Perinatal hepatocyte/hepatoma hybrids: construction

Microsomal monoacylglycerol acyltransferase is a developmentally expressed enzyme that catalyzes the synthesis of sn-1,2-diacylglycero1 from sn-2-monoacylglycero1 and palmitoyl-CoA. The activity is present in liver from fetal and suckling rats but is absent in the adult. In order to obtain a stable permanent cell line that expresses this activity, Fa0 rat hepatoma cells and hepatocytes from 8-day-old baby rats were hybridized and clones were selected. Two hybrids (HA1 and HA7) expressed monoacylglycerol acyltransferase activity. Like fetal hepatocytes, but unlike hepatocytes from postnatal rats, the HA cells had high rates of [ 'Clacetate incorporation into glycerolipids, cholesterol, and cholesteryl esters, and they secreted triacylglycerol into the media. Monoacylglycerol acyltransferase specific activity increased 2.5-fold as the cells divided in culture, suggesting growth-dependent regulation. The specific activities of glycerol-P acyltransferase, the committed step of the microsomal pathway of glycerolipid synthesis, and diacylglycerol acyltransferase, the activity unique to triacylglycerol biosynthesis, were comparable to the levels of the corresponding activities in fetal hepatocytes. Addition of insulin or dexamethasone to the media increased the incorporation of [ '4C]oleate into triacylglycerol about 1.7-fold within 2 h, but had little effect on ['%]oleate incorporation into phospholipid. These hormonally responsive rat-hepatomdhepatocyte hybrids reflect the fetal stage of hepatocyte development in five major aspects of lipid metabolism: sterol, fatty acid, and triacylglycerol biosynthesis, glycerolipid secretion, and the presence of the developmentally expressed monoacylglycerol pathway. -Coleman, R.A., and E. S-G. Bades. Perinatal hepatocyte/hepatoma hybrids: construction of clones that express the developmentally regulated monoacylglycerol acyltransferase activity. J. Lipid Res. 1990. 31: 2257-2264. Supplementary key words acylglycerol acyltransferase * lipid metabolism * diacylglycerol fetal hepatocytes hybrid cells monoglycerolipid biosynthesis begins with the acylation of glycerol-3-P to form phosphatidic acid which is at the branchpoint of the formation of the anionic phospholipids and sn-1,2-diacylglycerol (2). Diacylglycerol is the immediate precursor for the quantitatively most prominent glycerolipids: triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine. Diacylglycerol can also be formed via minor catabolic pathways that include the regulated hydrolysis of phosphatidylinositol and phosphatidylcholine by phospholipase C. When diacylglycerol is formed via these latter catabolic routes in the plasma membrane, however, its role is thought to be limited to that of a second messenger and activator of protein kinase C. Monoacylglycerol acyltransferase provides an alternate, and perhaps major, pathway of diacylglycerol synthesis during the perinatal period. The activity, which catalyzes the acylation of sn-2-monoacylglycerol, has a specific activity in postnatal rat liver about 300-fold higher than that of glycerol-3-P acyltransferase, the committed step of the glycerol-P pathway (1, 3). The activity is present in hepatocytes from perinatal rats, fetal guinea pigs, and embryo chicks, suggesting that it plays an important role in the developing animal (1, 4, 5). It remains unclear as to why this alternate route of diacylglycerol synthesis is required during perinatal life, and how the unusual developmental pattern is regulated. Long-term study of the regulation of this and other pathways of neonatal lipid metabolism have been precluded, not only Hepatic monoacylglycerol acyltransferase is a developmentally expressed microsomal enzyme whose specific acadult (1). In most eukaryotic cells, the major pathway of Abbreviations: DMEG, Dulbecco's modified Eagle medium with 4.5 g glucosell; H M G C d , hydroxymethylglutaryl-Cd; FBS, fetal bovine serum; EDTA, (ethylenedinitril0)-tetraacetic acid; HGPRT, hypoxanproteins. tivity is 700-fo1d higher in postnatal rat liver than in the thine-guanine phosphoribosyltransferase; VLDL, low density lipoJournal of Lipid Research Volume 31, 199