The Regulation of Lipogenesis by Cyclic Nucleotides in Intact Hepatocytes Prepared by a Simplified Technique

Abstract

Abstract An improved, simplified procedure is described for the isolation of liver cells from rat or chicken. Livers were perfused in situ and then incubated in a solution of collagenase and hyaluronidase. Greater than 50% of the liver mass was converted into a suspension of intact parenchymal cells which excluded a vital stain and were undamaged when viewed with the electron microscope. The cells actively incorporated labeled precursors into lipids and proteins without specific cofactor requirements in both simple, ionic, and complex culture media. The chicken hepatocytes synthesized lipids and proteins for several days in suspension culture, but suspended rat hepatocytes had minimal biosynthetic activity after 24 hours. Both dibutyryl cyclic AMP (0.1 mm) and adenosine 3' : 5'-monophosphate (cyclic AMP) (0.1 mm) produced a similar striking inhibition of [1-14C]acetate incorporation into lipids in chicken hepatocytes. Dibutyryl cyclic AMP was less inhibitory to lipid synthesis by rat liver cells and cyclic AMP had no effect on this process. The inhibitory actions of dibutyryl cyclic AMP were probably not due to isotopic dilution of the label within the cells. Neither cyclic nucleotide had a lipolytic effect in vitro on hepatocyte lipids prelabeled in vivo. The inhibitory action of dibutyryl cyclic AMP on lipogenesis was significantly greater than the effect of twice the concentration of sodium butyrate, and 5'-AMP did not diminish hepatic lipid synthesis. It is postulated that hepatic lipid synthesis is inhibited specifically by cyclic nucleotides, and that hormonal stimulation of adenylate cyclase regulates hepatic lipid synthesis.