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 end products (AGE) was first developed to qualitatively detect and quantify the AGE formed in rat tendon and aortic collagen. Individual collagen samples were extracted by extensive pepsin and collagenase digestion. The amount of AGE was measured by competitive ELISA and results were expressed as AGE U/mg collagen. Diabetic rats showed a significant increase in AGE content in aortic collagen at 20 weeks (n=6, 206.6 ± 16.7 U/mg collagen) compared with that measured at 4 and 12 weeks (n=6, 110 ± 12.8 U/mg collagen, and n=13, 184.9 ± 12.3 U/mg collagen at 4 and 12 weeks, respectively; p < 0.001 between 20 weeks and 4 weeks; p < 0.01 between 20 weeks and 12 weeks). The amount of AGE in tendon collagen of diabetic rats increased from 1.9 ± 0.38 U/mg at 4 weeks to 11.2 ± 6.1 U/mg collagen at 20 weeks, 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 to 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, 184.9 ± 12.3 U/mg) and diabetic rats treated for 12 weeks with insulin (DI; n=6, 133.9 ± 10.7 U/mg), or phlorizin (DP; n=6, 132.4 ± 8.9 U/mg), or by a combination of insulin/phlorizin (DIP; n=6, 124.3 ± 6.5 U/mg). 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, 104.6 ± 14.9 U/mg) of the same age (D, DI, DP, DIP vs. C, p < 0.001). Comparison between the mean levels of glycated hemoglobin (D: 5.62 ± 0.38 % vs.C: 1.7 ± 0.05 %) and mean AGE levels in the studied group of animals yielded a very good exponential correlation (r = 0.89, p < 0.001). Glycation-derived late-stage collagen modification was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and by immunoblot