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.
Animal ; Cell Membrane/enzymology ; Cell Membrane/metabolism ; Liver Neoplasms, Experimental/metabolism* ; Male ; Methyldimethylaminoazobenzene* ; Microsomes, Liver/enzymology ; Microsomes, Liver/metabolism ; Monosaccharide Transport Proteins/isolation & purification ; Monosaccharide Transport Proteins/metabolism* ; Rats ; Rats, Inbred Strains ; p-Dimethylaminoazobenzene*/analogs & derivatives

OBJECTIVETo explore the effect of puerarin injection on the amount of GLUT4 protein at the plasma membrane in insulin-resistant rat skeletal muscle.

METHODThe rat model of insulin resistance (IR) was made by being fed with high-fat diet. The animals were divided into three groups (ten in each group): group I: controls; group II: Insulin-resistant rats; group III: Insulin-resistant rats + Puerarin treatment. Insulin-resistant rats were injected with 100 mg puerarin injection per kg body weight through abdominal cavity once a day for 4 weeks. Fasting blood glucose and fasting serum insulin levels were measured before and after Puerarin treatment, respectively. Insulin treatment was achieved by intraperitoneal injection of insulin (1 unit insulin per kg body weight.) 15 minute before killing the animals. The right hindlimb skeletal muscle was rapidly dissected. Then the expression of GLUT4 protein at the plasma membrane in all the animals was assessed with Western blot method.

RESULTThe GLUT4 content at the plasma membrane in insulin-resistant rats skeletal muscle was significantly lower (about 31%) than that of controls (P < 0.01). Puerarin Injection partly corrected fasting blood glucose (from 6.17 +/- 0.67 mmol x L(-1) to 5.54 +/- 0.35 mmol x L(-1)) and fasting serum insulin levels (from 17.09 +/- 2.02 mU x L(-1) to 11.86 +/- 1.35 mU x L(-1)) and increased the GLUT4 content at the plasma membrane by 1.18-fold in insulin-resistant rats skeletal muscle.

CONCLUSIONPuerarin Injection can ameliorate IR, and the mechanism may be involved in increasing cell-surface level of GLUT4 through decreasing fasting blood glucose and fasting serum insulin levels, improving GLUT4 trafficking and intracellular insulin signaling.

Animals ; Cell Membrane ; metabolism ; Glucose Transporter Type 4 ; Injections ; Insulin Resistance ; Isoflavones ; administration & dosage ; isolation & purification ; pharmacology ; Male ; Monosaccharide Transport Proteins ; metabolism ; Muscle Proteins ; metabolism ; Muscle, Skeletal ; metabolism ; pathology ; Plants, Medicinal ; chemistry ; Pueraria ; chemistry ; Rats ; Rats, Sprague-Dawley