should be mentioned, however, that the mode of action of aminoguanidine remains controversial: some investigators believe that to a significant extent, its effects are due to inhibition of nitric oxide synthase rather than to inhibition of browning (127).
In studies analogous to those with aminoguanidine described above, recent work by our group has demonstrated that the cytotoxicity toward cultured retinal capillary cells that develops in LDL exposed to glycation or oxidation can be entirely prevented by pre-supplementation of the LDL with vitamin E (α-tocopherol) (128). α-Tocopherol was more effective than aminoguanidine in protecting LDL in our model system. In the studies using vitamin E, plasma was supplemented in vitro with α-tocopherol before the isolation of LDL. The LDL α-tocopherol content attained was similar to that achieved in vivo by typical dietary supplemental doses. α-Tocopherol is the most important lipid-soluble antioxidant in plasma. It is an amphiphilic molecule located in the outer layers of lipoprotein particles, and it transfers reactive free radicals generated in the lipid-rich cores of the particles to aqueous antioxidant systems, primarily to ascorbate (vitamin C). Its efficacy in preventing the development of cytotoxicity in LDL suggests that exposure to glucose (50 mmol/1 for 3 days in this work) and consequent glycation, even in an antioxidant environment, confers increased oxidative stress on the lipoprotein particles. Such stress might be mediated by the presence of glucose auto-oxidation products in glucose solutions before their addition to LDL. As discussed below, these data suggest that long-term treatment with α-tocopherol or low doses of aminoguanidine, or related drugs yet to be developed, may play a future role in the prevention of diabetic retinopathy.
Glycation of VLDL, HDL, and lipoprotein (a)
Increased glycation of VLDL and HDL apoproteins has been demonstrated in diabetes (129). As with LDL, it is believed to affect cellular interactions, function, and metabolism of these particles. VLDL from both type 1 and type 2 diabetic, normolipemic patients stimulated increased cholesteryl ester synthesis and accumulation in cultured human monocyte-macrophages (89, 130), but the effect was less marked than with LDL. Subtle alterations in apoprotein composition are present in VLDL in diabetes (131), so metabolic differences cannot be attributed to increased glycation with any confidence. Any investigation of the effects of glycation of VLDL is hampered by problems in measuring the separate degrees of modification of its various apoproteins. These are known to differ because of differing plasma residence times. Use of VLDL glycated in3 vitro is unsatisfactory because the differential glycation of its different apoproteins occurring in vivo cannot be reproduced. Increased glycation of VLDL apoproteins may therefore have important consequences, but this has not yet been convincingly proven.
Similar problems afflict studies on the effects of glycation of HDL. Nevertheless, when HDL was even mildly glycated in vitro, its clearance from plasma in guinea pigs was accelerated (132). This effect is the opposite of that observed with glycated LDL and might partially explain the low plasma levels of HDL in diabetic patients. Duell et al. (133) have demonstrated impaired high-affinity binding of glycated HDL to fibroblasts and a diminished capacity to remove cholesterol from peripheral cells (134). Enhanced glycation of HDL may therefore contribute to the acceleration of atherosclerotic disease in diabetic patients.
Lipoprotein(a) [Lp(a)] has an unusual apoprotein, apo(a), which has structural homology with plasminogen. It may contribute to macrovascular disease by competitively inhibiting the activation of plasminogen, and because it is a cholesterol-rich particle (135). In the DCCT, levels were shown to be decreased by intensive management (136). There is evidence that plasma levels of Lp(a) are elevated in the presence of diabetic complications (137, 138). However, a recent study investigating the effects of in vitro glycation of Lp(a) found no evidence that glycation increases its atherogenicity, at least in terms of its interaction with macrophages (139).
LDL modification and the immune system
Modification of LDL may alter its structure sufficiently to provoke an immune response, and the enhanced modifications occurring in diabetes may make this mechanism for vascular disease particularly pertinent in this condition (140–145). LDL glycated in vitro in the presence of cyanoborohydride to produce heavily modified particles was found to be strongly immunogenic (141). However, as detailed above, such LDL contains glucitol-lysine, a product of “reductive glycation” not present in vivo. FL, which does form in vivo, was not recognized by the antibody raised against reductively glycated LDL. FL itself proved to be a much less potent immunogen, but nevertheless, in vitro glycated LDL— and even control LDL, which always contains some FL—competed for the resulting antibody. The presence of antibodies against “true” glycated LDL had no effect on its rate of clearance from plasma (146). Nevertheless, the existence of such antibodies, even at low levels, is probably important, because LDL immune complexes (LDL-ICs) are known to stimulate foam cell formation and to be potently atherogenic (reviewed in 142, 144, 145). This atherogenicity is thought to be mediated through a variety of interactions between LDL-IC and vascular cells in processes involving alterations in cytokine release, coagulant activity, vascular permeability, and vascular growth factors.
In diabetes, the formation of LDL-IC is likely to be enhanced by the presence of more severely modified (glycoxidized) lipoproteins sequestered in vessel walls, which may stimulate in situ formation of ICs (147). It is currently unclear whether significant formation of LDL-IC occurs before, or only after, significant vascular disease has developed. Higher titers of antibodies against oxidized LDL have been demonstrated in patients with type 2 diabetes, but it is not clear in this study whether or not the nondiabetic control group had vascular disease (142). The role of LDL-IC in the accelerated atherosclerosis of diabetes has been reviewed recently (142, 144, 145). Recent work from Baynes’s group (44) suggests that CML is the dominant epitope for antibody formation against glycoxidation products. Because CML is also a lipoxidation product, it is conceivable that it may also be an important epitope on modified LDL.
Consequences of Glycation of Other Plasma Constituents
Glycation of albumin and diabetic nephropathy
Glycation of albumin has been implicated in the pathogenesis of vascular disease, especially in glomerular damage, in diabetes. Inhibition of proliferation of aortic endothelial cells in culture was demonstrated in the presence of in vitro glycated albumin, and this effect could be inhibited by antibodies that react specifically with Amadori products in albumin (148). Similar effects were observed using renal mesangial cells (149); and changes in collagen gene expression by these cells, which could also be inhibited by the specific antibody, were observed (150). Finally, administration of the specific antibody to diabetic mice was found to prevent the development of nephropathy (151) and to inhibit retinal capillary basement membrane thickening (152).
Glycation and antithrombin III activity
Glycation of antithrombin III is thought to impair its thrombin-inhibiting activity (153), potentially enhancing thrombosis. The inhibition of antithrombin III activity is reversible by an excess of sodium heparin. Consistent with this effect of glycation, an inverse correlation has been described between antithrombin III activity and both HbA1c and plasma glucose, independent of plasma concentrations of antithrombin III (154). In contrast, in vitro glycation of fibrinogen apparently has no effect on its function and therefore does not appear to promote thrombosis (155).
GLYCATION, GLYCOXIDATION, AND LIPOXIDATION OF STRUCTURAL PROTEINS
Lens Crystallins
Lens crystallins are of interest because they are among the longest-lived proteins in the body and are therefore pertinent to studies of the aging process and glycoxidation/lipoxidation in diabetes. Also, premature cataract formation is a feature of diabetes (156–158). Glucose levels in the aqueous humor (159) and within the lens (160) are normally much lower than plasma levels (50% and 20% of plasma levels, respectively). Low intralenticular glucose concentrations combined with antioxidant mechanisms may protect crystallins in the normal lens from modification. Consistent with this, dietary antioxidant therapy can inhibit the development of acute sugar (or “metabolic”) cataracts in diabetic animals (161, 162), and lipid peroxidation products are increased in diabetic compared with nondiabetic senile-type cataracts (the common variety occurring in diabetic patients) (163). However, these observations do not prove that increased oxidative stress is important in the formation of senile-type cataracts in diabetes. Acute metabolic cataracts, particularly in animal studies, are of questionable relevance: they develop very differently, and are morphologically distinct, from the senile-type cataract most common in human diabetic patients. The increased lipid peroxidation may be explained by increased permeation of lipids into the lens following disruption of the blood-aqueous and blood-retinal barriers in diabetes, though such permeation could, in itself, increase oxidative stress in the lens. Breakdown of the blood-aqueous and blood-retinal barriers in diabetes may result in disproportionate increases in glucose levels both in aqueous humor and in the lens compared with plasma. There is evidence that such large changes in glucose levels do indeed occur: DiMattio et al. (164) observed that permeation of glucose from the plasma to both the aqueous and vitreous compartments is abnormally high in diabetic rats. Also, damage to the ocular vasculature, with increased permeability documented by vitreous and aqueous humor nuorophotometry, has been identified as an early abnormality in diabetes (165, 166).
Levels of FL and glycoxidation products have been measured in lens crystallins from nondiabetic individuals over a wide age range and from diabetic and nondiabetic patients with cataracts (23, 167–169). In the nondiabetic patients, FL changed little with advancing age (168), but CML steadily increased. These changes are similar to those observed with insoluble skin collagen, described above. Lens crystallin FL was increased more than fourfold in patients with cataracts if diabetes was present. This increase was disproportionate to increases in plasma glucose or HbA1 (both less than twofold), which is consistent with abnormal entry of glucose into the lens in diabetes (169). No increase in lens crystallin FL was observed in nondiabetic cataract patients, suggesting that the barriers to glucose in these patients must remain intact. These findings are consistent with a role for increased intralenticular glucose or FL in accelerated cataractogenesis in diabetic patients.
The glycoxidation products CML and pentosidine were measured in the same lens crystallins. Age-corrected pentosidine levels were increased in cataract patients with diabetes compared with those without diabetes, as might be expected. However, no difference in CML levels between diabetic and nondiabetic senile cataract levels were observed. In fact, age-corrected CML levels were similar in both groups of cataract patients to those in nondiabetic, noncataractous control subjects. The increase in pentosidine suggests that enhanced glucose-mediated cross-linking does occur in lens crystallins in diabetic patients and could contribute to accelerated cataract formation. This concept is supported by a study identifying AGEs immunologically in the lenses of diabetic rats (170). The fact that CML levels were not affected by the presence of either diabetes or cataracts, however, suggests that lenticular CML must be formed predominantly from species other than glucose. Ascorbate is a potential candidate, since levels are high in the lens, but against this, pentosidine can also be formed from ascorbate. Of relevance here is that another product of ascorbate-mediated modification of lens protein, a pyrrole formed from L-threose (a degradation product of ascorbate), was recently identified (171). CML could theoretically be derived mainly from lipids, via lipoxidation of crystallins, but this seems unlikely: again, one might expect increased levels in diabetes. Consistent with an alternative origin for CML in lens proteins, lens crystallin CML accumulates with age in normal individuals more rapidly than does skin collagen CML (41), even though proteins in the lens are exposed to lower ambient glucose levels. Irrespective of its origin, CML is always formed in reactions involving free radical oxidation. The lower-than-expected levels of CML in cataractous lenses from diabetic patients thus weighs against the presence of increased oxidative stress in the lens in senile-type diabetic cataracts.
In conclusion, the type of cataract commonly seen in older diabetic patients is morphologically similar to, but biochemically distinct from, cataracts occurring in nondiabetic patients. Increased glycation and glucose-mediated cross-linking may accelerate cataract formation in diabetes, but the role of oxidative stress is less clear.
Collagen
Collagen is a ubiquitous and important structural component of the body. Its slow turnover means that it may be subjected to glycoxidative stresses for prolonged periods. In relation to diabetes, investigators have studied glycoxidation products primarily in skin and tendon collagen. The nature of the glycation and browning reactions, however, makes it reasonable to extrapolate alterations observed in these collagens to different collagens in other sites. Glycoxidation occurring in type 1 collagen in skin is therefore considered likely to be reflected in other collagens, including those in vascular basement membranes. Increased fluorescence of collagen from human arterial tissue in diabetes has been observed (172). Similar changes may also affect other connective tissue proteins, e.g., elastin, fibronectin, and laminin. The effects of glycation on laminin have been reviewed in detail by Charonis and Tsilibary (173).
Effects of aging and diabetes on the physical properties of collagens
Collagen becomes progressively more insoluble, thermally stable, and resistant to enzymatic attack with advancing age (174). The changes are predictable and allow reasonably accurate determination of the age of sample donors (175). These age-related changes are thought to be caused by the formation of stable cross-links, many of which are derived from glucose via the browning process. Not surprisingly, therefore, the biochemical aging of collagen is accelerated in diabetes. Hamlin et al. (176) found that three diabetic patients whose true ages were 33, 41, and 44 years had collagen that, by its digestibility, behaved as though patients were 84, 103, and 106 years old, respectively.
Consequences of in vitro modification of structural proteins
To determine the effect of collagen glycation on its physical properties, experiments were performed using collagen modified in vitro. Incubation of collagen with glucose for 1 week under oxidative conditions had no effect on resistance to proteolytic digestion (177), but it did increase the stability of rat tail tendons (178). With longer glycoxidative incubations, however, spectrophotometric changes in another long-lived protein, lens crystallin, occurred that resembled those observed in vivo with advancing age (70), and thermal stability of basement membrane collagens increased (179). More recent studies have shown that although collagen cross-linking depends on the presence of glucose, it can be completely inhibited by antioxidant conditions (39). Thus, in the presence of glucose, antioxidant conditions inhibited formation of CML, pentosidine, and collagen fluorescence but had no effect on the formation of FL. These in vitro studies suggest that the increased cross-linking and fluorescence and the parallel accumulation of the specific products CML and pentosidine are dependent on the presence of an oxidant environment as well as the on presence of glucose.
Glycation, glycoxidation, and lipoxidation of collagen in diabetes
In 1979, Rosenberg et al. (180) were the first to study collagen glycation (FL content) in animals with and without diabetes. They found a significant increase in glycation of aortic collagen in diabetic compared with nondiabetic rats, and they suggested that it might be relevant to accelerated atherogenesis in diabetes. Increased glycation of collagen in the presence of diabetes was later observed in many tissues: rat glomerular basement membranes (181), human tendon and skin (18, 19, 182–184), and human aorta, glomerular basement membrane, and tendon (185, 186).
As already summarized, in nondiabetic subjects, collagen glycation increases only slightly between the ages of 20 and 80 years (17, 18). It is increased in diabetes but does not correlate with age or duration of disease. It does correlate with HbA1 (19), and especially with HbA1 averaged over a period of several years (18). It decreases promptly after a short period (4 months) of improved glycemic control (20). All of these findings suggest that collagen glycation is in relatively short-term equilibrium with ambient glucose concentrations.
In contrast to FL, glycoxidation/lipoxidation products in insoluble collagen accumulate continuously throughout life (17, 18, 32). Their rate of accumulation is accelerated in diabetic patients (18, 176, 187, 188), so that even a young diabetic patient may have levels higher than those in the oldest nondiabetic subject (18, 176). This excessive accumulation in diabetic patients may depend not only on duration of diabetes and average glycemic control, but also on individual variations in oxidative stress (18). Such variations are suggested by the wide range of levels of glycoxidation products among elderly nondiabetic subjects (Figs. 5 and 6). Since these subjects have similar long-term glycemia, the variations may be attributed to differences in oxidative stress or in antioxidant defenses. Overall, however, as outlined above, there is no evidence from these studies of a general increase in oxidative stress in diabetes (18): the increased accumulation of glycoxidation products in collagen from diabetic patients can be explained by the increased glycation alone (taking into account duration of diabetes) without invoking an increase in oxidative stress. Nevertheless, the accumulation of glycoxidation products, whether or not diabetes is present, is thought to be irreversible (20): the products may be removed only by catabolism, which is itself inhibited by their presence. This is ominous if, as is suspected (see below), excessive accumulation of these products plays a role, directly or indirectly, in the development of diabetic complications. The consequences of hyperglycemia for individual diabetic patients may therefore be modulated by their ability to resist oxidative stress: those with poor antioxidant defenses may be particularly vulnerable to developing complications. More optimistically, the dual origin of glycoxidation products (from both glycemic and oxidative stresses) may provide a means to predict, soon after the onset of diabetes, those patients who are particularly susceptible to complications. In addition, more therapeutic options may be developed: controlling oxidative stress as well as hyperglycemia may be a rational preventive measure to inhibit the development of complications.
Early-stage collagen glycation products and complications
Early studies sought an association between FL levels in skin collagen and diabetic complications (19, 184). It was believed that collagen FL levels might represent a very long-term index of glycemic control (much longer than HbAlc). It is now clear that this is not the case. As discussed above, collagen FL levels appear to reflect only relatively recent glycemia. In vitro, early glycation does little to alter the physicochemical properties of collagen. In studies of skin collagen, our group found no association between FL levels and the presence or severity of limited joint mobility in type 1 diabetic patients (19). Vishwanath et al. (184) confirmed this, also showing that collagen FL levels are not associated with retinopathy, nephropathy, or arterial stiffness. Most evidence, therefore, suggests that FL in collagen does not contribute directly to the development of complications. The possibility remains that it may contribute indirectly by generating the later, more permanent products of advanced glycation. Consistent with this, and in contrast to the conclusion reached above, a later study by our group found skin collagen FL levels to be independently associated with the presence of retinopathy and early nephropathy (microalbuminuria) (45). The latter observation is congruent with the work of Ziyadeh and others (148–152), discussed above, concerning glycated albumin and diabetic nephropathy.
Glycoxidation/lipoxidation products in collagen and complications
The accumulation of glycoxidation products in collagen alters its physicochemical properties and is therefore more likely than FL to cause significant functional abnormalities. Monnier and colleagues found associations between advanced glycation of collagen (fluorescence [187, 188] and pentosidine content [189]) and the severity of diabetic retinopathy, joint stiffness, and arterial stiffness. An association between collagen browning and diabetic nephropathy was also found by Makita et al. (190), and our group has shown that levels of collagen fluorescence and specific glycoxidation products are associated with severity of retinopathy (Fig. 8) and with the presence of early nephropathy (Fig. 9) (45). In the latter study, which controlled for age and duration of diabetes, independent associations were found between skin collagen CML and retinopathy and early nephropathy, and between pentosidine and early nephropathy (45). AGEs have been identified immunohistochemically in skin, where levels were found to correlate with severity of nephropathy and retinopathy and to increase in the earliest stages of these conditions (191, 192). Using the same techniques, AGEs have been identified in coronary arteries in diabetic patients, with particularly high levels in atheromatous lesions, a finding consistent with a contributory role in the accelerated atheroma of diabetes (193). AGEs have also been identified immunologically in atheromatous lesions in nondiabetic rabbits (194). Because atheromatous plaques are lipid-laden and because CML appears to be a potent epitope (44), it seems likely that the antibodies in these assays may be recognizing CML formed predominantly as a lipoxidation product.
Figure 8.
Maillard products in skin collagen and retinopathy status. Grade 0, no retinopathy; grade 1, background retinopathy; grade 2, proliferative retinopathy. Bars show mean ± 1 SD in each category. FL, CML, pentosidine, and total fluorescence all increased progressively with increasing severity of retinopathy (P<0.05, P< 0.001, P<0.05, P< 0.01, respectively). Using logistic regression to control for duration of diabetes, FL, CML, and total fluorescence remained independently associated with retinopathy. Reprinted from McCance et al. (45) with permission from the American Society for Clinical Investigation.
Figure 9.
Maillard products in skin collagen and albumin excretion rate. N, normoalbuminuria (AER24 < 20 μg/min); A, albuminuria (AER24 (20 μg/min). Bars show mean (1 SD in each category. FL, CML, pentosidine, and total fluorescence were all increased in the presence of early nephropathy (P < 0.05, P < 0.001, P <0.01, P< 0.01, respectively). Using logistic regression to control for duration of diabetes, FL, CML, and pentosidine remained independently associated with early nephropathy. Reprinted from McCance et al. (45) with permission from the American Society for Clinical Investigation.
Glycoxidation of vascular wall structural proteins in the development of diabetic complications: possible mechanisms
Several mechanisms have been proposed: some have substantial supporting evidence, others are more speculative.
Abnormal vascular rigidity and tone
Monnier et al. (187) showed an association between increased skin collagen fluorescence in type 1 diabetic patients and both arterial stiffness (assessed in vivo) and elevated systolic and diastolic blood pressures. Oxlund et al. (195) demonstrated increased aortic stiffness in patients with type 1 diabetes at autopsy but did not determine the level of glycoxidation products. Decreased elasticity and compliance of arteries and arterioles in diabetes may be due, at least in part, to increased glucose-mediated cross-linking. This may contribute to the development of hypertension, and arterial stiffness and hypertension combined may result in abnormal shear stresses affecting the endothelium, predisposing to injury and atherogenesis. In smaller vessels, the same effects may contribute to the development of diabetic retinopathy and nephropathy. The presence of collagen glycoxidation products appears to quench the activity of nitric oxide (endothelium-derived relaxing factor) both in vitro and in vivo (196). This may result in impaired endothelium-mediated vasodilation and possibly in abnormalities in vascular tone and abnormalities of flow, perfusion, and blood pressure, which may contribute to arterial and arteriolar damage.
Covalent binding of plasma constituents
Endothelial injury leads to permeation of plasma constituents into the vessel wall, where they may become bound covalently to connective tissue glycoxidation products. Brownlee et al. (113) found increased binding of LDL to glycated compared with control collagen, and cross-linking of LDL to aortic collagen was increased 2.5-fold in diabetic compared with nondiabetic animals. Once trapped in a high-glucose environment in the vessel wall, the LDL particles are subjected to extensive glycoxidative modification, with further increases in atherogenicity. Free radical chain reactions in the trapped LDL may damage not only its own constituent lipids but also neighboring structural proteins and cells. It has been shown, for instance, that products of lipid peroxidation stimulate cross-linking of collagen (197), and recent work from our group suggests they may directly mediate cross-link formation (31). These interwoven mechanisms may lead to various vicious cycles in the diabetic milieu, leading to damage of arteries and small vessels and later to in situ formation of lipoprotein-ICs, further accelerating foam cell formation.
The receptor for AGEs
Monocyte-macrophages are strongly implicated in the development of atherosclerotic lesions. AGEs in vessel walls are chemotactic to these cells and induce them to migrate through the vascular endothelium (198). A specific receptor for AGEs on monocyte-macrophages was identified as distinct from other scavenger receptors by Vlassara et al. in 1986 (199). Macrophages expressing this receptor can phagocytose protein molecules and also entire cells with glycoxidation products on their surface (200). Consistent with this, AGEs in vessel walls have been localized immunologically to intracellular sites in macrophages, smooth muscle cells, and the foam cells derived from these cells (201). Two receptors for AGEs on endothelial cells have been characterized in detail (202), and one was cloned in 1992 (203). Many of the consequences of increased AGE formation in diabetes are thought to be mediated through interactions with various receptors (204). AGE/receptor interactions in macrophages induce release of cytokines, tumor necrosis factor-a, and interleukin-1 (205), which in turn mediate growth and remodeling and may accelerate the atherosclerotic process. These interactions may also induce prothombotic tissue factor by macrophages, an action that can be inhibited by antioxidants (206). In T-cells, AGE/receptor interactions may induce synthesis of interferon-7, which may enhance immune-mediated mechanisms of tissue injury (207). In rat renal mesangial cells, AGEs induce increased collagen production, an effect that appears to be mediated by transforming growth factor-β and platelet-derived growth factor (208). Receptor-mediated actions of AGEs have also been implicated in the development of diabetic retinopathy (119, 209, 210). Infusion of AGEs in rabbits produced a variety of vascular changes. In endothelial cells, these included increased expression of vascular cell adhesion molecule 1 (VCAM-1) and intercellular adhesion molecule 1, effects seen predominantly in areas showing evidence of developing atheroma (211). The induction of VCAM-1 has been shown to be dependent on AGE/receptor interactions, and VCAM-1 antigen is elevated in diabetic plasma (212). In further support of the significance of these interactions, it has been shown that blockade of the AGE receptor can inhibit AGE-induced impairment of endothelial barrier function and consequent hyperpermeability. Inhibition of AGE formation using antioxidants has a similar effect (213).
Renal impairment
Accumulation of glycoxidation products in skin collagen is associated with the development of renal impairment in diabetes (45, 187, 189, 190), suggesting a possible causative role in diabetic nephropathy. In support of this hypothesis, infusion of AGE-modified albumin in rodents induces renal changes (glomerular sclerosis, albuminuria) analogous to those seen in diabetic renal disease (214). It also induces glomerular upregulation of mRNA for laminin and collagen (215), proteins that accumulate in glomerulosclerosis. Diabetic nephropathy, and other forms of renal disease, are characterized by a considerable increase in the expression of the AGE receptor in a wide variety of renal cell types, many of which do not normally express this receptor (216). In addition to these kidney-specific effects, if a generalized collagen abnormality is a common underlying mechanism for both microalbuminuria and atheroma, it may partly explain the identification of microalbuminuria as a risk factor for macrovascular disease. The hypertension and lipid abnormalities characteristic of renal impairment may further contribute to the development of atherosclerosis.
Glycoxidation of collagen and diabetic complications: alternative hypotheses
The studies discussed above demonstrate associations between the levels of both FL and glycoxidation products in collagen and the presence or severity of diabetic complications. The data are compatible with, but do not prove, the hypothesis that these products may contribute to the development of complications, including accelerated macrovascular disease. FL, thought to be the relatively harmless precursor of damaging glycoxidation products, may contribute indirectly, or increased FL and the presence of complications may be related only insofar as both arise from a common origin—either prolonged hyperglycemia itself or other associated metabolic derangements. Glycoxidation products significantly alter the properties of collagen, and a direct causative role in the development of complications is easier to envisage, but the case is not proven. Glycoxidation products may simply reflect long-term glycoxidative (and in some cases lipoxidative) stress, and this stress may cause disease primarily by its cumulative effects on substrates other than collagen. Alternatively, complications may arise in association with hyperglycemia through mechanisms not related to glycoxidation, in which case glycoxidation products in collagen would represent only a record of past events.
A CARBONYL STRESS HYPOTHESIS FOR THE DEVELOPMENT OF DIABETIC COMPLICATIONS
The above considerations may be synthesized into a new carbonyl stress hypothesis for the pathogenesis of diabetic complications. This states 1) that reactive carbonyl-containing compounds are generated by free radical oxidation of both carbohydrates (mainly glucose) and lipids; 2) that the rate of formation of these compounds is a function of substrate concentration (glucose or lipids) and of inherent levels of oxidative stress/antioxidant defenses; and 3) that modifications of macromolecules by carbonyl-containing compounds contribute to the development of diabetic complications. The hypothesis is consistent with the findings described above relating both to short- and long-lived proteins. In the case of collagen, it appears that the rate of the formation of glycoxidation or lipoxidation products (a measure of combined glycoxidative and lipoxidative stress)—rather than their absolute levels—may be important in determining the risk of complications. This is consistent with the obvious fact that elderly, nondiabetic patients, despite having high levels of these products, do not develop diabetic complications. According to the hypothesis, combinations of oxidative stress affecting both carbohydrates and lipids, coupled with hyperglycemia, mediate tissue damage. Reactive carbonyl-containing compounds are generated, and the advanced products accumulate to provide a permanent “record” of cumulative carbonyl stress in long-lived proteins such as insoluble collagen. The changes occurring in collagen may be important in themselves, but the real damage may result from the chronic effect of these stresses on other substrates, e.g., on lipids in cell membranes and in plasma lipoproteins, and on other macromolecules, both extra- and intracellular. Thus, diabetic patients who inherently (i.e., unrelated to their diabetes) have poor antioxidant defenses may be particularly vulnerable to complications, and oxidative stress may be regarded as a modulator of the consequences of hyperglycemia.
The carbonyl stress hypothesis is further supported by preliminary, unpublished data from our group. Analysis of levels of skin collagen CML and pentosidine in type 1 diabetic patients (described in 45) revealed that among those with diabetes for more than 10 years, those with severe retinopathy had experienced faster overall accumulation rates of CML and pentosidine than expected (controlling for long-term glycemia and duration of diabetes). Conversely, those with little or no retinopathy had lower-than-expected rates of accumulation of CML and pentosidine. The subset of patients whose data could be analyzed in this way was small; nevertheless, the difference in rates of accumulation came close to statistical significance (P < 0.06).
PRACTICAL IMPLICATIONS: REDUCING CARBONYL STRESS
If carbonyl stress is indeed a significant risk factor in the development of macro- and microvascular complications of diabetes, then its reduction would clearly be desirable. Reducing carbonyl stress would mitigate recurrent damage to short-lived species, such as LDL, and cumulative damage to long-lived species, such as collagen. Means to inhibit the glucose-mediated, lipid-mediated, and “oxidative” components of carbonyl stress may be considered separately.
Reducing Glucose-Mediated Stress
The most obvious measure to decrease glycation is to optimize glycemic control, thereby rninimizing FL formation and reducing concentrations of glucose vulnerable to auto-oxidation. In so doing, existing FL levels may be reduced: improved glycemic control has been shown to reduce FL content within a few months, even in long-lived proteins (20). Decreased FL should decrease the subsequent formation of glycoxidation products. Optimal glycemic control is already an established goal in the management of diabetic patients.
Reducing Lipid-Mediated Stress
The degree of lipoxidative modification of proteins and other macromolecules may be minimized by decreasing the substrate available for oxidative damage and making the substrate more resistant to oxidation. This may be achieved by optimizing the plasma lipid profile by dietary and pharmacological means and by using dietary measures to minimize oxidizability of the fatty acid constituents of lipoproteins and cell membranes. Reaven et al. (217) have shown that a diet in which monounsaturated fats are substituted for polyunsaturated or saturated fats results in LDL that is less susceptible to oxidative damage.
Reducing Oxidative Stress
At present, there is little direct evidence concerning the efficacy of any treatment aimed at reducing oxidative damage to proteins and lipids in diabetes. Vitamin C (ascorbate) is believed to be the most important aqueous antioxidant (218). Plasma levels of vitamin C and platelet levels of vitamin E, the most important fat-soluble free radical scavenger, tend to be abnormally low in diabetic patients (219, 220). Dietary supplements of these vitamins therefore appear to provide a cheap, low-risk intervention. However, under some circumstances, vitamin C can act as a prooxidant (221), and there are insufficient grounds to recommend its routine use in diabetic patients. In the case of vitamin E (α-tocopherol), there is significant circumstantial evidence, but no direct evidence, in favor of its use in diabetic patients to reduce oxidative stress and perhaps to slow atherogenesis and the development of microvascular complications. Unfortunately, these same advantages, by leading to widespread use, are likely to impede investigation of the efficacy of such treatments. Also, under some circumstances, vitamin E may have prooxidant effects (222). Probucol may be effective in reducing lipid peroxidation (223) and may therefore have a protective effect in the vessel wall (224–226), but it seems to have few advantages over vitamin E. Butylated hydroxytoluene may have similar effects (227, 228). Coenzyme Q (ubiquinone), which detoxifies the tocopheroxyl radical (the oxidation product of vitamin E) can also inhibit LDL oxidation and may also have a role to play, but this role has not been clearly defined at present (229). Other agents to inhibit the toxic consequences of lipid peroxidation or to inhibit the oxidation itself may be developed as a result of improved understanding of the chemistry involved (230).
Scavenging Reactive Carbonyls
Aminoguanidine acts as a scavenger of reactive carbonyl groups, especially dicarbonyl compounds (e.g., glyoxal formed by oxidative decomposition of FL, Schiff base, or fatty acids, or 3-deoxyglucosone formed by decomposition of FL), species which may mediate advanced carbonyl reactions. It can therefore prevent the formation of glycoxidation and lipoxidation products and interrupt vicious cycles of oxidative damage. In vitro, aminoguanidine can inhibit collagen cross-linking (121) and lipid peroxidation (231, 232). In cell culture, recent studies by our group (described above) have shown that at concentrations as low as 1 μmol/1, aminoguanidine can significantly inhibit the cytotoxicity that develops in LDL when it is exposed to glycoxidative stress (233). The same low concentrations can inhibit the toxicity of simulated hyperglycemia (25 mmol/1 glucose) toward retinal vascular cells (T.J.L., A.J.J., unpublished observations). These results suggest that the observed toxicities may be mediated by oxidation products of LDL and glucose, respectively, and that these products are present at very low concentrations. In vivo, aminoguanidine inhibits the development (126, 234) and progression (235) of diabetic retinopathy in streptozotocin-diabetic rats and can also inhibit the development of diabetic nephropathy (123, 236) and neuropathy (125, 237). Multicenter Phase III studies are now underway to assess the efficacy of aminoguanidine in diabetic nephropathy in humans, and other similar agents with much greater potency are currently being developed (238). Another class of experimental agents, the leumedins (N-(fluorenyl-9-methoxycarbonyl) amino acids [239]), have also shown promise in inhibiting LDL modification (240).
Possibilities for the Direct Removal of AGEs From Serum or Dialysis Fluid
AGEs in serum are not effectively removed by hemodialysis. Recent increases in knowledge concerning AGE receptor recognition have implicated lysozyme, a naturally-occurring protein to which AGEs are initially bound (241). This information has led to experiments using matrix-bound lysozyme as a means of clearing AGEs from serum and dialysis fluid, and initial results are encouraging (242). Finally, compounds that may cleave existing AGE-mediated cross-links in proteins are under investigation (243).
In the future, a combination of the measures outlined above may delay the onset and slow the progression of both macro- and microvascular complications of diabetes. Indeed, they may also be useful in other conditions in the nondiabetic population, e.g., atherosclerosis (244), Alzheimer’s, and other neurodegenerative diseases (245), as well as other age-related conditions, even including the aging process itself.
Acknowledgments
T.J. Lyons gratefully acknowledges support from the NIH (R29 Grant EY10697), the American Diabetes Association, and the Diabetes Research and Wellness Foundation. A.J. Jenkins is supported by a Lions SightFirst/American Diabetes Association Training Grant and by a Research Grant from the Juvenile Diabetes Foundation International.
Glossary
- AGE
advanced glycation end product
- apo
apolipoprotein
- CML
Nɛ-carboxymethyllysine
- DCCT
Diabetes Control and Complications Trial
- FL
fructoselysine
- HNE
hydroxynonenal
- IC
immune complex
- Lp(a)
lipoprotein(a)
- MDA
malondialdehyde
- VCAM-1
vascular cell adhesion molecule 1
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