Advanced nonenzymatic tissue glycosylation: cell-mediated interactions implicated in the complications associated with diabetes and aging.

Blood Purif 1990;8:223-232

© 1990 S. Karger AG, Basel 0253-5068/90/0084-022352.75/0

Advanced Nonenzymatic Tissue Glycosylation: Cell-Mediated Interactions Implicated in the Complications Associated with Diabetes and Aging Helen Vlassara The Rockefeller University, New York, N.Y., USA

Key Words. Nonenzymatic glycosylation • Advanced glycosylation endproducts • Macrophage • Advanced glycosylation endproducts receptor • Cachectin/tumor necrosis factor ■Interleukin 1 • Tissue remodeling • Diabetes • Aging

Introduction A number of age-related tissue changes are consistent with biochemical protein al terations mediated by glucose. Nonenzy matic glycosylation, the reaction of free amino groups of proteins with ambient glu cose without the aid of enzymes, is such a process, leading to the formation of a revers ible ketoamine called Amadori product, and occurs on many proteins throughout the body (fig. 1) [1-3]. However, from this early

and reversible Amadori product, a family of diverse irreversible products form more slowly, following a sequence of further reac tions and rearrangements [3, 4]. These late adducts, called advanced glycosylation endproducts (AGEs), tend to accumulate on long-lived structural proteins such as colla gen and basement membrane proteins, lead ing to structural and functional alterations [4]- These are mostly related to increased cross-linking between AGE protein mole cules, as well as with other unmodified pro-

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Abstract. Tissue and cell surface proteins modified nonenzymatically by glucose are shown to be highly active in protein cross-linking and have been implicated in tissue damage. The production of such protein-glucose interactions called advanced glycosylation endpro ducts (AGE) are recently shown to be processed by macrophages through a recently charac terized high-affinity receptor. Coupling of AGE proteins to their AGE receptor results in TNF and 1L-1 synthesis and secretion. This suggests that AGE may act as a signal for growthpromoting factor secretion in a coordinated replacement process during tissue remodeling. A disturbance of this balance may lead to pathologic proliferative response such as in the vasculopathy of diabetes and aging. Since peritoneal surface proteins can be modified by AGE after exposure to high-glucose, a similar pathogenetic process may be involved in the peritoneal fibrosis associated with chronic peritoneal dialysis.

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a

b

Ki . _____ Schiff base " K-,

K,

Glucose + NH,-protein

x2 _ _ Amadori product X-2

k2

^ Schiff base —

!

Amadori product

' K_,

i KAge proteins (Brown fluorescent pigment which cross-links proteinsi

teins by covalent trapping [5-7], The first isolation of an AGE, called 2-furoyl-4(5)-(2furanyl)-lH-imidazole (FFI) [8] was fol lowed by two novel adducts called 1-alkyl-2-formyl-3,4-diglucosyl-pyrroles (AFGP) which have been isolated and characterized more recently with the aid of NMR and mass spectroscopy [9]. There are two types of cross-links responsible for the hyperglycemic aggregation and cross-linking of proteins; ox idation of previously unexposed sulfhydryl groups by the nonenzymatic glycosylation of lysine amino groups leading to disulfide bond formation, and AGE formation owing to the long half-life of the protein [10, 11]. These two mechanisms may act synergistically in the diabetic as well as the aging lens to accelerate cataract formation [10, 12]. The relationship between diabetic and agerelated tissue deterioration and accelerated protein cross-linking has been under active investigation in our and other laboratories. Formation of AGEs, for instance, has been shown to promote immunoglobulin ac cumulation within the nerve, as a result of long-term exposure of peripheral nerve my elin proteins to glucose [7], IgG trapping on

Fig. 1. Schematic representation of the formation of early reversible (t 1/2 of days or weeks) (a) and ad vanced irreversible (b) nonenzymatic glycosylation products.

peripheral nerve myelin from diabetic pa tients was shown to be almost 14 times, and IgM 4 times greater than those of nondia betic subjects. This process may contribute to the peripheral neuropathy of aging and chronic diabetes. In contrast, the insignifi cant amount of trapped immunoglobulin in brain myelin may partly explain the rarity of central diabetic neuropathy. In addition, recent studies have shown that LDL is covalently bound by glyco sylated collagen more than 3 times as much as by normal collagen at a constant LDLcholesterol level (fig. 2), suggesting a role for glycosylated connective tissue components in atherogenesis [6]. This immobilization of otherwise short-lived LDL particles on gly cosylated wall protein can promote fibrous plaque lipid aggregation and, by preventing its diffusion out of the intima, contribute to the AGE formation on the lipoprotein itself. The observation that generated reactive groups of AGE-modified structural proteins can trap innocent bystander soluble proteins and render them destructive offers impor tant insight into several well-known aging and diabetes-related tissue changes such as


Glucose + NH -protein

Advanced Nonenzymatic Tissue Glycosylation

Collagen advanced glycosylation products, fluorescence/mg

Fig. 2. Covalent binding of l25I-LDL by nonenzymatically glycosylated and control collagen as a func tion of LDL protein concentration. Data are ex pressed as the mean ± SEM of these experiments. Other proteins can be trapped by AGE modified con nective tissue, e.g. albumin, immunoglobulins, fibrin and complement.

prevent glomerular basement membrane thickening have been shown in preliminary data of longer-term studies [Brownlee et al., unpubl. data].

A Novel Receptor-Mediated Processing Mechanism for AGE

Recently, the AGE products forming on long-lived proteins, such as collagen, myelin proteins, etc. in vitro or in vivo, have been


basement membrane thickening, and lipid accumulation within the vessel wall [4, 15, 16]. Also diabetic and age-dependent changes in genetic material may be initiated by the accumulation of AGEs on long-lived nucleic acids since DNA, like proteins, react with glucose, especially in the presence of lysine [13, 14], The recently acquired knowledge on the significance of the biochemical and biologi cal properties of AGE may finally allow much awaited for studies correlating the ex tent of complications to the extent of timeaveraged exposure of long-lived proteins to glucose over months and years. This may help identify and monitor carefully those subsets of patients who are inherently more susceptible to tissue damage. However, it is thought that the ability of the already formed glycosylated proteins such as collagen to con tinue trapping and cross-linking soluble pro teins, even in the absence of free glucose, may allow protein cross-linking to progress, even after correction of hyperglycemia, and may not prevent the development of compli cations in those patients with years of dia betes. Fortunately, prevention of AGE forma tion by a novel compound called aminoguanidine HC1, has recently been found to act by selectively blocking reactive carbonyl formation on early glycosylation products [17], While studies in humans focusing on pharmacokinetics and toxicity are only be ginning , the agent’s effects on early vascular lesions in vivo have been examined in aorta and kidney of diabetic animals. A 4-fold reduction in AGE content, as well as crosslinked plasma proteins onto extracellular matrix, has been demonstrated in diabetic rats treated with aminoguanidine for 4 months [17, 18]. Indications of its ability to

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shown to be recognized and endocytosed by mouse peritoneal macrophages [19-21], and human monocytes [22]. This was first found using in vitro glycosylated myelin proteins and mouse-elicited peritoneal macrophages and was shown to be specific for the AGE, while neither unmodified bovine serum al bumin (BSA) nor unmodified myelin was recognized [19]. Only protein modification by AGE results in macrophage uptake, while Amadori product formation alone does not [ 19]. The important question as to whether there was a specific macrophage/monocyte receptor which recognized a distinct AGE or family of AGEs, accumulating on long-lived proteins after prolonged exposure to glucose, was pursued using AGE-BSA as a probe [21 ]. AGE-BSA was specifically bound to cells at 4 °C, and taken up and degraded at 37 °C, in a concentration-dependent saturable man ner. Scatchard analysis of AGE-BSA binding data indicated that there are approximately 1.5 X 105 receptors per cell, with an affinity constant (Ka) of 1.75 X 107A/_1. Similarly, human monocytes have 1.4 X 105 sites per cell, but a K, of 1.7 X 106 M~' [33]. In addition specific binding of AGE-BSA to the macrophage receptor was competi tively inhibited by BSA which had been chemically coupled to a synthetic analogue of AGE, FFI-BSA [8]. This suggested that the AGE receptor recognizes a specific type of AGE structure having important homol ogy with FFI. In vivo a diverse group of AGE may form on proteins during long-term exposure to glucose [24, 25], and a number of these may bind to the AGE receptor with specific individual affinities. Using RAW 264.7 cells (a murine macro phage-like cell line) as a source of AGE receptor we have recently been able to solu

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bilize and study its ligand specificity [26], We have subsequently isolated a 90-kD pro tein from the membrane fraction of RAW 264.7 cells, utilizing an FFI affinity column that we have synthesized [27], By combining several sequential FPLC chromatographic steps we have been able to isolate the protein to homogeneity (fig. 3). We have further shown that human eryth rocytes having AGE attached to their surface can be specifically bound and ingested by human monocyte AGE receptors in vitro (fig. 4) [22], while AGE-RBC survival in vivo was markedly shortened [22]. These studies indicated that the AGE receptor mechanism may be in part responsible for specific recognition and removal of cells with prolonged half-life, likely to be modi fied by glucose as they age.

Regulation of Age Receptor Function

As suggested by the studies described above, the AGE protein receptor could con ceivably play an important role in the regula tion of extracellular protein turnover, and primarily the vessel wall proteins. Since AGE formed on extravascular matrix colla gen can trap LDL [6] which can subse quently undergo AGE modification, the AGE-receptor may normally play a role in the removal of atherogenic material. In aging, however, as well as in diabetes, the efficiency of this removal system is not com plete, as indicated by the continuous in crease of AGE formation with age and/or hyperglycemia [4], The rate at which agingassociated vascular changes take place may be profoundly affected by hormones and other physiological mediators [28, 29]. Since such factors might influence the net amount


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Fig. 3. SDS-PAGE of purified AGE-binding pro tein obtained from the macrophage-like murine tu mor cell line RAW 264.7. The 90-kD protein is of similar molecular weight to that obtained from hu man monocytes by cross-linking studies.

creased degradation of the endocytosed AGE-BSA (25-30%) [31]. In contrast, macrophages from the hyper insulinémie but equally hyperglycemic C57B1/6J (db/db) mice exhibited a marked reduction in both the number of AGE recep tors (6 X 104/cell) as well as in binding affin ity (4 X 106A/_I)> with a 50% reduction in the amount of AGE-BSA degraded [31]. Thus, it appears that insulin and not high glucose, or the accumulation of AGE pro teins, may be a causative agent for this mod ulation of the AGE receptor. These observa tions have led us to the conclusion that an alteration of the AGE receptor can be


of cross-linking AGE proteins in the tissues, we have begun to study the effects of glucose and insulin on the macrophage AGE recep tor [31]. Elevated glucose or insulin concen trations in vitro failed to demonstrate any short-term effect on the AGE receptor num ber or function. Therefore, binding and deg radation studies of labeled AGE-BSA by mu rine peritoneal macrophages from experi mentally induced and genetically hypo- and hyperinsulinémie diabetic mice were carried out. Macrophages from hypoinsulinemic al loxan-induced diabetic animals showed a 2to 3-fold increase in AGE receptor number per cell as compared to control mice. A sim ilar increase was observed with macrophages from C57BL/KsJ (db/db) mice. The binding affinity for both of these groups of animals was approximately the same as the control animals, and was accompanied by an in-

Fig. 4. Electron scanning micrograph of human AGE (FFI)-modified RBC bound by human periph eral monocytes at 4°C . Micrograph made by David M. Phillips of the Population Council.

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prompted by the in vivo exposure of the macrophage to variable insulin concentra tions. It is not clear whether this reflects the necessity for exposure of an insulin-sensitive macrophage precursor or that hyperinsulinemia generates a more proximal regulatory signal in vivo. However, these studies pro vided evidence for a specific insulin-sensi tive mechanism that may modulate the properties and function of the macrophage AGE receptor. Factors that reduce its effi ciency, such as in insulin-resistant diabetic patients, may play a major role in determin ing the total amount and rate of hypergly cemia-accelerated glucose-modified protein accumulation in blood vessel walls. In non diabetic subjects, such a mechanism may explain the well-documented association be tween hypocaloric intake or fasting (where insulin levels are reduced), and retardation of aging in animal models [32], A 4- to 6-fold increase in maximum bind ing and degradation of nonenzymatically glycosylated albumin (AGE-BSA) by macro

phage/monocytes previously exposed to tu mor necrosis factor (TNF) was also demon strated as compared to nonexposcd cells, while there was no effect on the binding and degradation of unmodified BSA (fig. 5] [33]. These effects were completely blocked in the presence of an anti-TNF antibody [33]. These data suggest that AGE-induced TNF may normally play an important regulatory role in the macrophage removal of glucose modifications forming or. long-lived tissue proteins, such as in vessel walls. Finally, the effect of aging on the macro phage AGE receptor was recently evaluated in young (6-month-old) and old (2.5-yearold) mice. A greater than 2-fold decrease in both receptor number and binding affinity was found in cells from the old group as compared to the young group of animals in preparation. These data suggest that aging in itself may adversely affect the AGE receptor efficiency, which may compound the tissue damage by preventing the removal of crosslinked glycosylated proteins [43].


Fig. 5. Maximal specific binding (a), intracellular accumulation (b), and degradation (c) of either l25IAGE-modified BSA (25 pg/ml) or l25I-normaI BSA (25 pg/ml) by mu rine peritoneal resident macro phages after preincubation with the indicated agents for 48 h at 37 °C. Control wells contained cells prein cubated in medium with serum alone, for the same period of time before receiving the appropriate ra dioactive ligand. Data are ex pressed as the means ± SEM of four independent experiments each done in triplicate.

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Age-Receptor-Mediated Induction of Monokines

Fig. 6. Detection of cachectin/TNF secretion by human monocytes in response to AGE proteins.

Table 1. Detection of IL-1 production of human monocytes in response to AGE proteins Ligand added

IL-ip, pM

AGE-BSA (250 pg/ml) Nl-BSA (250 pg/ml) IFN-t (1 ng/ml) LPS (0.2 ng/ml)

93.5 ±29.9 16.2 ± 1.6 none detected 372.0 ±36.4

This difference presumably reflects the abil ity of interferon-Y to exert a priming effect on human monocytes, which augments their subsequent cachectin/TNF secretory re sponse [39, 40]. In order to determine whether the observed appearance of cachec tin/TNF was associated with the induction of new mRNA or translation of cryptic ca chectin/TNF message [41], Northern blot analysis of monocyte mRNA for cachectin/


Recently we have begun to examine the process by which removal of AGE protein might be coordinated with its replacement by newly synthesized material. We have found that in response to the binding of AGE proteins the macrophage synthesizes and re leases the monokines cachectin TNF (fig. 6) and interleukin 1 (IL-1) (table 1) [34], The two monokines cachectin/TNF and IL-1 have diverse biological activities, many of which are shared by both, and which have been extensively reviewed [35, 36], Of rele vance here is the fact that both have been reported to have growth factor-like [37], and angiogenic activities [38], In these studies, normal human peripheral blood monocytes were incubated in medium containing hu man interferon-y (1 ng/ml, 50 units/ml) and polymyxin (100 ng/ml), in the presence of either normal BSA (Nl-BSA, 250 pg/ml), in vitro glycosylated BSA (Glu-BSA, 250 pg/ml or G6P-BSA, 250 pg/ml), or chemically syn thesized FFI-BSA (150 pg/ml). Following in cubation, cachectin/TNF was measured in the media by an enzyme-linked immunosorbant assay using a purified monoclonal anticachectin antibody. Figure 6 records the amount of material made by each of the experimental groups. Macrophages incu bated with unmodified albumin (Nl-BSA) released only minimal levels of cachectin/ TNF. In contrast, medium from macro phages incubated with each of the three types of AGE-BSA contained approximately 10 times the amount of cachectin/TNF found with Nl-BSA. In the absence of interferon-y, human monocyte supernatants con tained approximately one-half the amount of cachectin/TNF in response to AGE protein.

Fig. 7. Potential mechanisms by which AGE-mediated synthesis and secretion of cachectin/TNF and 1L-1 might contribute to the regulation of normal tis sue remodeling.

TNF was performed. A small background amount of message was detected in the monocyte preparations that had been incu bated with Nl-BSA, while cells incubated with the three AGE-BSA preparations had significantly increased amounts of message [34]. These results suggested that the AGE proteins increase both the cellular levels of cachectin/TNF mRNA and the amount of secreted cytokine in noncytotoxic amounts. AGE proteins are the first reported endoge nously produced materials that, although not inflammatory in origin, can induce this po tent cytokine. Under identical experimental conditions, the amount of cell-associated and extracellu lar IL-1 released by macrophages in response

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to AGE-BSA was measured (table 1) [34]. Total IL-1p was significantly higher in the AGE-BSA preparations than the Nl-BSA or the interferon-? controls. Since cachectin/ TNF can prompt the release of IL-1, it is not clear whether the AGE-BSA is acting directly or through the release of the cachectin/TNF. Even if its production represents a secondary response to cachectin/TNF, IL-1 still shares and would therefore amplify the activities of cachectin/TNF relating to tissue growth and remodeling. The finding of cytokine release by macro phages in response to AGE proteins is of great significance since it offers an explana tion to several observations noted previously in association with these cytokines. The abil ity of cachectin/TNF and IL-1 to stimulate mesenchymal cells to synthesize and release collagenase and other extracellular proteases could reflect their role in initiating local degradative events. Similarly the ability of cachectin/TNF and IL-1 to prompt a synthetic/ proliferative response in cells such as fibro blasts, and the release of other growth factors might be viewed as their role in the repair process. Such a role for cachectin/TNF and IL-1 might further explain why these genes have been so highly conserved in mam mals. The in vivo modification of matrix pro teins by time-dependent formation of glu cose-derived AGEs may thus constitute a unique biologic time clock which signals macrophages to secrete cachectin/TNF, IL1, and possibly other cytokines, which in turn influence both the degradation as well as the proliferation of tissue components (fig. 7). Since the macrophage has been found to produce a number of monokines [42], the coordinated response of this remod eling system is of critical importance. A dis


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turbance of this system in states such as in normal aging, where AGE accumulation is increased as a function of time, may explain at least in part the excessive proliferative response characteristic of several aging tis sues. In addition, excessive fibrotic changes observed in relation to chronic peritoneal dialysis - where the peritoneal tissues are exposed chronically to excessive amounts of glucose-containing dialysis fluid - may be explained on the basis of AGE-induced pro liferative response.

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35 Dinarello CA: Interleukins, tumor necrosis factor (cachectin), and interferons as endogenous pyro gens and mediators of fever; in Pick E (ed): Lymphokines. New York, Academic Press, 1987, pp 1-27. 36 Le J, Vilcek J: Tumor necrosis factor and interleu kin 1: cytokines with multiple overlapping biolog ical activities. Lab Invest 1987;56:234-248. 37 Sugarman BJ, Aggarwal BB, Hass PE, Figari IS, Palladino MA Jr, Shepard HM: Recombinant hu man tumor necrosis factor-a: effects on prolifera tion of normal and transformed cells in vitro. Science 1985;230:943-945. 38 Frater-Schroeder M, Risau W. Hallman R, Gautschi P, Bohlen P: Tumor necrosis factor type a, a potent inhibitor of endothelial cell growth in vi tro, is angiogenic in vivo. Proc Natl Acad Sci USA 1987;84:5277-5281. 39 Nathan CF, Murray HW, Wiebe ME, Rubin BY: Identification of interferon-x as the lymphokine that activates human oxidative metabolism and antimicrobial activity. J Exp Med 1983; 158:670689. 40 Pace JL, Russell SW, Schreiber RD. Altman A. Katz DH: Macrophage activation: priming activ ity from a T-cell hybridoma is attributable to interferon-y. Proc Natl Acad Sci USA 1983;80: 3782-3786. 41 Beutler B, Krochnin N, Milsark IW, Luedke C, Cerami A: Control of cachectin (tumor necrosis factor) synthesis: mechanisms of endotoxin resis tance. Science 1986;232:977-980. 42 Nathan CF: Secretory products of macrophages. J Clin Invest 1987;79:319-326. 43 Vlassara H, Harrison H: Effects of aging on AGEreceptor, in preparation.

Accepted: March 14, 1990 Dr. Helen Vlassara Laboratory of Medical Biochemistry The Rockefeller University 1230 York Ave New York, NY 10021 (USA)


2 1 Vlassara H, Brownlee M, Cerami A: High-affinity receptor-mediated uptake and degradation of glu cose-modified proteins: a potential mechanism for the removal of senescent macromolecules. Proc Natl Acad Sci USA 1985;82:5588-5592. 22 Vlassara H, Valinsky J, Brownlee M, Cerami C, Nichimoto S, Cerami A: Advanced glycosylation products on erythrocyte cell surface induce recep tor mediated phagocytosis by macrophages; a model of turnover of aging cells. J Exp Med 1987; 166:539-549. 23 Vlassara H: Peripheral neuropathy and aging. Age 1988;11:14-18. 24 Reynolds TM: Chemistry of nonenzymatic browning: I. Adv Food Res 1963;12:1-52. 25 Reynolds TM: Chemistry of nonenzymatic browning: II. Adv Food Res 1965;14:167-283. 26 RadoffS, Vlassara H, Cerami A: Characterization of a solubilized cell surface binding protein on macrophages specific for proteins modified nonenzymatically by advanced glycosylation end products. Arch Biochem Biophys 1988:263:418423. 27 RadofFS, Vlassara H: Isolation of advanced glyco sylation endproduct cell membrane-binding pro tein from murine RAW 264.7 cells. Diabetes, in press. 29 Fox PL, DiCorleto PE: Modified low density lipo proteins suppress production of a platelet-derived growth factor-like protein by cultured endothelial cells. Proc Natl Acad Sci USA 1986;83:47744778. 30 Warren MK, Vogel SN: Opposing effects of gluco corticoids on interferon-y-induced murine macro phage Fc receptor and la antigen expression. J Immunol 1985;134:2462-2469. 31 Vlassara H, Brownlee M, Cerami A: Specific mac rophage receptor activity for advanced glycosyla tion end products inversely correlates with insulin levels in vivo. Diabetes 1988;37:456-461. 32 Harrison DE, Archer JR, Astle CM: Effects of food restriction on aging: Separation of food in take and adiposity. Proc Natl Acad Sci USA 1984; 81:1835-1838. 33 Vlassara H, Moldawer L, Chan B: Upregulating AGE-receptor. J Clin Invest, in press. 34 Vlassara H, Brownlee M, Manogue ICR, Dinarello CA, Pasagian A: Cachectin/TNF and IL-1 in duced by glucose-modified proteins: role in nor mal tissue remodeling. Science 1988;240:1546— 1548.

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