APPENDIX

APPENDIX I

(This abstract is published in the symposium of ' the Frontline of Biomembrane Study ---- 97' China Young Biological Researcher Symposium ' , Dec. of 1997, Beijing )

The Conformational Change of Glucose Transporter on Erythrocyte Membrane of Non-Insulin Dependent Diabetes Mellitus

Hu Xiaojian Zhang Zhihong Peng Feng Zhou Hanqing

Non-insulin dependent diabetes mellitus (NIDDM), caused by some diabetegenes and the environment factors such as diet and exercise, is characterized of impaired glucose-stimulated insulin secretion and impaired insulin-mediated glucose uptake. Comi R.J. & Hamilton H. reported that the intrinsic activity of erythrocyte glucose transporter (GluT1) reduced in diabetes running (Horm. Metab. Res. 1994, 26, p26). We want to know whether there is the functional abnormality in NIDDM patients� erythrocytes and what is the conformational change in GluT1.

With a photometic method of recording the red cell suspendation absorption at 660nm during the course of glucose transport, we investigated the transport character of NIDDM patients '  GluT1. The maximal velocity of 200mM glucose trans-zero entry was decreased compared to the normal controls at 35¡æ (30.24 +/- 5.29mmol/L/min, 19 patients compared to 45.1 +/- 0.97mmol/L/min, 9 controls; t-test, P<0.001). However, the rate of 60mM glucose cis-infinite exit exhibit no significantly difference (230.07 +/- 37.66 mmol/L/min, 23 patients compared to 233.96 +/- 25.2mmol/L/min, 10 controls).

Phloretin, an inhibitor of glucose transport binding on the outer domain, was used to study the abnormality of GluT1 in NIDDM patients. It was observed that the inhibitory constant was increased significantly after phloretin treatment in the glucose entry kinetics experiment. Considered of above results, we suspected that this functional abnormality would be related to GluT1 conformational change after ligands binding in NIDDM patients.

After the patients '  erythrocytes were made into stripped white ghost, fluorescence quenching experiment was performed. Glucose, cytochalasin B and phloretin are specifically bound to the glucose transporter, and can quench the fluorescence of tryptophan residues in GluT1. The abnormality of fluorescence quenches in erythrocyte membrane of patients was observed with a Hitachi M850 fluorescence spectrophotometer. The tendency of trpytophane moved from hydrophilic environment to hydrophobic environment was decreased in patients ' eythrocytes as binding with glucose, and the opposite tendency was observed as 2-15umol/L cytochalasin B and 0.5-4umol/L phloretin instead of glucose.

These results indicated that the decrease in glucose entry in the erythrocyte membranes of NIDDM patients was due to the GluT1 structure change ---- mostly the outer domain of the glucose transporter.