PubMed · 1545861
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S L Chuck, V R Lingappa. 1992-03-12. Apolipoprotein B intermediates.. https://doi.org/10.1038/356115b0
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Most truncations of apoB associated with hypobetalipoproteinemia (HBL) result from frame shift mutations of the apoB gene that give rise to premature stop codons and truncations of C-terminal sequences. The "natural" truncation, apoB-48, arises from a stop codon by cotranscriptional editing of intestinal apoB-100 mRNA. We hypothesized that mutant apoB mRNA would be normally edited and that only those apoB truncations shorter than apoB-48 would be expressed in enterocytes, because translation of mRNAs giving rise to longer truncations would be interrupted by the apoB-48 stop codon. Duodenal mucosal biopsies from HBL and normolipidemic subjects were incubated with [35S]methionine, apoB was immunoprecipitated and bands were visualized by autoradiography. Biopsies of three subjects heterozygous for apoB-54.8 or apoB-89 synthesized virtually only apoB-48. By contrast, the biopsy of a subject heterozygous for apoB-40 synthesized both apoB-48 and apoB-40. Thus, enterocytes in HBL edit the mutant mRNAs similarly to the apoB mRNA of normal enterocytes and the small intestine of heterozygotes with truncations longer than apoB-48 produce only apoB-48, as the apoB-48 stop codon terminates translation proximal to the mutant stop codon. By contrast, intestines of heterozygotes with truncations shorter than apoB-48 produce the truncated apoB because the mutant stop codon stops translation before the apoB-48 stop codon. In conclusion, only the liver secretes apoB truncations larger than apoB-48, whereas shorter truncations are secreted by both liver and intestine.
Non-esterified fatty acids (NEFAs) and insulin are important factors in the control of lipoprotein secretion, but the mechanism of action is unclear. The present study was undertaken to determine whether insulin and NEFAs modulated hepatic secretion of triacylglycerol and apolipoprotein B (apo-B) by regulation of hepatic intracellular apo-B content. The experiments were performed with the human hepatoblastoma cell line Hep G2, for periods of up to 72 h in the presence and absence of NEFAs and insulin. Higher concentrations of eicosapentanoate (EPA) sustained for 72 h decreased cellular protein content (at 250 microM) or caused cell death (at 750 microM), and this effect was not observed with the other NEFAs studied, whereas 75 microM-EPA did not affect cell viability. Compared with the absence of NEFA, 75 microM-EPA did not alter the intracellular triacylglycerol content, but decreased the intracellular content of apo-B by 47% (P < 0.01) and decreased secreted triacylglycerol and secreted apo-B by 13% (P < 0.05) and 21% (P < 0.01) respectively, after 72 h. However 250 microM-oleate increased the intracellular triacylglycerol by 36% (P < 0.01), intracellular apo-B by 22% (P < 0.05) and secreted triacylglycerol and apo-B by 20-30% (P < 0.05-0.01). Insulin decreased secreted triacylglycerol and apo-B in the presence of each NEFA studied by 20-30%. There was no correlation between the changes in intracellular triacylglycerol and the rate of secretion. However, when the secreted triacylglycerol or apo-B was plotted against intracellular apo-B content a significant correlation was observed (r = 0.89, P < 0.001 for both analyses). Apo-B mRNA levels did not change after 72 h incubation with oleate or EPA. These results demonstrate that EPA can be toxic to hepatocytes and that NEFAs and insulin control secretion of triacylglycerol and apo-B by regulation of the intracellular apo-B concentration, thus controlling assembly of apo-B with triacylglycerol to form lipoproteins.