Temporal association between lens protein glycation and cataract development in diabetic rats

In an attempt to shed more light on the relation between the glycation process and structural protein alterations, we followed the formation of glycated products in the lenses of hyperglycaemic Wistar rats during a period of 5 months following alloxan diabetes inducement. The study groups included non-diabetic (control), untreated diabetic rats (D), and diabetic rats receiving insulin alone or in combination with phlorizin, an inhibitor of renal tubular glucose transport. Lenses were removed at 4 and 20 weeks, and advanced glycation products in alkalisoluble lens proteins were determined by their characteristic spectrofluorescence (emission at 385 nm with excitation of 335 nm). In 20-week untreated diabetic rats as compared to control rats, a significant increase was observed in the fluorescence level (3.25 +/- 1.02 vs 1.61 +/- 0.17 FU/mg, p < 0.001), while in 4-week animals the increase was from 1.26 +/- 0.11 FU/mg in controls to 1.80 +/- 0.25 FU/mg in diabetics (P < 0.001). Daily treatment of 20-week diabetic rats with insulin alone (2.46 +/- 0.48 FU/mg) or in combination with phlorizin (2.30 +/- 0.26 FU/mg) did not significantly influence lens fluorescence level. The amount of glucosebound ketoamine linkage was estimated after acid hydrolysis as released 5-hydroxymethylfurfural (HMF). In 20-week controls, it was slightly higher than in 4-week controls (0.57 +/- 0.31 vs 0.41 +/- 0.20 nmol HMF/mg, respectively). The diabetic group showed a significant increase, however. In 4-week diabetics, a level of 1.07 +/- 0.36 nmol HMF/mg was found, while in 20-week animals the glycated protein amount rose to 2.46 +/- 0.79 nmol HMF/mg. In addition to the increases in glycated content with continuing diabetic hyperglycaemia, significant changes in protein composition of alkali-soluble lenses developed. The SDS-PAGE pattern showed an appearance of protein polymers of heterogeneous size (C 4 weeks 3.0 +/- 1.1% vs C 20 weeks 17.9 +/- 2.9%, D 4 weeks 7.3 +/- 2.1% vs D 20 weeks 19.8 +/- 3.6%) and the proteins of high molecular weight (HMW) failed to penetrate into the gel. Only a small amount of these HMW proteins was present in controls (C 20 weeks 2.5 +/- 1.2%) and short-term diabetes (D 4 weeks 0.8 +/- 0.2%), whereas in long-term untreated diabetes there was a dramatic increase (D 20 weeks 30.5 +/- 3.2%) with a corresponding decrease in other peaks. All diabetic animals from this group had macroscopically detectable cataractous lenses. Treatment with insulin or insulin/phlorizin followed the HMW protein level of the untreated animals (28.2 +/- 4.0% or 27.08 +/- 3.3% vs 30.52 +/- 3.32%).