Abstract
The purpose of the present study was to investigate the synthesis of very low density lipoprotein in primary monolayer culture of hepatocytes prepared from streptozotocin-induced diabetic rats. Adult rat hepatocytes were cultured in serum free culture medium, HI/WO5/BA2000 (International Scientific Industries Inc., Cary, Ill.). Liver cells were isolated using collagenase according to the modified method of Berry and Friend. Approximately 2.5-3.0×106 isolated liver cells in a final volume of 2.5ml of culture media supplemented with Gentamicin were inoculated into plastic culture dishes (diameter=60mm)(Falcon Plastics, Oxnard, Ca.) which had been coated with calf skin collagen (type III) and fibronectin (Collaborative Res.). The culture dishes were placed in a humidified incubator under 5% CO2/95% air at 37°C. After 4 hours of culture, the inoculated cells adhered to the bottom of the culture dishes and the culture medium was changed to remove cell debris and loosely attached cells. Conditioned medium was concentrated by Diaflo® ultrafiltration membrane YM 10 approximately 10 times and concentrated medium was ultracentrifuged sequentially at the densities of 1.006, 1.063 and 1.210g/ml. Lipoprotein fractions were dialyzed against 0.15 M NaCl with 3 mM EDTA, pH 7.4. Cholesterol (Ch), triglyceride (TG), phospholipid (PL) and protein in VLDL, LDL and HDL were measured. Time dependent increase of concentration of VLDL-TG in the culture medium was observed. VLDL-TG in the culture medium after 12 and 24 hours incubation were 8.2±1.2 and 23.1±1.9μg/mg cell protein, respectively. Electron-microscopic features of VLDL particles in the culture medium were comparable to those of plasma VLDL. Synthesis of VLDL in the cultured hepatocytes prepared from streptozotocin-induced diabetic rats was approximately 1/10 of control rats (0.6 vs 8.2μg/mg cell protein at 12 hours incubation). These observations suggest that synthesis of VLDL in the liver does not play a significant role in increased concentrations of VLDL observed in the insulin-deficient diabetic rats.
