Secretion of nascent lipoproteins by isolated rat hepatocytes.

Abstract

The nature of the nascent lipoproteins secreted by suspensions of isolated rat hepatocytes incubated in a lipid-deficient medium was investigated. Samples of the concentrated medium after 12 and 24 h of incubation were resolved by gel filtration and demonstrated that lipoproteins were secreted with a wide spectrum of particle sizes. Particles corresponding to sizes of serum very low density lipoproteins (VLDL) and low density lipoproteins (LDL) had similar levels of apolipoproteins (apo) B and E as serum VLDL when determined by electroimmunoassay, suggesting that the liver cell secretes a "small" VLDL under these conditions and not an LDL particle as present in the serum. Lipid analyses of the secreted triglyceride-rich particles show them to be similar in composition to serum VLDL with the exception of their cholesterol ester content, which was much lower in the hepatocyte-secreted VLDL. Incorporation of 3H-labelled amino acids into the VLDL apoproteins from the incubation medium after 24 h was determined after ultracentrifugal isolation (d < 1.063 g∙mL−1) and urea–gel electrophoresis, and found to be 70% and 22% of the total applied radioactivity for apo B and apo E, respectively. The lack of immunochemicaily detectable apo C-II and C-III in the isolated nascent VLDL and the lack of significant radioactive incorporation confirmed their visual absence from the gels. Further purification of the VLDL apo E by immunoaffinity chromatography showed it to consist of two narrowly separated bands on 7 M urea – polyacrylamide gels. Apo B was secreted only with particles having mean diameters of greater than 194 Å. In contrast, 75% of the total secreted apo E was associated with fractions of smaller particle diameters. This apo E (LpE) was almost equally distributed in two peaks corresponding to a particle size of approximately 100 Å and a molecular weight of < 60 000, respectively. Only 35% of the total apo E was found in the comparable fractions when hepatocytes from hypercholesterolemic rats were used. Thus, normal hepatocytes secrete a significant proportion of apo E, as a low molecular weight, essentially lipid-free form. The apparent secretion rates, for the apoproteins (mean ± SEM) for hepatocytes from normal rats, were 77.0 + 10.6 μg∙h−1∙g cell protein−1 (apo B) and 71.7 ± 8.6 μg∙h−1∙g cell protein−1 (apo E) after 24 h.