SPHINGOLIPID BIOSYNTHESIS DE-NOVO BY RAT HEPATOCYTES IN CULTURE - CERAMIDE AND SPHINGOMYELIN ARE ASSOCIATED WITH, BUT NOT REQUIRED FOR, VERY-LOW-DENSITY LIPOPROTEIN SECRETION

Sphingolipids are constituents of liver and lipoproteins, but relative ly little is known about their synthesis and secretion. Incubation of rat hepatocytes with [C-14]- or [H-3]serine labeled the long-chain bas e backbones of mainly ceramide and sphingomyelin. Most of the labeled sphingolipids were associated with the cells; however, 1-5% (the major ity of which was ceramide) was released into the medium as part of ver y low density Lipoproteins (VLDL). Since this is the first report that Lipoproteins contain ceramide, lipoproteins were isolated from rat pl asma, and the ceramide contents were (per mg of protein): 6.5 nmol for VLDL (d < 1.018), 0.6 nmol for low density lipoproteins (1.018 < d < 1.063), 0.2 nmol for high density lipoproteins (1.063 < d < 1.18), and 0.1 nmol for the albumin fraction; the lipoproteins also contained 0. 1-0.4 nmol of free sphingosine/mg of protein. A number of factors affe cted the secretion of radiolabeled sphingolipids: 1) addition of palmi tic acid, but not stearic or oleic acid, enhanced secretion due to an increase in long-chain base synthesis de novo. 2) Choline deficiency c aused a 42% reduction in the secretion of radiolabeled sphingomyelin, but this was due to an effect on VLDL secretion rather than a decrease in sphingolipid synthesis. Removal of choline was examined because pr evious studies (Yao, Z, M., and Vance, D. E. (1988) J. Biol. Chem. 263 , 2998-3004) have shown that choline deficiencies depress phosphatidyl choline synthesis and lipoprotein secretion. 3) Incubation of the cell s with fumonisin B-1, a mycotoxin inhibitor of sphinganine (sphingosin e) N-acyltransferase, reduced overall sphingolipid synthesis and secre tion by 90%, but had no effect on the secretion of apoB, phosphatidylc holine, or cholesterol. Ah together, these findings demonstrate that r at hepatocytes synthesize ceramide and sphingomyelin de novo and incor porate them into both cellular membranes and secreted VLDL, but normal sphingolipid synthesis is not required for lipoprotein secretion.