engleski

Rat tissue collagen modified by advanced glycation : correlation with duration of diabetes and glycemic control

Collagenous proteins are especially prone to nonenzymatic glycation, because they contain several dibasic amino acid residues with free amino groups, have a very slow turnover rate, and are exposed to ambient levels of glucose. The aim of this study was to determine the time-dependent course of advanced glycation process in diabetic rats in relation to glycemic control and duration of diabetes, compared to age-matched controls. Immunochemical assay with antibodies to advanced glycation endproducts (AGE) was first developed to qualitatively detect and quantify the AGEs formed in rat tendon and aortic collagen. Individual collagen samples were extracted by extensive pepsin and collagenase digestion. The amount of AGEs was measured by competitive ELISA and results were expressed as AGE U/mg collagen. Diabetic rats showed a significant increase in AGEs content on aortic collagen at 20 weeks (n=6, 206.6ą16.7 U/mg collagen) compared with that measured at 4 and 12 weeks (n=6, 110ą12.8 U/mg collagen, and n=13, 184.9ą12.3 U/mg collagen at 4 and 12 weeks, respectively)(p<0.001 between 20 wk and 4 wk ; p<0.01 between 20 wk and 12 wk). The amount of AGEs on tendon collagen of diabetic rats increased from 1.9ą0.38 at 4 wk to 11.2ą6.1 U/mg collagen at 20 wk, p<0.001. Considerable disparity was observed in the intensity of glycation between aortic and tendon collagen. AGE-content per mg of aortic collagen was several-fold that found in tendon collagen (p<0.001). To investigate the effect of glycemic control on the advanced glycation process, total aortic AGE-collagen content was compared between untreated diabetic rats ((D) n=13, 184.9ą12.3) and diabetic rats treated for 12 weeks with insulin ((DI) n=6, 133.9ą10.7), or phlorizin ((DP) n=6, 132.4ą8.9), or by a combination of insulin/phlorizin ((DIP) n=6, 124.3ą6.5). In spite of therapy used, all groups of diabetic animals had a significantly higher aortic AGE-collagen content than those in the nondiabetic control group (C: n=8, 104.6ą14.9) of the same age (D, DI, DP, DIP vs C, p<0.001). Comparison between the mean levels of glycated hemoglobin (D:5.62ą0.38% vs C:1.7ą0.05%) and mean AGE levels in the study group of animals yielded a very good exponential correlation (r=0.89, p<0.001). Glycation-derived late-stage collagen modification was detected by SDS-PAGE, and by immunoblotting confirmed to contain an AGE-structure(s). Our study provided strong immunochemical evidence of AGE formation in vivo during hyperglycemia, and of their temporal association with structural alterations of extracellular matrix proteins. The advanced glycation process is retarded and reduced in intensity but not completely abolished by glycemia regulation with or independently of insulin.

diabetes; advanced glycation end products; aortic collagen; tendon collagen; ELISA

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