Influence of triacylglycerol biosynthesis rate on the assembly of apoB-100-containing lipoproteins in Hep G2 cells.

Borén, Jan; Rustaeus, Sabina; Wettesten, Margit; Andersson, Maria; Wiklund, A; Olofsson, Sven-Olof

doi:10.1161/01.atv.13.12.1743

Cited by 75 publications

(49 citation statements)

References 42 publications

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“…However, when the assembly process is inhibited, this VLDL-precursor will be degraded in the cell. In agreement with our previous results (20), the present observations indicate that the assembly process plays a critical role in the sorting of apoB-100; apoB-100 that is not recruited to the assembly process will gradually be degraded intracellularly.…”

Section: Discussionsupporting

confidence: 93%

“…The apoB-100 radioactivity that was present in the carbonate extract of the microsomes banded during gradient ultracentrifugation in fractions with a higher density than VLDL, with the dominating amount present in the density region of HDL (20) (Fig. 1, chase 1).…”

Section: Resultsmentioning

confidence: 99%

“…This suggests that, as in the case of apoB-48, MTP also plays a role in the early stages of apoB-100 lipoprotein assembly, but not in the later stages. One drawback with the HepG2 cells is that they do not assemble any substantial amount of bona fide VLDL (20), but rather an LDL-size particle rich in triacylglycerol. The assembly of this particle occurs to a significant degree cotranslationally (28).…”

Section: Discussionmentioning

confidence: 99%

“…There is a possibility that at least part of apoB-100 is post-translationally translocated to the lumenal side of the membrane (20,30,31). MTP may participate in such a translocation indirectly by allowing this part of the protein to properly fold as it enters the interior of the endoplasmic reticulum.…”

Section: Discussionmentioning

confidence: 99%

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The Microsomal Triglyceride Transfer Protein Catalyzes the Post-translational Assembly of Apolipoprotein B-100 Very Low Density Lipoprotein in McA-RH7777 Cells

Rustaeus

Stillemark

Lindberg

et al. 1998

Journal of Biological Chemistry

118

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In cells in which the lipoprotein assembly process had been inactivated by brefeldin A (BFA), membrane-associated apoB-100 disappeared without forming lipoproteins or being secreted, indicating that it was degraded. Reactivation of the assembly process by chasing the cells in the absence of BFA, gave rise to a quantitative recovery of the membrane-associated apoB-100 in the very low density lipoprotein (VLDL) fraction in the medium. These results indicate that the membrane-associated apoB-100 can be converted to VLDL.A new method was developed by which the major amount (88%) of microsomal apoB-100 but not integral membrane proteins could be extracted. The major effect of this method was to increase the recovery of apoB-100 that banded in the LDL and HDL density regions, suggesting that the membrane-associated form of apoB-100 is partially lipidated. We also investigated the role of the microsomal triglyceride transfer protein (MTP) in the assembly of apoB-100 VLDL using a photoactivatable MTP inhibitor (BMS-192951). This compound strongly inhibited the assembly and secretion of apoB-100 VLDL when present during the translation of the protein. To investigate the importance of MTP during the later stages in the assembly process, the cells were preincubated with BFA (to reversibly inhibit the assembly of apoB-100 VLDL) and pulse-labeled (؉BFA) and chased (؉BFA) for 30 min to obtain full-length apoB-100 associated with the microsomal membrane. Inhibition of MTP after the 30-min chase blocked assembly of VLDL. This indicates that MTP is important for the conversion of full-length apoB-100 into VLDL. Results from experiments in which a second chase (؊BFA) was introduced before the inactivation of MTP indicated that only early events in this conversion of full-length apoB-100 into VLDL were blocked by the MTP inhibitor. Together these results indicate that there is a MTP-dependent "window" in the VLDL assembly process that occurs after the completion of apoB-100 but before the major amount of lipids is added to the VLDL particle. Thus the assembly of apoB-100 VLDL from membrane-associated apoB-100 involves an early MTP-dependent phase and a late MTP-independent phase, during which the major amount of lipid is added.

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Section: Discussionsupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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The Microsomal Triglyceride Transfer Protein Catalyzes the Post-translational Assembly of Apolipoprotein B-100 Very Low Density Lipoprotein in McA-RH7777 Cells

Rustaeus

Stillemark

Lindberg

et al. 1998

Journal of Biological Chemistry

118

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“…Regulated translocation of apoB across the endoplasmic reticulum could explain the posttranslational control of apoB secretion. In several studies, the amount of apoB that is secreted is less than the amount that is synthesized (5,(7)(8)(9)(10)(11)(12)(13)(14), leading to the conclusion that the unaccounted for apoB is degraded intracellularly. The consistent observation that in different types of cultured cells and perfused organs obtained from several species, a diverse group of hormones, nutritional states, stimulatory and inhibitory lipids, and mutations in the coding region of apoB alter the rate of apoB secretion by reciprocal changes in its rate of intracellular degradation suggests that this unusual regulatory mechanism of secretion is of general importance (reviewed in Refs.…”

mentioning

confidence: 99%

In HepG2 Cells, Translocation, Not Degradation, Determines the Fate of the de Novo Synthesized Apolipoprotein B

Bonnardel¹,

Davis²

1995

Journal of Biological Chemistry

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Previous studies show that translocation and degradation of apolipoprotein B (apoB), two processes occurring on or within the endoplasmic reticulum, determine how much de novo synthesized apoB is secreted. We determined which of these processes regulates the intracellular fate of apoB by examining whether degradation determines how much apoB is translocated or if translocation determines how much apoB is degraded. 35 S]methionine-labeled proteins), its effect on apoB was specific. Pulsechase studies showed that ALLN dramatically reduced the first-order rate of removal of [ 35 S]methionine-labeled apoB from the cell but did not effect its rate of secretion. The finding that ALLN caused the intracellular accumulation of incompletely translated chains of apoB suggests that at least some of the degradation occurs at the ribosomal level. Moreover, 85% of the apoB that accumulated in isolated microsomes in response to ALLN was accessible to exogenous trypsin, indicating this pool of apoB was incompletely translocated. The combined data suggest that translocation, not degradation, determines the intracellular fate of de novo synthesized apoB. apoB 1 is the major structural protein responsible for the assembly of triglyceride-rich lipoproteins by the intestine and liver (1-3). After secretion into blood plasma by the liver, VLDL triglycerides are rapidly hydrolyzed into free fatty acids, which are taken up by peripheral tissues where they are utilized for energy and anabolic purposes. The remaining VLDL remnants, containing apoB100, are then either removed by the liver or converted into LDL, a major risk factor for atherosclerosis (reviewed in Ref. 4). Because of the importance of hepatic secretion of VLDL apoB in determining blood levels of LDL, a major goal of our research has been directed toward identifying the regulatory factors and processes.We have made two observations, both of which have shown unusual characteristics governing the hepatic secretion of apoB: 1) a large amount of de novo synthesized apoB is not secreted but is degraded intracellularly (5) and 2) the translocation of apoB across the endoplasmic reticulum is unusually inefficient (6). These results led us to hypothesize that as a result of incomplete translocation, apoB is diverted from the secretory pathway into a degradative pathway that occurs in the endoplasmic reticulum (3, 6). Regulated translocation of apoB across the endoplasmic reticulum could explain the posttranslational control of apoB secretion. In several studies, the amount of apoB that is secreted is less than the amount that is synthesized (5,(7)(8)(9)(10)(11)(12)(13)(14), leading to the conclusion that the unaccounted for apoB is degraded intracellularly. The consistent observation that in different types of cultured cells and perfused organs obtained from several species, a diverse group of hormones, nutritional states, stimulatory and inhibitory lipids, and mutations in the coding region of apoB alter the rate of apoB secretion by reciprocal changes in its rate of intracellular ...

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Factors affecting the regulation of apo b secretion by liver cells

Hahn¹,

Goldberg²

1995

Clinical Laboratory Analysis

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The concentration of apo B is an important risk factor for atherosclerosis, and thus its reduction is associated with a reduction in CHD mortality. In order to reduce apo B concentrations effectively, we must understand how plasma apo B concentration is regulated. Apo B is synthesized, assembled, and secreted by the liver, controlling this process will reduce the number of particles that eventually enter the plasma compartment. The assembly of apo B into a VLDL particle is a complex process which occurs through several stages: peptide synthesis, translocation, accumulation of lipid, and transport through the secretory pathway. Multiple control points regulate the synthesis and secretion of apolipoproteins. Modulation of transcription, translation and intracellular degradation represent independent regulatory mechanisms. The ability of the lipoprotein to bind cotranslationally to lipid appears to be crucial to the formation of a secreted particle. This process may be regulated solely by MTP, or may be modified by the activity of the lipid-synthesizing enzymes. A great deal of evidence supports the role of TG and CE synthesis, although the relative importance of these two lipids is a source of major controversy. In summary, all the lipoprotein components can be limiting for apo B and VLDL synthesis when their availability is substantially decreased. The rate-limiting component in vivo has still not been identified. By understanding how lipoprotein synthesis and assembly are regulated, it should become possible to design new ways of altering these processes in a beneficial manner.

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Influence of triacylglycerol biosynthesis rate on the assembly of apoB-100-containing lipoproteins in Hep G2 cells.

Cited by 75 publications

References 42 publications

The Microsomal Triglyceride Transfer Protein Catalyzes the Post-translational Assembly of Apolipoprotein B-100 Very Low Density Lipoprotein in McA-RH7777 Cells

The Microsomal Triglyceride Transfer Protein Catalyzes the Post-translational Assembly of Apolipoprotein B-100 Very Low Density Lipoprotein in McA-RH7777 Cells

In HepG2 Cells, Translocation, Not Degradation, Determines the Fate of the de Novo Synthesized Apolipoprotein B

Factors affecting the regulation of apo b secretion by liver cells

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