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 (T(2)D-; plasma glucose 12.6 +/- 1.0 mmol/l) and another following 10 days of intensive insulin treatment (T(2)D+; 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 differences 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 T(2)D- 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 glycemic control in diabetic patients.