Lipoprotein secretion by isolated rat hepatocytes: characterization

Lipoprotein production by freshly isolated rat hepatocytes in suspension was studied during short (1 -3 hr) incubation periods. The hepatocytes release very low density (d < 1.01 g/ml) lipoprotein (VLDL) particles, which as a group a) contain triacylglycerols, phospholipids, free cholesterol, and cholesteryl esters in molar proportions of 100:21:8:4; b) have a mean diameter 1.5-fold larger than those of plasma VLDL; c) have a similar electrophoretic mobility as plasma VLDL; and d ) carry apoproteins B and E as major, and apoproteins AI and C as minor, protein components. These apoproteins in the secreted VLDL can be newly synthesized during the incubation, as indicated by the incorporation of [‘4C]leucine. The secretion of VLDL by the hepatocytes is inhibited by addition of glucagon or dibutyryl cyclic AMP, and stimulated by added palmitate; thus, as in the whole liver, the secretory process is under hormonal or substrate control also in the isolated cells condition. Phospholipids and free cholesterol are also released as components of particles with higher densities, ranging from 1.031.08 g/ml, and from 1.10 to 1.24 g/ml. Colchicine and cycloheximide, while strongly suppressing VLDL secretion, inhibit the release of these other particles to a lesser extent (d 1.031.08 g/ml) or not at all (d > 1.10 g/ml). These particles with higher densities have not been positively identified; the latter group is dissimilar to the high density lipoprotein, which occurs in rat liver perfusates, or to rat bile micelles.-KKempen, H. J. M. Lipoprotein secretion by isolated rat hepatocytes: characterization of the lipid-carrying particles and modulation of their release. J. Lipzd Res. 1980. 21: 671-680. Supplementary key words lipid composition . apoprotein composition . apoprotein synthesis . density gradient centrifugation . bile Lipoprotein synthesis and secretion by isolated liver parenchymal cells has been investigated previously, using either freshly isolated hepatocytes (1-3) or hepatocytes maintained for a few days in suspension (4), or monolayer (5 ,6 ) cultures. In these studies, very low density lipoprotein (VLDL) was found to accumulate in the incubation medium; it differed to a varying degree from mature plasma VLDL in both lipid and apoprotein compositions. For instance, for rat hepatocytes the active synthesis and secretion of apoproteins B and E could be demonstrated, whereas that of apoprotein C was very low or absent (4, 6). This is in contrast to the situation in the perfused rat liver, which produces a VLDL with a complement of apoprotein C similar to that of rat plasma VLDL (7-9). Furthermore, the content of cholesteryl ester in the VLDL from cultured hepatocytes was found to be lower than that of plasma VLDL (6). Although the secretion of HDL has been described for the perfused rat liver (8lo), documentation concerning HDL production by isolated hepatocytes is as yet lacking. In the study by Edwards, Lemongello, and Fogelman (3) the secreted lipid material was separated in two density regions (below and above 1.074 g/ml), but except for the cholesterol content the particles in the higher density region were not further characterized. In the present study with freshly isolated rat hepatocytes, the nature of the released lipid-carrying particles has been assessed by lipid and apoprotein analysis, agarose electrophoresis, and density gradient centrifugation. In addition, the effect of various agents, known to influence the secretion of VLDL from the perfused liver, on the lipid release in the various density fractions was investigated. MATERIALS AND METHODS