The effects of fatty acids on apolipoprotein B secretion by human hepatoma cells (HEP G2)

Abstract

We have investigated the effect of fatty acids on the rate of apolipoprotein B (apo B) secretion by human hepatoma cells (Hep G2). When Hep G2 cells were maintained in tissue culture flasks oleic acid up to 0.4 mM increased apo B secretion in a dose-dependent manner, whereas increases in triacylglycerol (TG) were smaller and dose dependency was less evident. In the absence of oleic acid, apo B accumulating in the tissue culture medium was predominantly in lipoproteins of higher density than very low density lipoproteins (VLDL). However, when the rate of secretion was stimulated with oleic acid the apo B-containing lipoproteins became lower in density. We postulated that there was a high rate of lipolysis of newly secreted VLDL by Hep G2 cells, which would account both for the relatively smaller effect of oleic acid on TG as opposed to apo B accumulating in the culture medium and the predominance of apo B in lipoproteins of a higher density than VLDL, which became less evident when VLDL secretory rates were stimulated by oleic acid. To test this hypothesis, cultured Hep G2 cells were transferred to columns containing Cytodex beads, permitting their continuous perfusion with culture medium so that newly secreted VLDL did not remain in contact with the cells. Apo B recovered from the perfusate was largely in VLDL range lipoproteins and the TG measured in the perfusate indicated that the true secretory rate of TG-rich lipoproteins was substantially higher than had been reflected by TG accumulating in culture medium left in contact with cells. Apo B measured in the culture medium of Hep G2 cells may thus be a better reflection of VLDL secretion, even though it is contained in higher density lipoproteins due to removal of TG by lipolysis. The effects of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) on apo B (apo B) secretion by Hep G2 cells maintained in tissue culture flasks were next investigated. SFA (0.4 mM), with the exception of stearic acid (C18:0), increased apo B secretion. Lauric acid (C12:0) increased apo B secretion by 32%, myristic acid (C14:0) by 41% (P<0.005), palmitic acid (C16:0) by 154% (P<0.025), and arachidic acid (C20:0) by 186% (P<0.005). The effect of MUFA (0.4 mM) was to increase apo B secretion, oleic acid (C18:1) by 239% ((P<0.0005) and palmitoleic acid (C16: 1) by 125% (P<0.005). Of the PUFA investigated, linolenic acid (C18:3) (0.4 mM) did not have any significant effect on apo B secretion, whereas linoleic acid (C18:2) (0.4mM) arachidonic acid (C20:4) (0.1 mM) and eicosapentaenoic acid (C20:5) (0.1 mM) caused significant increases of 164, 171 and 171%, respectively (P<0.005). The fatty acids studied increased intracellular TG and cholesteryl ester concentrations to varying extents. The increase in intracellular TG produced by the different fatty acids correlated with the rate of apo B secretion (r=0.6; P<0.05). In this human hepatoma cell line, with the exception of the saturated fatty acids, the rate of secretion of apo B-containing lipoproteins does not follow the same pattern as changes in circulating low density lipoprotein (LDL) concentrations reported with dietary manipulation in man. If our findings reflect the in vivo situation, we suggest that whilst the dietary effects of SFA on serum LDL may in part be determined by the hepatic apo B secretory rate, the effects of MUFA and PUFA must be largely mediated through a catabolic effect rather than an effect on hepatic secretion. The marked increase in apo B secretion with the more highly polyunsaturated fatty acids, such as eicosapentaenoic acid, may also explain why they do not lower circulating LDL, despite reports of their apparently favourable effect on LDL-receptor mediated clearance.