Synthesis and secretion of lipolytic enzymes by cultured chicken hepatocytes

G L Jensen, D L Baly, P M Brannon, A Bensadoun
1980 Journal of Biological Chemistry  
The synthesis and secretion of hepatic triglyceride lipase and lipoprotein lipase were evaluated in nonproliferating monolayer cultures of chicken hepatocytes. Cells were maintained in an Arg-deficient Dulbecco's modified medium that contained 5% heat-inactivated chicken serum and 5 pg/ml of bovine insulin. Lipolytic activity was determined as a function of pH with acetone cell powder extracts. A predominant peak of lysosomal lipase activity (pH 4 to 7) was observed, but a discrete alkaline pH
more » ... ptimum of activity was not readily detected. A potent lysosomal lipase inhibitor, chlorpromazine, did not alter hepatic triglyceride lipase and lipoprotein lipase activities. Chlorpromazine enabled resolution of a discrete alkaline pH optimum (pH 7 to 10) of lipolytic activity. Partial inhibition of lipolytic activity in this alkaline range was observed with chlorpromazine, which suggested the presence of residual lysosomal lipase activity in this pH interval. Alkaline lipolytic activities were measured by chlorpromazine resistance in cell acetone powder extracts from cells harvested at different time points. Heparin-Sepharose affinity chromatography indicated the presence of hepatic triglyceride lipase and lipoprotein lipase activities in both cell powder extract and culture media. A specific radioimmunoassay detected lipoprotein lipase enzyme protein in both cell powder extract and culture media. Colchicine and tunicamycin inhibited the secretion of lipoprotein lipase protein. The incorporation of ['HILeu into lipoprotein lipase enzyme protein was monitored by specific radioimmunoadsorption. The 3Hlabeled protein, dissociated from the immunoadsorbent and subjected to sodium dodecyl sulfate electrophoresis or isoelectric focusing, showed migration identical with highly purified lipoprotein lipase. These findings indicate that maintenance cultures of chicken hepatocytes synthesize lipoprotein lipase and demonstrate the secretion of hepatic triglyceride lipase and lipoprotein lipase. Triacylglycerol lipase activity is found in liver homogenate (1) and in constituent fractions of plasma membrane (2, 3 ) , cytosolic (2-4), microsomal (2-5), and lysosomal origin (2-8). All fractions show alkaline pH optima (pH 8.0 to 9.5) for lipolytic activity, except the lysosomal preparation, which has an optimum of pH 4 to 6. A number of distinct triacylglycerol lipases may contribute to these lipolytic activities.
pmid:7440534 fatcat:gnvz2nselzeuxjodnripcjqkp4