A study on the regulation of translocation of glucose transporters during hepatocarcinogenesis induced by 3'-Me DAB

The mechanism of glucose transported (GT) expression on the plasma membranes of hepatoma cells in rats induced by 3'-methyl-4-dimethylaminoazobenzene (3'-MeDAB) was studied. Cytochalasin B binding to plasma membrane fractions from control and 3'-MeDAB group in the absence of cold cytochalasin B showed 9,825 +/- 925 and 30,165 +/- 625 dpm/mg membrane protein. Scatchard plot analysis showed that the GTs present on the plasma membrane fractions in control and 3'-Me DAB groups were 5.0 and 16.0 pmol/mg membrane protein and their Kd values were 151 and 157 nM, respectively. These results suggest that the numbers of GTs in plasma membrane were increased in the 3'-Me DAB group compared to the control group. In contrast, the amounts of GTs in low density microsomal (LDM) fractions measured by a photoaffinity labeling technique using [3H]-cytochalasin B were 31,207 and 11,702 dpm/mg protein in the control and 3'-Me DAB group, respectively. These results suggest that GTs were translocated from LDM to plasma membranes during carcinogenesis. To confirm these results by an independent method 10% SDS-polyacrylamide gel electrophoresis was carried out. Gel slice No. 13 corresponding to MW of 45 kDa from plasma membrane fractions showed increased radioactivities in the 3'-Me DAB group compared to the control group. However, LDM fractions of the 3'-Me DAB group showed decreased radioactivities compared to the control group. Western blot analysis using anti-human RBC GT antibody present in the plasma membranes and LDM fractions from control and 3'-Me DAB groups did not show any significant difference, indicating low cross-reactivity between them. These results indicate that increased glucose transport seems to be more likely due to reciprocal redistribution of GTs between plasma membrane and LDM fractions.

A Study of the Regulation of the Glucose Transporter in the Plasma Membranes of Hepatoma Cells Induced by 3'-Me DAB

5'-nucelotidase and glucose-6-phosphatase are liver plasma and microsomal membranes markers and their respective activities were determined. In the liver homogenate, the activities of 5'-nucleotidase were 0.58 +/- 0.08 and 0.29 +/- 0.07 micromols/mg protein/10min in the control and 3'-methyl-4-dimethyl aminoazobenzene (3'-Me DAB) groups respectively. The enzyme activities m the partially purified plasma membranes were 2.15 +/- 0.25 and 1.31 +/- 0.23 micromols/mg protein/10min in the control and 3'-Me DAB groups respectively. The glucose-6-phosphatase activities in the homogenates of the control and 3'-Me DAB groups were 0.23 +/- 0.10, and 0.45 +/- 0.25 micromols/mg protein/10min, and in the microsomal fraction, 1.14 +/- 0.32, and 0.63 +/- 0.11 micromols/mg protein/10min, respectively, The concentrations of glucose carrier in the plasma membranes from the control and 3'-Me DAB group were 25, and 35 pmols/mg membrane protein, respectively, and the Ka values for cytochalsin B in each group were 5.20 X 109. and 5.14 X 109ml/mol, respectively. However in the microsomal fraction, no significant differences of glucose carrier were found between the control and 3'-Me DAB groups from the DEAE Sephadex A-50 ion exchange chromatography, fractions I and ll were obtained. Electrophoretic analysis of fraction I revealed a major protein band with a molecular weight of 45,000 and minor bands with MWs of 50,000, 55,000 and 15,000. Following AcA 34 gel filtration, a major protein band with a MW of 45,000 was obtained. From these results, it can be concluded that the glucose carrier protein was increased on plasma membrane of hepatoma induced by 3'-Me DAB, and the carrier protein showed similar molecular weight to other glucose carrier found in the RBC, muscle cells and adipocyte.