Identification of amadori-modified plasma proteins in type 2 diabetes and the effect of short-term intensive insulin treatment

OBJECTIVE - Growing evidence supports that nonenzymatic glycation products may cause hyperglycemia-induced diabetes complications. Amadori-modified proteins are the intermediate products of nonenzymatic glycation and constitute the forms of glycated proteins in diabetes. The objective of the current study was to utilize two-dimensional gel electrophoresis, Western blot, and mass spectrometry to identify Amadori-modified plasma proteins in type 2 diabetic patients with poor glycemic control and assess the impact of short-term insulin treatment on the glycation of these proteins. RESEARCH DESIGN AND METHODS - We compared eight type 2 diabetic subjects (aged 56 +/- 3 years and BMI 29.7 +/- 0.9 kg/m(2)) with an average diabetes duration of 8.5 years (range 3-19) with equal numbers of weight-matched (aged 56 +/- 2 years and BMI 30.1 +/- 10.0 kg/m(2)) and lean (aged 58 2 years and BMI 25 +/- 00.5 kg/m(2)) nondiabetic subjects who have no first-degree relatives with diabetes. Two separate blood samples were collected from the type 2 diabetic subjects, one following 2 weeks of withdrawal of all antidiabetic medications (T2D-; plasma glucose 12.6 +/- 1.0 mmol/l) and another following 10 days of intensive insulin treatment (T2D+; plasma glucose 5.5 +/- 0.2 mmol/l). Plasma proteins were separated using single and two-dimensional gel electrophoresis. Western blot analysis was performed, and several proteins, which reacted with the Amadori-antibody (1-deoxyfructosyl lysine), were identified by tandem mass spectrometry. RESULTS - No significant difference's in the glycation of proteins between the obese and lean groups were noted, but type 2 diabetic patients had several proteins with higher glycation than the control groups. We identified 12 plasma proteins with reduced reaction to the anti-Amadori antibody upon intensive insulin treatment. A significant (P < 0.03) difference in Amadori modification was observed between the T2D- and control subjects for all these proteins except the Ig light chain. Insulin treatment reduced Amadori modification of albumin (23.2%, P < 0.02), fibrin (34.6%, P < 0.001), Ig heavy chain constant region (20.7%, P < 0.05), transferrin (25.4%, P < 0.04), and Ig light chain (13%, P < 0.02). In addition, western blot analysis of two-dimensional gel electrophoresis identified alpha-fibrinogen precursor, beta-fibrinogen precursor, fibrinogen gamma-B chain precursor, hemopexin, vitamin D binding protein, and serine protease inhibitor as proteins with a reduced reaction to anti-Amadori antibody upon intensive insulin treatment. CONCLUSIONS - The current approach offers the opportunity to identify Amadori modification of many proteins that may cause functional alterations and offers the potential for monitoring short-term glycerine control in diabetic patients.