ASSEMBLY AND SECRETION OF VLDL IN NONDIFFERENTIATED CACO-2 CELLS STABLY TRANSFECTED WITH HUMAN RECOMBINANT APOB48 CDNA

Intestinal cells secrete apoB48-containing very low density lipoprotei ns (VLDLs) and chylomicrons for the transport of biliary and dietary l ipids. The molecular mechanisms regulating the assembly of intestinal lipoproteins are not known due to a lack of reliable and specific cell culture models. Caco-2 (a human colon carcinoma) cells have been used to study intestinal lipid metabolism. These cells have been shown to secrete both apoB100- and apoB48-containing triglyceride (TG)-rich lip oproteins only after differentiation into enterocyte-like cells. To st udy lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constit utive cytomegalovirus promoter. Pulse-chase analysis revealed that the majority (>50%) of apoB48 synthesized was degraded intracellularly in the presence or absence of oleic acid. Transfected nondifferentiated cells secreted lipoproteins with flotation densities similar to those of plasma HDL or LDL when cultured in serum-free or serum-containing m edia, respectively. Incubation of cells with media containing serum an d oleic acid resulted in the secretion of VLDL-like particles. Secreti on of VLDL was inhibited (>80%) by triacsin C due to >60% inhibition o f oleate-induced TG synthesis. However, inhibition of cholesteryl este r synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipopr oteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measur ing TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, whic h corresponds to 30% to 60% of the MTP activity present in liver-deriv ed cells. To determine whether MTP activity was required for lipoprote in assembly, transfected cells were incubated in the presence of the M TP inhibitor CP-10,447. This compound completely abolished the secreti on of apoB. These data show that the transfected cell lines secrete li poproteins of different densities under different culture conditions a nd that the assembly of larger VLDL particles requires active TG synth esis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the am ount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.