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The relative roles of advanced glycation, oxidation and aldose reductase inhibition in the development of experimental diabetic nephropathy in the Sprague-Dawley rat.
Soulis-Liparota T, Cooper ME, Dunlop M, Jerums G
Department of Medicine, University of Melbourne, Austin Hospital, Victoria, Australia.
Diabetologia (Germany) Apr 1995, 38 (4) p387-94

Advanced glycation is an important pathogenic mechanism in the development of diabetic complications. However, other biochemical processes, such as the polyol pathway or lipid and protein oxidation which can interact with advanced glycation can also yield tissue fluorescence and may also be implicated in the genesis of diabetic microangiopathy. Aminoguanidine is an inhibitor of advanced glycation, but it is not known if all of its effects are mediated by this mechanism. The present study explores the relative contributions of aldose reductase, oxidative stress and advanced glycation on the development of aortic and renal fluorescence and urinary albumin excretion in streptozotocin diabetic rats. The study groups included non-diabetic (control), streptozotocin diabetic rats and diabetic rats receiving aminoguanidine, the anti-oxidants butylated hydroxytoluene and probucol and the aldose reductase inhibitor, ponalrestat. Serial measurements of glycaemic control and urinary albumin excretion were performed every 8 weeks. At 32 weeks, animals were killed, tissues removed and collagen extracted for measurement of fluorescence. Diabetic rats had increased fluorescence in aorta, glomeruli and renal tubules. Aminoguanidine prevented an increase in fluorescence at all three sites suggesting that diabetes-related tissue fluorescence is predominantly due to advanced glycation. Ponalrestat retarded fluorescence in aorta only and butylated hydroxytoluene attenuated fluorescence at the renal sites but not in the aorta. Diabetic rats had increased renal cortical sorbitol levels. Ponalrestat normalized renal cortical sorbitol levels but aminoguanidine did not affect this parameter. The only agent to decrease plasma thiobarbituric acid reactive substances was butylatedhydroxytoluene. Diabetic rats developed albuminuria over the 32-week period.

Cloning and expression of cytokine-inducible nitric oxide synthase cDNA from rat islets of Langerhans.
Karlsen AE, Andersen HU, Vissing H, Larsen PM, Fey SJ, Cuartero BG, Madsen OD, Petersen JS, Mortensen SB, Mandrup-Poulsen T, et al
Steno Diabetes Center, Gentofte, Denmark.
Diabetes 1995 Jul;44(7):753-8

An inducible nitric oxide (NO) synthase isoform (iNOS) is specifically induced in the beta-cells of interleukin (IL)-1 beta-exposed rat islets, suggesting a role for NO in the pathogenesis of type I diabetes. The aim of this study was to clone and characterize iNOS cDNA from cytokine-exposed islets. Neither NO production nor iNOS transcription could be detected in rat islets or in rat insulinoma RIN-5AH beta-cells cultured in the absence of cytokines. Addition of IL-1 beta alone or in combination with tumor necrosis factor-alpha induced a concentration- and time-dependent expression of the iNOS gene and associated NO production (measured asnitrite) from both islets and RIN cells. iNOS transcripts were cloned by reverse transcriptase-polymerase chain reaction from the cytokine-exposed rat islets and RIN cells, and DNA sequence analysis revealed a near 100% identity to the recently published iNOS cDNA cloned from cytokine-exposed rat hepatocytes and smooth muscle cells. Recombinant rat islet iNOS was transiently and stably expressed in human kidney 293 fibroblasts, and the high enzymatic activity was inhibited by addition of the L-arginine analogs, N omega-nitro-L-arginine methyl ester and aminoguanidine. Two-dimensional gel electrophoresis revealed the recombinant iNOS as aseries of spots with the expected molecular mass of 131 kDa and pI values in the range of 6.8 to 7.0. In conclusion, the IL-1 beta-induced iNOS cloned and expressed from rat islets and RIN cells is encoded by the same transcript as the iNOS induced in other cell types.

Aminoguanidine does not inhibit the initial phase of experimental diabetic retinopathy in rats.
Hammes HP, Ali SS, Uhlmann M, Weiss A, Federlin K, Geisen K, Brownlee M
Third Medical Department, Justus-Liebig-University of Giessen, Germany.
Diabetologia (Germany) Mar 1995, 38 (3) p269-73

We have previously shown that long-term administration of aminoguanidine, an inhibitor of advanced glycosylation product formation, reduces the extent of experimental diabetic retinopathy in the rat by 85%. In order to determine whether the residual retinopathy that developed despite aminoguanidine was attributable to advanced glycation endproduct formation, a time-course study was performed in three different groups of male Wistar rats: non-diabetic controls (NC), streptozotocin-diabetic controls (DC) and streptozotocin-diabetic rats treated with aminoguanidine HCL, 50 mg/100 ml drinking water (D-AG). Eyes were obtained at 24, 32, 44 and 56 weeks of diabetes/treatment duration and morphologic evaluation was done on retinal digest preparations. At 56 weeks, retinal basement membrane thickness was additionally measured. After 24 weeks of diabetes, the number of acellular capillaries was significantly elevated in DC (44.6 +/- 5.7/mm2 of retinal area, NC 19.6 +/- 4.9; p < 0.001) and increased continuously over time (DC56 weeks 87.4 +/- 15.1; p < 0.001 vs DC24 weeks). In contrast, acellular capillaries in D-AG increased over the first 24 weeks and then remained constant for the rest of the study (D-AG 24 weeks 35.7 +/- 5.18; p < 0.01 vs NC 24 weeks and NS vs DC 24 weeks; D-AG 56 weeks 42.0 +/- 6.20; p NS vsD-AG 24 weeks). (ABSTRACT TRUNCATED AT 250 WORDS)

Neurotoxicity of advanced glycation endproducts during focal stroke and neuroprotective effects of aminoguanidine.
Zimmerman GA, Meistrell M 3rd, Bloom O, Cockroft KM, Bianchi M, Risucci D, Broome J, Farmer P, Cerami A, Vlassara H, et al
Department of Surgery, North Shore University Hospital, Manhasset, NY 11030, USA.
Proc Natl Acad Sci U S A (United States) Apr 25 1995, 92 (9) p3744-8

Cerebral infarction (stroke) is a potentially disastrous complication of diabetes mellitus, principally because the extent of cortical loss is greater in diabetic patients than in nondiabetic patients. The etiology of this enhanced neurotoxicity is poorly understood. We hypothesized that advanced glycation endproducts (AGEs), which have previously been implicated in the development of other diabetic complications, might contribute to neurotoxicity and brain damage during ischemic stroke. Using a rat model of focal cerebral ischemia, we show that systemically administered AGE-modified bovine serum albumin (AGE-BSA) significantly increased cerebral infarct size. The neurotoxic effects of AGE-BSA administration were dose- and time-related and associated with a paradoxical increase in cerebral blood flow. Aminoguanidine, an inhibitor of AGE cross-linking, attenuated infarct volume in AGE-treated animals. We conclude that AGEs may contribute to the increased severity of stroke associated with diabetes and other conditions characterized by AGE accumulation.

Agmatine and spermidine reduce collagen accumulation in kidneys of diabetic db/db mice.
Marx M, Trittenwein G, Aufricht C, Hoeger H, Lubec B
Department of Pediatrics, University of Vienna, Austria.
Nephron (Switzerland) 1995, 69 (2) p155-8

In the present study, we tested the hypothesis whether agmatine and spermidine, metabolites of arginine metabolism, share the pharmacological activities of arginine reducing collagen accumulation in the diabetic kidney. Eleven db/db mice were administered agmatine and 12 db/db mice spermidine (50 mg/kg body weight). Ten db/db mice received no treatment as negative controls and 10 db/db mice were treated with aminoguanidine (50mg/kg body weight) as positive controls. Mean kidney OH-proline content reflecting kidney collagen content and mean CML concentration were significantly higher but acid solubility of collagen significantly lower in the untreated group than in the treated groups. Agmatine, although missing the alpha-amino group and the carboxyl group, and spermidine, although missing the guanidino group, thus still revealed the arginine activity. We hypothesize that the strongly nucleophilic structure of polyamines common to all active compounds is able to block reactive carbonyls.

Mechanism of autoxidative glycosylation: identification of glyoxal and arabinose as intermediates in the autoxidative modification of proteins by glucose.
Wells-Knecht KJ, Zyzak DV, Litchfield JE, Thorpe SR, Baynes JW
Department of Chemistry and Biochemistry, University of South Carolina, Columbia 29208.
Biochemistry (United States) Mar 21 1995, 34 (11) p3702-9

Glycation and oxidation reactions contribute to protein modification in aging and diabetes. Formation of dicarbonyl sugars during autoxidation of glucose is the hypothetical first step in the autoxidative glycosylation and subsequent browning of proteins by glucose [Wolff, S. P., & Dean, R. T. (1987) Biochem. J. 245, 243-250]. In order to identify the dicarbonyl sugar(s) formed during autoxidation of glucose under physiological conditions, glucose was incubated in phosphate buffer (pH 7.4) at 37 degrees C under air (oxidative conditions) or nitrogen with transition metal chelators (antioxidative conditions). Dicarbonyl compounds were analyzed spectrophotometrically and by HPLC after reaction with Girard-T reagent. Carbohydrates were analyzed by gas chromatography-mass spectrometry. Both dicarbonyl sugar and arabinose concentrations increased with time and glucose concentration in incubations conducted under oxidative conditions; only trace amounts of these products were detected in glucose incubated under antioxidative conditions. HPLC analysis of adducts formed with Girard-T reagent indicated that glyoxal was the only alpha-dicarbonyl sugar formed on autoxidation of glucose. Glyoxal and arabinose accounted for > or = 50% of the glucose lost during a 21 day incubation. Neither glucosone nor its degradation product, ribulose, was detectable. Reaction of glyoxal with RNase yielded the glycoxidation product, N epsilon-(carboxymethyl)lysine, while arabinose is a source of pentosidine. Our results implicate glyoxal and arabinose as intermediates in the browning and crosslinking of proteins by glucose under oxidative conditions. They also provide a mechanism by which antioxidants and dicarbonyl trapping reagents, such as aminoguanidine, limit glycoxidation reactions and support further evaluation of these types of compounds for inhibition of chemical modification and crosslinking of proteins during aging and diabetes.

Nitric oxide synthesis and the effect of aminoguanidine and NG-monomethyl-L-arginine on the onset of diabetes in the spontaneously diabetic BB rat.
Wu G
Department of Animal Science, Texas A&M University, College Station TX 77843-2471
Diabetes (United States) Mar 1995, 44 (3) p360-4

Nitric oxide (NO) synthesis and the effect of aminoguanidine (AG) and NG-monomethyl-L-arginine (NMMA) (inhibitors of NO synthase) on the onset of diabetes were studied in the spontaneously diabetic BB rat. To measure in vivo NO production, 20 male 50-day-old diabetes-prone BB (BBdp) rats and age-matched non-diabetes-prone BB (BBn) rats were individually placed in metabolism cages. The animals had free access to a casein-based semipurified diet and deionized and double-distilled water. Urine excretion was collected every other day for 70 days, and urinary excretion of nitrate was measured as an index of in vivo NO synthesis. The urinary excretion of nitrate was enhanced by 150-200% in BBdp rats 4-6 days before the onset of diabetes, compared with aged-matched BBn rats. There was no difference in urinary excretion of nitrate between BBn rats and those BBdp rats that did not develop diabetes by the age of up to 120 days. To determine a role of NO in the development of spontaneous diabetes, 40-day-old male BBdp rats (30 rats per group) received daily subcutaneous injections of NMMA (acetate salt) (5 mg/kg body wt) or equal amounts of acetate (control) or oral administration of AG (0 or 3 g/l of drinking water) for 80 days. Both NMMA and AG delayed the onset of diabetes in BBdp rats by 13-15 days without altering the rate of incidence of diabetes.

The pharmacokinetics of aminoguanidine in end-stage renal disease patients on hemodialysis.
Foote EF, Look ZM, Giles P, Keane WF, Halstenson CE
Department of Medicine, Hennepin County Medical Center, Minneapolis, MN 55404.
Am J Kidney Dis (United States) Mar 1995, 25 (3) p420-5

Aminoguanidine is an investigational agent that may slow or prevent many diabetes-related complications. Since the elimination of aminoguanidine is dependent on renal function, its pharmacokinetics was investigated in eight chronic renal failure patients maintained on hemodialysis. Each patient received 300 mg of aminoguanidine hydrochloride during both an interdialytic and an intradialytic period. During the interdialytic period, the maximum aminoguanidine concentration (Cmax) and time to reach Cmax was 4.5 micrograms/mL and 1.5 hours, respectively. The terminal elimination hallife in these patients was prolonged (37.9 hours). The renal clearance was 2.1 mL/min. Only 8.7% of the administered dose was recovered unchanged in the urine, which is markedly reduced from what is recovered in urine in subjects with normal renal function. There was a positive correlation between the renal clearance of aminoguanidine and the patients' residual renal function (P < 0.05). During hemodialysis, the hallife of aminoguanidine was shortened to 3.9 hours. The hemodialysis clearance of aminoguanidine was 203.6 mL/min. After cessation of hemodialysis, a significant rebound in plasma aminoguanidine concentrations (mean, 39%) was observed. Thus, the dose of aminoguanidine hydrochloride will need to be significantly reduced in patients with end-stage renal disease. Given the interdialytic and intradialytic pharmacokinetics of aminoguanidine, three times weekly dosing after each hemodialysis session is suggested.

Effect of aminoguanidine on the impaired nitric oxide-mediated neurotransmission in anococcygeus muscle from diabetic rats.
Way KJ, Reid JJ
Department of Pharmacology, University of Melbourne, Parkville, Victoria, Australia.
Neuropharmacology (England) Nov 1994, 33 (11) p1315-22

The contribution of advanced glycation end-product (AGE) formation to alterations in nitrergic neurotransmission caused by 8-week streptozotocin-induced diabetes has been examined in the rat anococcygeus muscle. Relaxant responses to nitrergic nerve stimulation (0.5-5 Hz, 10-sectrain), to nitric oxide (NO; 0.1-3 microM), to the NO donor, sodium nitroprusside (SNP; 5-500 nM), and to the cell-permeable analogue of cyclic guanosine monophosphate (cGMP), 8-bromo-cGMP (15 and 30 microM), were significantly smaller in muscles from diabetic rats than from control rats.Pretreatment with aminoguanidine hemisulphate (1 milligram drinking water) to inhibit AGE formation, did not alter the relaxant responses to nitrergic nerve stimulation, NO or SNP in tissues from control rats, or responses to NO or SNP in tissues from diabetic rats, however relaxations to nitrergic nerve stimulation were further reduced in tissues from diabetic rats. In anococcygeus muscles from untreated animals, a 20-min exposure to aminoguanidine (1 mM) in vitro had no effect on relaxations to nitrergic nerve stimulation. The results suggest that diabetes impairs nitrergic transmission in the rat anococcygeus at least partly through alterations in the cGMP-relaxation pathway. The impaired neurotransmission does not appear to be related to the formation of AGEs.

Interleukin 1 beta induces diabetes and fever in normal rats by nitric oxide via induction of different nitric oxide synthases.
Reimers JI, Bjerre U, Mandrup-Poulsen T, Nerup J
Steno Diabetes Center, Gentofte, Denmark.
Cytokine (United States) Sep 1994, 6 (5) p512-20

Substantial in vitro evidence suggests that nitric oxide may be a major mediator of interleukin 1 (IL-1) induced pancreatic beta-cell inhibition and destruction in the initial events leading to insulin-dependent diabetes mellitus. Using NG-nitro-L-arginine methyl ester, an inhibitor of both the constitutive and the cytokine inducible forms of nitric oxide synthase, andaminoguanidine, a preferential inhibitor of the inducible form of nitric oxide synthase, we investigated the impact of inhibiting nitric oxide production on food-intake, body weight and temperature, blood glucose,plasma insulin, glucagon, corticosterone and leukocyte- and differential-counts in normal rats injected once daily for 5 days with interleukin 1 beta (IL-1 beta) (0.8 microgram/rat = 4.0 micrograms/kg). Inhibition of both the constitutive and the inducible forms of nitric oxide synthase prevented IL-1 beta-induced fever, hyperglycaemia, hypoinsulinemia, and hyperglucagonemia, and partially prevented lymphopenia and neutrophilia, but had no effect on IL-1 beta-induced anorexia and changes in plasma corticosterone. Preferential inhibition of the inducible form of nitric oxide synthase using two daily injections of 5 mg/rat of aminoguanidine prevented IL-1 beta-induced hyperglycaemia and hypoinsulinaemia, and slightly reduced the pyrogenicity of IL-1 on 3 out of 5 days. Higher doses of aminoguanidine (100 mg/rat) prevented lymphopenia and neutrophilia. We conclude that nitric oxide produced by the inducible form of nitric oxide synthase, mediates the IL-1 beta-induced inhibition of insulin release and that the effect of IL-1 beta on temperature, pancreatic alpha-cells, and leukocyte differential counts seems to be mediated by nitric oxide produced by the constitutive form of nitric oxide synthase.

Reversal of diabetes by intrapancreatic injection of aminoguanidine liposomes.
Ricordi C, Behboo R, Klibanov A, Singal A, Huang L
University of Pittsburgh Transplantation Institute, Pennsylvania.
Transplant Proc (United States) Dec 1994, 26 (6) p3479

No abstract.

The reaction of methylglyoxal with aminoguanidine under physiological conditions and prevention of methylglyoxal binding to plasma proteins.
Lo TW, Selwood T, Thornalley PJ
Department of Chemistry and Biological Chemistry, University of Essex, Colchester, UK
Biochem Pharmacol (England) Nov 16 1994, 48 (10) p1865-70

Increased formation of methylglyoxal in clinical diabetes mellitus and metabolism by the glyoxalase system has been linked to the development of clinical complications of diabetes: retinopathy, neuropathy and nephropathy. Aminoguanidine has been proposed as a prophylactic agent for preventive therapy of diabetic complications. Methylglyoxal reacted with aminoguanidine under physiological conditions to form two isomerictriazines, 3-amino-5-methyl-1,2,4-triazine and 3-amino-6-methyl-1,2,4-triazine. The mean second order rate constant for the reaction of methylglyoxal with aminoguanidine, kMG.AG = 0.39 +/- 0.06 M-1 sec-1 at pH 7.4 and 37degrees. Under these conditions, no methylglyoxal bisguanylhydrazone was detected. Aminoguanidine prevented the irreversible modification of human plasma protein by a physiological concentration of methylglyoxal (1microM); the median inhibitory concentration IC50 value of aminoguanidine was 203 +/- 16 microM (N = 28). The scavenging of methylglyoxal by aminoguanidine may contribute to the beneficial effects of aminoguanidine in the prevention of vascular pathogenesis in diabetes.

Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats.
Vlassara H, Striker LJ, Teichberg S, Fuh H, Li YM, Steffes M
Picower Institute for Medical Research, Manhasset, NY 11030.
Proc Natl Acad Sci U S A (United States) Nov 22 1994, 91 (24) p11704-8

High levels of tissue advanced glycation end products (AGEs) that result from the spontaneous modification of proteins by glucose occur in diabetes and aging. To address the potential pathogenic role of AGEs in the glomerulosclerosis of diabetes or nephrosclerosis of aging, doses of AGE-modified rat albumin (25 mg per kg per day, i.v.) sufficient to elevate circulating AGE levels to the range of diabetic serum were administered daily to healthy rats alone or in combination with the AGE inhibit oraminoguanidine. After 5 months, the AGE content of renal tissues in AGE-treated rats rose to 50% above controls (P < 0.025), whereas serum contained 2.8-fold greater AGE levels (P < 0.025). Light and electronmicroscopy of kidneys from AGE-treated rats revealed a more than 50% increase in glomerular volume compared to controls (P < 0.001), significant periodic acid/Schiff reagent-positive deposits, basement membrane widening, and mesangial extracellular matrix increase and indicated significant glomerulosclerosis compared to untreated (P < 0.002) or albumin-treated controls (P < 0.002). These changes were associated with significant loss of protein (P < 0.005) and albumin (P < 0.002) in the urine of AGE-treated rats compared to controls. Cotreatment with aminoguanidine markedly limited both the structural and functional defects. These in vivo data demonstrate that AGEs influence glomerular structure and function in a manner leading to glomerulosclerosis. The effects are AGE-specific, as they are ameliorated by a pharmacological AGE inhibitor, aminoguanidine.

Active and passive mechanical properties of isolated arterioles from STZ-induced diabetic rats. Effect of aminoguanidine treatment.
Hill MA, Ege EA
Department of Physiology, Eastern Virginia Medical School, Norfolk 23501.
Diabetes (United States) Dec 1994, 43 (12) p1450-6

Studies were performed to examine the effect of experimental diabetes (4-6 weeks duration) on both the passive elastic and active myogenic properties of isolated skeletal muscle arterioles. Studies were conducted on untreated streptozotocin (60 mg/kg)-induced diabetic rats and in similar rats treated daily with either amino-guanidine (25 mg/kg) ormethylguanidine (25 mg/kg). First-order cremaster muscle arterioles were isolated, cannulated, and pressurized in the absence of intraluminal flow.Video microscopy was used to determine relationships between arteriolar diameter and intraluminal pressure both in the active and passive (o mmol/lCa(2+)-2 mmol/l EGTA superfusated) tes. The measurements were used to calculate active myogenic responses, arteriolar distensibility, and stress-strain relationships. Under passive conditions, arterioles from untreated diabetic animals appeared to be stiffer and less distensible compared with similar arterioles from control animals. Under active conditions, i.e., in the presence of extracellular Ca2+, arterioles from the untreated diabetic group showed impaired myogenic reactivity as evidenced by a significant (P < 0.001) reduction in the negative slope of the pressure-diameter relationship over a physiological range of intraluminal pressures. Chronic treatment with aminoguanidine prevented the diabetes-induced changes in the active and passive properties of the isolated arterioles while treatment with methylguanidine appeared ineffective. Vasodilator responses to topically applied acetylcholine (10(-8) to 5 x 10(-6) mol/l) were significantly impaired in diabetic animals irrespective of treatment with aminoguanidine. The data indicate that experimental diabetes is associated with a decreased passive distensibility, or stiffening, of skeletal muscle arterioles that, in addition, may contribute to impaired active myogenic responses.

Effects of aminoguanidine on insulin release from pancreatic islets.
Tasaka Y, Nakaya F, Matsumoto H, Omori Y
Tokyo Women's Medical College, Diabetes Center, Japan.
Endocr J (England) Jun 1994, 41 (3) p309-13

Aminoguanidine (AG) is a potential therapeutic agent for preventing the generation of advanced glycation end products in diabetes mellitus. In this study, the effect of AG on insulin secretion was investigated in in vitro rat pancreatic islets. The islets were aseptically isolated and cultured in tissue culture medium 199 for 48 h with or without AG. After the culture, batches of 10 islets were incubated in Krebs-Ringer bicarbonate buffer containing 3.3 mM or 16.7 mM glucose. Islets previously exposed to 0.18 mM AG or 0.45 mM AG showed similar insulin release to control islets at a 16.7 mM glucose concentration, but high glucose-stimulated insulin release was inhibited in the islets exposed to 1.8 mM. In the perifusion experiment, insulin release caused by 16.7 mM glucose from the islets previously exposed to 1.8 mM AG was not significantly different from that of the control islets. However, culture of the islets with higher AG concentrations, 4.55 mM and 9.1 mM, significantly inhibited glucose-stimulated insulin release (< 0.02 and 0.002, respectively). These results suggest that AG at high concentrations impairs pancreatic B-cell response to a high concentration of glucose.

TNF-alpha and IFN-gamma potentiate the deleterious effects of IL-1 beta on mouse pancreatic islets mainly via generation of nitric oxide.
Cetkovic-Cvrlje M, Eizirik DL
Department of Medical Cell Biology, Uppsala University, Sweden.
Cytokine (United States) Jul 1994, 6 (4) p399-406

Cytokines may be important mediators of beta-cell damage in early insulin-dependent diabetes mellitus. In order to further characterize the mechanism(s) of action of cytokines on insulin-producing cells, mouse pancreatic islets were exposed for 48 h to IL-1 beta, IFN-gamma or TNF-alpha, alone or in combinations. The three cytokines induced islet nitric oxide (NO) production, an effect most marked when islets were exposed to the three cytokines together. In parallel with NO production, IL-1 beta+IFN-gamma+TNF-alpha impaired islet function, as judged by decreased islet DNA and insulin content, decreased glucose metabolism and decreased glucose-induced insulin release. Aminoguanidine, an inhibitor of NO production, prevented all the above described suppressive effects of the cytokines, with exception of depletion in islet insulin content. In parallel experiments, insulin-producing RIN cells were exposed for 6 h to the same cytokines. Both IL-1 beta and TNF-alpha, but not IFN-gamma, induced NO production and expression of the mRNA encoding for the inducible form of the enzyme NO synthase (iNOS). These effects were most pronounced when combinations of IL-1 beta+IFN-gamma or IL-1 beta+IFN-gamma+TNF-alpha were used. As a whole, the data suggest that combinations of cytokines induce higher amounts of NO generation by mouse pancreatic islets than each of the cytokines isolated. An important source of islet NO production are probably the beta-cells, as pointed by data obtained with an insulinoma cell line. Most of the deleterious effects of the cytokines of mouse islets are prevented by blocking NO production, suggesting that NO is the main mediator of cytokine-induced beta-cell damage.

Modification of low density lipoprotein by advanced glycation end products contributes to the dyslipidemia of diabetes and renal insufficiency.
Bucala R, Makita Z, Vega G, Grundy S, Koschinsky T, Cerami A, Vlassara H
Picower Institute for Medical Research, Manhasset, NY 11030.
Proc Natl Acad Sci U S A (United States) Sep 27 1994, 91 (20) p9441-5

Atherosclerosis develops rapidly in patients with diabetes or renal insufficiency. Plasma lipoprotein profiles are frequently abnormal in these conditions and reflect an elevation in the level of the apoprotein B (ApoB)-containing components very low density lipoprotein (VLDL) and low density lipoprotein (LDL). High levels of circulating advanced glycation end products (AGEs) also occur in diabetes and end-stage renal disease (ESRD). These products arise from glucose-derived Amadori products and include AGE-modified peptides (AGE-peptides) which result from the catabolism of AGE-modified tissue proteins. AGE-peptides have been shown to crosslink protein amino groups and to accumulate in plasma as a consequence of renal insufficiency. To address potential mechanisms for the dyslipidemia of diabetes and ESRD, we investigated the possibility that circulating AGEs react directly with plasma lipoproteins to prevent their recognition by tissue LDL receptors. AGE-specific ELISA showed a significantly increased level of AGE-modified LDL in the plasma of diabetic or ESRD patients compared with normal controls. AGE-LDL formed readily in vitro when native LDL was incubated with either synthetic AGE-peptides or AGE-peptides isolated directly from patient plasma. LDL which had been modified by AGE-peptides in vitro to the same level of modification as that present in the plasma of diabetics with renal insufficiency exhibited markedly impaired clearance kinetics when injected into transgenic mice expressing the human LDL receptor. These data indicate that AGE modification significantly impairs LDL-receptor-mediated clearance mechanisms and may contribute to elevated LDL levels in patients with diabetes or renal insufficiency. This hypothesis was further supported by the observation that the administration of the advanced glycation inhibitor aminoguanidine to diabetic patients decreased circulating LDL levels by 28%.

Chemistry of the fructosamine assay: D-glucosone is the product of oxidation of Amadori compounds.
Baker JR, Zyzak DV, Thorpe SR, Baynes JW
Department of Clinical Biochemistry, Green Lane Hospital, Auckland, New Zealand.
Clin Chem (United States) Oct 1994, 40 (10) p1950-5

The chemistry of the fructosamine assay was studied by using the Amadori compound, N alpha-formyl-N epsilon-fructose-lysine (fFL), an analog of glycated lysine residues in protein. Previously (Clin Chem 1993;39:2460-5), we reported that free lysine was formed from fFL at 70% yield during incubation with alkaline nitroblue tetrazolium (NBT) under the conditions routinely used for the fructosamine assay (sodium carbonate buffer, pH10. 35 at 37 degrees C). Here, we show that D-glucosone is the primary carbohydrate oxidation product formed from Amadori compounds in the fructosamine assay. Glucosone, which decomposes under alkaline assay conditions with a hallife of < 30 min, reaches a maximum concentration of approximately 50% of the initial fFL concentration after 10 min of incubation. Like fFL, glucosone reduces NBT to the purple monoformazan dye, but its decomposition is not accelerated by the presence of NBT. The dicarbonyl-trapping reagent, aminoguanidine, inhibits the fructosamine assay by approximately 25% when fFL is the substrate, but by nearly 100% with glucosone as substrate. Studies with serum samples from diabetics and nondiabetics indicate that glucosone formation does not have a significant effect on the clinical usefulness of the fructosamine assay; however, corrections for glucosone formation may be required when the assay is used for estimating the extent of glycation of proteins.

Creatine reduces collagen accumulation in the kidneys of diabetic db/db mice.
Lubec B, Aufricht C, Herkner K, Hoeger H, Adamiker D, Gialamas H, Fang-Kircher S, Lubec G
Department of Paediatrics, University of Vienna, Austria.
Nephron (Switzerland) 1994, 67 (2) p214-7

In the present study, we tested the hypothesis whether creatine, a metabolite of arginine metabolism, shares the pharmacological activities of arginine reducing collagen accumulation in the diabetic kidney. Ten db/db mice were given, for 3 months, a solution containing a daily dosage of creatine of 50 mg/kg body weight. Eleven db/db mice served as controls. At the end of the 3-month study period, the mean N-carboxymethyllysine concentration in the untreated group was significantly higher than in the treated group (0.163 +/- 0.18 versus 0.096 +/- 0.017 nmol/mumol hydroxyproline, p < 0.001). Collagen accumulation was also significantly higher in the untreated than in the treated group (2.21 +/- 0.24 versus 1.68 +/- 0.22 mumol hydroxyproline/100 mg kidney weight, p < 0.001). We conclude that creatine led to a significant reduction in collagen type IV accumulation resembling arginine or aminoguanidine action. We do suggest that the guanidino group common to both compounds is able to block reactive carbonyls.

Increase in 3-deoxyglucosone levels in diabetic rat plasma. Specific in vivo determination of intermediate in advanced Maillard reaction.
Yamada H, Miyata S, Igaki N, Yatabe H, Miyauchi Y, Ohara T, Sakai M, Shoda H, Oimomi M, Kasuga M
Second Department of Internal Medicine, Kobe University School of Medicine, Japan.
J Biol Chem (United States) Aug 12 1994, 269 (32) p20275-80

A specific assay of 3-deoxyglucosone (3-DG) was developed in our laboratory to help elucidate the relationship between advanced Maillard reaction and diabetic complications. 3-DG is known as a highly reactive intermediate of the reaction in vitro and a precursor of advanced glycosylation end products such as pyrraline and pentosidine, which have been previously detected in vivo. 3-DG was converted to a stable compound, 2-(2,3,4-trihydroxybutyl)-benzo[g]quinoxaline, by reacting with 2,3-diaminonaphthalene. Since the derivative had a characteristic UV spectrum, it was determined at 268 nm by high performance liquid chromatography. This method was sensitive enough to detect 10 ng/ml (61.7nM) of 3-DG in vitro. A slight modification to this method allowed in vivodetection of small amounts of 3-DG. Plasma free 3-DG levels were significantly higher in streptozotocin-induced diabetic rats compared with controls (918 +/- 134 nM versus 379 +/- 69 nM, p < 0.001) and were suppressed with the administration of aminoguanidine, an inhibitor of Maillard reaction. Plasma pyrraline levels in diabetic rats also increased in parallel with elevated 3-DG levels but were only marginally suppressed by administration of aminoguanidine. Our results indicate that 3-DG ispresent in vivo under normal conditions and that its level increases indiabetic subjects. Determination of 3-DG represents a good tool to predict development and progression of diabetic complications and to assess the efficiency of inhibitors to Maillard reaction.

L-arginine reduces heart collagen accumulation in the diabetic db/db mouse.
Khaidar A, Marx M, Lubec B, Lubec G
Department of Paediatrics, University of Vienna, Austria.
Circulation (United States) Jul 1994, 90 (1) p479-83

BACKGROUND: Diabetic cardiomyopathy presents with significant collagen accumulation; decreased solubility, increased glucose-mediated abnormal cross-linking, free radical cross-linking, or glucose-induced increased transcription of collagen is incriminated. In a previous study, we reducedcollagen accumulation in the kidneys of diabetic mice by treatment with oral arginine. This observation led us to examine the effect of arginine oncardial fibrosis.

METHODS AND RESULTS: Twenty-nine db/db spontaneouslydiabetic mice were used in the experiments. Sixteen were given L-arginine(free base, in tap water, 50 mg/kg body wt per day) for 4 months. At theend of the experiment, we determined total collagen content of total ventricular tissue, acid solubility, carboxymethyllysine, O-tyrosine, glutathione, blood glucose, and fructosamine as parameters for glycemic control. Heart collagen level was significantly (P = .0001) reduced in the experimental group (mean, 0.24 +/- 0.05) compared with the control group (mean, 0.49 +/- 0.10 mumol hydroxyproline per 100 mg heart tissue). Significantly more collagen could be eluted from heart samples of the experimental group (P = .02). Carboxymethyllysine and O-tyrosine did not differ when related to heart weight. Glutathione level was significantly higher in the untreated group (P = .003). Parameters of glycemic control did not differ between the groups.

CONCLUSIONS: Our findings clearly indicate that L-arginine reduced total heart collagen and increased acid solubility of heart collagen. Both findings are compatible with the cross-linking hypothesis. The data for carboxymethyllysine, O-tyrosine, and glutathione would rule out the glycoxidation hypothesis and, therefore, free radical cross-linking. The postulated mechanism of action is most likely the blocking of reactive carbonyl functions by L-arginine in analogyto amino guanidine activity. Correlations of collagen with glycemic control, however, point to an association of glucose with collagen metabolism, a phenomenon documented in cell cultures at the transcriptional level.

Reactive glycosylation endproducts in diabetic uraemia and treatment of renal failure.
Makita Z, Bucala R, Rayfield EJ, Friedman EA, Kaufman AM, Korbet SM, Barth RH, Winston JA, Fuh H, Manogue KR, et al
Picower Institute for Medical Research, Manhasset, NY 11030.
Lancet (England) Jun 18 1994, 343 (8912) p1519-22

In diabetes and ageing, glucose-derived advanced glycosylationend products (AGEs) cross-link proteins and cause vascular tissue damage. Elimination of circulating low-molecular weight AGE-modified molecules (LMW-AGEs) by the kidney is impaired in diabetic patients with end-stage renal disease, a group subject to accelerated atherosclerosis. We determined the effectiveness of current renal replacement treatments on elimination of serum LMW-AGEs in diabetic and non-diabetic patients with end-stage renal disease. Although diabetic patients receiving high-flux haemodialysis achieved 33% lower steady-state serum LMW-AGE than did those in conventional haemodialysis (p < 0.005), LMW-AGE concentrations remained 3.5-6 fold above normal, whether high-flux dialysis, conventional haemodialysis, or chronic ambulatory peritoneal dialysis were used. High-flux haemodialysis markedly reduced AGE during each treatment session (47.9% in the diabetic, p < 0.001 and 60.6% in the non-diabetic group, p <0.001) but concentrations returned to pre-treatment range within 3 hours. In contrast, normal LMW-AGE concentrations were maintained in patients with functioning renal transplants. We found that LMW-AGEs with an apparent molecular weight of 2000-6000 circulate and retain strong inherent chemical reactivity--when exposed to collagen in vitro, up to 77% attached covalently to form AGE-collagen, and the AGE-crosslink inhibitor aminoguanidine completely inhibited this reaction. The results suggest that LMW-AGEs comprise a set of chemically-reactive molecules that are refractory to removal by current dialysis treatments. Through covalent reattachment onto vascular matrix or serum components, LMW-AGEs may exacerbate vascular pathology associated with end-stage renal disease.

Cytokines suppress human islet function irrespective of their effects on nitric oxide generation.
Eizirik DL, Sandler S, Welsh N, Cetkovic-Cvrlje M, Nieman A, Geller DA, Pipeleers DG, Bendtzen K, Hellerstrom C
Department of Medical Cell Biology, Uppsala University, Sweden.
J Clin Invest (United States) May 1994, 93 (5) p1968-74

Cytokines have been proposed as inducers of beta-cell damage in human insulin-dependent diabetes mellitus via the generation of nitric oxide (NO). This concept is mostly based on data obtained in rodent pancreatic islets using heterologous cytokine preparations. The present study examined whether exposure of human pancreatic islets to different cytokines induces NO and impairs beta-cell function. Islets from 30 human pancreata were exposed for 6-144 h to the following human recombinant cytokines, alone or in combination: IFN-gamma (1,000 U/ml), TNF-alpha (1,000 U/ml), IL-6 (25U/ml), and IL-1 beta (50 U/ml). After 48 h, none of the cytokines alone increased islet nitrite production, but IFN-gamma induced a 20% decrease in glucose-induced insulin release. Combinations of cytokines, notably IL-1beta plus IFN-gamma plus TNF-alpha, induced increased expression of inducible NO synthase mRNA after 6 h and resulted in a fivefold increase in medium nitrite accumulation after 48 h. These cytokines did not impair glucose metabolism or insulin release in response to 16.7 mM glucose, but there was an 80% decrease in islet insulin content. An exposure of 144 h toIL-1 beta plus IFN-gamma plus TNF-alpha increased NO production and decreased both glucose-induced insulin release and insulin content. Inhibitors of NO generation, aminoguanidine or NG-nitro-L-arginine, blocked this cytokine-induced NO generation, but did not prevent the suppressive effect of IL-1 beta plus IFN-gamma plus TNF-alpha on insulin release andcontent. In conclusion, isolated human islets are more resistant to thesuppressive effects of cytokines and NO than isolated rodent islets. Moreover, the present study suggests that NO is not the major mediator of cytokine effects on human islets.

Amelioration of dermal lesions in streptozotocin-induced diabetic rats by aminoguanidine.
Bannai C, Yamazaki M, Matsushima Y, Kunika K, Itakura M, Okuda Y, Yamashita K
Department of Endocrinology and Metabolism, University of Tsukuba, Ibaraki, Japan
Diabetes Res (Scotland) 1992, 20 (4) p87-95

As aminoguanidine (AG) is known to prevent non-enzymatic glycosylation in various tissues, we have histologically and biochemically evaluated AG effects on the skin in control, SZ-diabetic and AG-treated (25 mg/kgbw/day, 10w) diabetic rats. HbA1c and plasma glucose levels in diabetic andAG-treated diabetic rats were maintained about two times higher than those in control rats during the 10 weeks of the experiment. Histological findings revealed that the dermis in diabetic rats was thin and edematous, associated with swelling and degeneration of collagen fibers. Necrobiotic changes were seen in the lower dermis. These changes were greatly improved in AG-treated diabetic rats. Skin glucose contents in diabetic andAG-treated diabetic rats were about 10 times higher than those in the controls, whereas there was no difference in the sorbitol contents between three groups. Dry weight of the skin and collagen content was wellcorrelated (r = 0.9044) and collagen represented 78.0 +/- 2.3% of the dryweight. By SDS-PAGE analysis of cyanogen bromide digests it was shown that high molecular weight peptides were increased in diabetic rats, but were decreased in AG-treated diabetic rats. The mean of glycosaminoglycan (GAG) contents of diabetic skin was 54% of that in the controls (1.58 +/- 0.09vs. 2.94 +/- 0.39 micrograms/mg dry weight, P < 0.0025), which increased significantly in AG-treated diabetic rats (1.75 +/- 0.07 microgram/mg dryweight, P < 0.01 vs. diabetic).

Glycation, glycoxidation, and cross-linking of collagen by glucose. Kinetics, mechanisms, and inhibition of late stages of the Maillard reaction.
Fu MX, Wells-Knecht KJ, Blackledge JA, Lyons TJ, Thorpe SR, Baynes JW
Department of Chemistry and Biochemistry, University of South Carolina, Columbia SC 29208
Diabetes (United States) May 1994, 43 (5) p676-83

The Maillard or browning reaction between sugar and protein contributes to the increased chemical modification and cross-linking of long-lived tissue proteins in diabetes. To evaluate the role of glycation and oxidation in these reactions, we have studied the effects of oxidative and antioxidative conditions and various types of inhibitors on the reaction of glucose with rat tail tendon collagen in phosphate buffer at physiological pH and temperature. The chemical modifications of collagen that were measured included fructoselysine, the glycoxidation products Nepsilon-(carboxymethyl) lysine and pentosidine and fluorescence. Collagen cross-linking was evaluated by analysis of cyanogen bromide peptides using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by changes in collagen solubilization on treatment with pepsin or sodium dodecylsulfate. Although glycation was unaffected, formation of glycoxidation products and cross-linking of collagen were inhibited by antioxidative conditions. The kinetics of formation of glycoxidation products proceeded with a short lag phase and were independent of the amount of Amadori adduct on the protein, suggesting that autoxidative degradation of glucose was a major contributor to glycoxidation and cross-linking reactions. Chelators, sulfhydryl compounds, antioxidants, and aminoguanidine also inhibited formation of glycoxidation products, generation of fluorescence, and cross-linking of collagen without significant effect on the extent of glycation of the protein. We conclude that autoxidation of glucose or Amadori compounds on protein plays a major role in the formation of glycoxidation products and cross-liking of collagen by glucose in vitro and that chelators, sulfhydryl compounds, antioxidants, and aminoguanidine act as uncouplers of glycation from subsequent glycoxidation and cross-linking reactions.

Aminoguanidine inhibits the development of accelerated diabetic retinopathy in the spontaneous hypertensive rat.
Hammes HP, Brownlee M, Edelstein D, Saleck M, Martin S, Federlin K
Third Medical Department, of Justus-Liebig-University of Giessen, Germany.
Diabetologia (Germany) Jan 1994, 37 (1) p32-5

Arterial hypertension has been identified as a major secondary risk factor for diabetic retinopathy. However, the mechanisms by which hypertension worsens retinopathy are unknown. Inhibition of advanced glycation product formation prevents the development of experimental diabetic retinopathy in normotensive diabetic rats. In this study the effect of hypertension on the rate of diabetic retinopathy development and the formation of arteriolar thrombosis was evaluated. We also evaluated the effect of aminoguanidine, an inhibitor of advanced glycation and product formation on retinal pathology of diabetic hypertensive rats. After 26 weeks of diabetes, hypertension accelerated the development of retinopathy despite a lower mean blood glucose level than in the non-hypertensive group (diabetic spontaneous hypertensive rats (SHR) 16.00 +/- 6.83 mmol/l; diabetic normotensive Wistar Kyoto rats (WKY) 34.9 +/- 3.64 mmol/l; p <0.0001). Diabetic SHR had nearly twice as many acellular capillaries as diabetic WKY (SHR diabetic: 91.9 +/- 7.5 acellular capillaries per mm2 of retinal area vs WKY diabetic: 53.7 +/- 8.5 acellular capillaries per mm2 of retinal area), and a 3.8-fold increase in the number of arteriolar microthromboses (SHR diabetic 23,504 +/- 5523 microns2 vs SHR non-diabetic 6228 +/- 2707 microns2). Aminoguanidine treatment of SHR diabetic rats reduced the number of acellular capillaries by 50%, and completely prevented both arteriolar deposition of PAS-positive material and abnormal microthrombus formation. These data suggest that hypertension-induced deposition of glycated proteins in the retinal vasculature plays a central role in the acceleration of diabetic retinopathy by hypertension.

Aminoguanidine reduces regional albumin clearance but not urinary albumin excretion in streptozotocin-diabetic rats.
Huijberts MS, Wolffenbuttel BH, Crijns FR, Nieuwenhuijzen Kruseman AC, Bemelmans MH, Struijker Boudier HA
Department of Internal Medicine, University Hospital Maastricht, The Netherlands.
Diabetologia (Germany) Jan 1994, 37 (1) p10-4

Advanced glycation end-product-formation is thought to play a role in the development of diabetic angiopathy. By altering the structure of different extracellular matrix components advanced glycation end-products might affect vascular and glomerular permeability. In this study we investigated the effect of treatment with an inhibitor of advanced glycation end-product-formation, aminoguanidine, on vascular permeability and the development of albuminuria in streptozotocin-induced diabetic rats. Male Wistar Rp rats were randomized into a control group, a diabetic group, and an aminoguanidine-treated diabetic group. After 8 weeks, 24-h urine collections were taken and rats were implanted with an arterial and avenous catheter. mean arterial blood pressure was determined by intra-arterial measurement. Regional albumin clearances were assessed in the eye, ileum, lung, skeletal muscle and skin using an isotope technique. Mean arterial pressure in the diabetic group was significantly lower in the control and aminoguanidine-treated groups (p < 0.02). Regional albumin clearances were significantly increased in all tissues of diabetic rats compared to control rats (p < 0.05). Aminoguanidine treatment of diabetic rats resulted in a significant decrease of regional albumin clearance in all tissues except the lung (p < 0.05, lung p = 0.07). The development of albuminuria in diabetic rats however, was not affected by aminoguanidine.

Aminoguanidine, an inhibitor of nitric oxide formation, fails to protect against insulitis and hyperglycemia induced by multiple low dose streptozotocin injections in mice.
Holstad M, Sandler S
Department of Medical Cell Biology, Uppsala University, Sweden.
Autoimmunity (Switzerland) 1993, 15 (4) p311-4

It has been suggested that pancreatic beta-cell destruction occurring during the process leading to insulin-dependent diabetes mellitus (IDDM) involves formation of nitric oxide (NO). We have presently studied the effect of aminoguanidine (AG), which has recently been reported to inhibit generation of NO induced by the cytokine interleukin-1 beta. AG currently counteracted IL-1 beta induced impairment of the glucose oxidation rate in rat pancreatic islets. Then we studied the effect of AG on the development of hyperglycemia and pancreatic insulitis in mice treated with multiple low dose injections of streptozotocin (40 mg/kg body-weight for five consecutive days). It was found that one daily intraperitoneal injection of AG (50 mg/kg body-weight) for 14 days failed to prevent the development of diabetes as well as insulitis following the streptozotocin injections. Furthermore, the mice treated with streptozotocin plus AG showed an increased mortality compared to mice treated with streptozotocin plus saline. Although the present data do not exclude a role for NO in IDDM, it raises concerns about the use of testing AG as therapeutic agent in IDDM.

Aminoguanidine: a drug proposed for prophylaxis in diabetes inhibits catalase and generates hydrogen peroxide in vitro.
Ou P, Wolff SP
Department of Medicine, University College London Medical School, Rayne Institute, U.K
Biochem Pharmacol (England) Oct 5 1993, 46 (7) p1139-44

Aminoguanidine (AG) has been proposed as a drug of potential benefit in prophylaxis of the complications of diabetes. We show here that AG irreversibly inhibits catalase with an efficacy similar to aminotriazole. AG also produces hydrogen peroxide, in a transition metal-catalysed process which may be partially dependent upon prior hydrolysis of AG to semicarbazide and hydrazine. These observations may be of importance in proposals for the long term administration of AG in diabetes.

Nitric oxide production in islets from nonobese diabetic mice: aminoguanidine-sensitive and -resistant stages in the immunological diabetic process.
Corbett JA, Mikhael A, Shimizu J, Frederick K, Misko TP, McDaniel ML, Kanagawa O, Unanue ER
Department of Pathology, Washington University School of Medicine, St. Louis, MO 63110.
Proc Natl Acad Sci U S A (United States) Oct 1 1993, 90 (19) p8992-5

The role of nitric oxide (NO.) in the development of immunologically induced diabetes was examined. Transfer of spleen cells obtained from diabetic female nonobese diabetic (NOD) mice to nondiabetic irradiated males induced diabetes 11-13 days after transfer. Islets isolated from recipient male mice produced NO. in a time-dependent fashion. The production of nitrite was initially detected at day 6 after transfer, with increasing levels by days 9 and 13. Under similar conditions glucose-induced insulin secretion by isolated NOD mouse islets was irreversibly reduced by approximately 40% at days 6, 9, and 13 after transfer of spleen cells. The number of islets harvested per pancreas by the 9th and 13th day after transfer was decreased by 20-25% as compared to controls. Treatment of male NOD mice with aminoguanidine, an inhibitor of the inducible form of NO. synthase, reduced the production of NO. in islets and delayed the development of diabetes by 3-8 days. The temporary inhibition by aminoguanidine was dependent on both inhibitor concentration and number of spleen cells transferred. These results indicate that NO. is produced in NOD islets as a result of an immunological diabetogenic process and suggests a role of this compound in the immunological diabetic process.

Inhibition of matrix-induced bone differentiation by advanced glycation end-products in rats.
Fong Y, Edelstein D, Wang EA, Brownlee M
Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York.
Diabetologia (Germany) Sep 1993, 36 (9) p802-7

Glycation of long-lived proteins is an inevitable consequence of aging that is accelerated in patients with diabetes mellitus. Treatment of demineralized bone matrix particles from 35-week-old normal Long-Evans rats with glycoaldehyde, a precursor of advanced glycation end-products, was used to assess the effects of bone-matrix glycation on the process of bone differentiation. Matrix was incubated in phosphate buffered saline alone, phosphate buffered saline containing glycolaldehyde, glycolaldehyde plus the advanced glycation product-inhibitor aminoguanidine, or glycolaldehyde plus the advanced glycation product-inhibitor sodium cyanoborohydride. Glycolaldehyde increased the matrix advanced glycation product content as measured by specific fluorescence more than two-fold, while inhibiting bone differentiation more than 90% as measured by in vivo 45CaCl2 uptake, alkaline phosphatase levels, and histology. In contrast, simultaneous incubation with the advanced glycation product-inhibitor aminoguanidine or sodium cyanoborohydride not only reduced fluorescence to normal, but also restored bone differentiation. Furthermore, the inhibition of bone differentiation by glycolaldehyde was not reversed by subsequent application of recombinant bone morphogenetic protein-2. These observations suggest that formation of advanced glycation products on bone matrix alters its ability to induce bone formation, and probably involves alterations of binding sites for extractable proteins with direct bone inductive properties such as bone morphogenetic protein-2. Decreased bone formation associated with aging and diabetes may result, in part, from advancedglycation product formation on matrix proteins.

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