Isolated liver parenchymal cells were prepared from adult rat liver by perfusing the livers according to the method of Berry M. N. and Friend D. S. with some modifications. The hepatocytes were cultured in the complete symthetic serum free culture media HI/WO5/BA2000 under 5% CO2/95% air at 37°C. The optimum conditions for cell attachment to Falcon plastic dishes (diameter: 60mm) were determined. When approximately (1.5-3.5)×106 cells per dish suspended in HI/WO5/BA2000 were incubated at 37°C for 4 hours, about 42% of cells inoculated were attached. The experiments were started after the first exchange of the culture media 4 hours after cell inoculation. During first 24 hours after cell inoculation, light microscopic study and the following metabolic function of the cells were investigated:
1. Total protein synthesis
2. Albumin synthesis
3. Hepatic triglyceride lipase (H-TGL) synthesis and its activities
4. Effect of insulin on amounts of H-TGL and its activities
The results of metabolic and morphological studies showed that integrity of cultured hepatocytes was well maintained at least for 24 hours after inoculation. A constant rate of total protein synthesis and albumin synthesis were observed up to 12 hours and then these rates were decreased. A constant rate of H-TGL synthesis which was determined by [14C] leucine incorporation to H-TGL precipitated by anti-H-TGL rabbit serum, was observed up to 12-24 hours. H-TGL activities were studied in adult rat hepatocytes cultured for 12 hours. A constant increase of H-TGL activity for 4 hours incubation and the plateau of the activity from 4-10 hours were observed and then the activity was decreased. The effect of insulin on H-TGL synthesis was studied in cultured hepatocytes prepared from adult normal and streptozotocin induced diabetic rats which were prepared by intravenous injection of streptozotocin (STZ), 65mg/Kg body weight according to the method of Junod A. et al. Addition of insulin into the culture media increased HTGL synthesis significantly in cultured hepatocytes prepared from STZ diabetic rats. A significant positive correlation was observed between [14C] leucine incorporation to H-TGL and log10 insulin concentration from 0 to 10μM. However, no effect of insulin on H-TGL synthesis in the concentration up to 10μM was seen in cultured hepatocytes prepared from normal rats. The results presented indicate that H-TGL is regulated partly by insulin for optimal expression and that H-TGL might be a key enzyme for pathogenic features of hyperlipoproteinemia observed in diabetic rats.
