Abstract
Previously, based on distinct requirement of microsomal triglyceride transfer protein (MTP) and kinetics of triglyceride (TG) utilization, we concluded that assembly of very low density lipoproteins (VLDL) containing B48 or B100 was achieved through different paths (Wang, Y. , McLeod, R. S., and Yao, Z. (1997) J. Biol. Chem. 272, 12272-12278). To test if the apparent dual mechanisms were accounted for by apolipoprotein B (apoB) length, we studied VLDL assembly using transfected cells expressing various apoB forms (e.g. B64, B72, B80, and B100). For each apoB, enlargement of lipoprotein to form VLDL via bulk TG incorporation was induced by exogenous oleate, which could be blocked by MTP inhibitor BMS-197636 treatment. While particle enlargement was readily demonstrable by density ultracentrifugation for B64- and B72-VLDL, it was not obvious for B80- and B100-VLDL unless the VLDL was further resolved by cumulative rate flotation into VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). BMS-197636 diminished B100 secretion in a dose-dependent manner (0.05-0.5 microM) and also blocked the particle enlargement from small to large B100-lipoproteins. These results yield a unified model that can accommodate VLDL assembly with all apoB forms, which invalidates our previous conclusion. To gain a better understanding of the MTP action, we examined the effect of BMS-197636 on lipid and apoB synthesis during VLDL assembly. While BMS-197636 (0.2 microM) entirely abolished B100-VLDL(1) assembly/secretion, it did not affect B100 translation or translocation across the microsomal membrane, nor did it affect TG synthesis and cell TG mass. However, BMS-197636 drastically decreased accumulation of [(3)H]glycerol-labeled TG and TG mass within microsomal lumen. The decreased TG accumulation was not a result of impaired B100-VLDL assembly, because in cells treated with brefeldin A (0.2 microgram/ml), the assembly of B100-VLDL was blocked yet lumenal TG accumulation was normal. Thus, MTP plays a role in facilitating accumulation of TG within microsomes, a prerequisite for the post-translational assembly of TG-enriched VLDL.
Highlights
Based on distinct requirement of microsomal triglyceride transfer protein (MTP) and kinetics of triglyceride (TG) utilization, we concluded that assembly of very low density lipoproteins (VLDL) containing B48 or B100 was achieved through different paths
A Unified VLDL Assembly Model for All ApoB Forms—Our previous study suggested that assembly of B100-VLDL and B48-VLDL took different paths [7]
B64, B72, and B80/B100 were found predominantly in fractions with densities resembling those of high density lipoproteins (HDL), low density lipoproteins (LDL), and VLDL, respectively (Fig. 1A, panels labeled –OA)
Summary
From the Lipoprotein and Atherosclerosis Group and the Departments of Pathology & Laboratory Medicine and Biochemistry, University of Ottawa Heart Institute, Ottawa, Ontario K1Y 4W7, Canada. BMS-197636 diminished B100 secretion in a dose-dependent manner (0.05– 0.5 M) and blocked the particle enlargement from small to large B100-lipoproteins These results yield a unified model that can accommodate VLDL assembly with all apoB forms, which invalidates our previous conclusion. Emerging evidence suggests that the demand for MTP is much greater for bulk TG incorporation during VLDL assembly than that for primordial particle formation [7, 21] These data raise the possibility that MTP may play a role other than assisting apoB translocation. We studied VLDL assembly using transfected cells expressing various apoB forms (e.g. B64, B72, B80, and B100) to inquire if there were dual mechanisms for the assembly of VLDL containing small or large apoB. We have found that MTP activity is crucial in the maintenance of a metabolically dynamic microsomal TG pool, which is essential for bulk TG incorporation during the final stage of B100-VLDL assembly
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