Assembly and Secretion of VLDL in Nondifferentiated Caco-2 Cells Stably Transfected With Human Recombinant ApoB48 cDNA

Abstract

Intestinal cells secrete apoB48-containing very low density lipoproteins (VLDLs) and chylomicrons for the transport of biliary and dietary lipids. 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 lipoproteins only after differentiation into enterocyte-like cells. To study lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constitutive 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 media, respectively. Incubation of cells with media containing serum and oleic acid resulted in the secretion of VLDL-like particles. Secretion of VLDL was inhibited (> 80%) by triacsin C due to > 60% inhibition of oleate-induced TG synthesis. However, inhibition of cholesteryl ester synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipoproteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measuring TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, which corresponds to 30% to 60% of the MTP activity present in liver-derived cells. To determine whether MTP activity was required for lipoprotein assembly, transfected cells were incubated in the presence of the MTP inhibitor CP-10,447. This compound completely abolished the secretion of apoB. These data show that the transfected cell lines secrete lipoproteins of different densities under different culture conditions and that the assembly of larger VLDL particles requires active TG synthesis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the amount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.

Differential, Tissue-specific, Transcriptional Regulation of Apolipoprotein B Secretion by Transforming Growth Factor β

“…As seen before, treatment of HepG2 cells with TGF-␤ decreased apoB secretion by ϳ25%. As expected, nondifferentiated Caco-2 cells secreted (54 Ϯ 7 ng/well) significantly smaller amounts of apoB than the differentiated cells (see above), in agreement with other studies (48,52). Nonetheless, treatment of these cells increased apoB secretion by 26% (Fig.…”

“…1). The amount of apoB secreted by the control Caco-2 and HepG2 cells was 944 Ϯ 59 and 1266 Ϯ 82 ng/well, respectively, in agreement with our earlier studies (44,48,(52)(53)(54). In Caco-2 cells, TGF-␤2 showed a concentrationdependent increase in the amount of apoB secreted into the basolateral medium.…”

Assembly and Secretion of Chylomicrons by Differentiated Caco-2 Cells

“…In contrast to enterocytes which mainly synthesize apoB48, Caco-2 cells mainly synthesize apoB100 (2,(5)(6)(7)(8)(9). In a previous study, we showed that the inability of these cells to assemble CM was not due to limited synthesis of apoB48, since overexpression of apoB48 did not result in the secretion of chylomicrons (10). Furthermore, lipid absorption and transport were not impaired in mice expressing only apoB100 (11,12).…”

Inhibiting Proteasomal Degradation of Microsomal Triglyceride Transfer Protein Prevents CCl4-induced Steatosis

“…Primary enterocytes were isolated and cultured as described (24). HepG2 and Caco-2 cell were cultured as described previously (25,26). Human apoB and apoAI secreted from cells were measured by enzyme-linked immunosorbent assay (23,27).…”