Intracellular events in the assembly of very-low-density-lipoprotein lipids with apolipoprotein B in isolated rabbit hepatocytes

Cartwright, Ian J.; Higgins, Joan A.

doi:10.1042/bj3100897

Cited by 42 publications

(64 citation statements)

References 48 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…5A, bottom) were found in the membranes with the remainder in the lumen in oleate-treated cells. (The high proportion of membrane-associated TG in hepatic microsomes was observed previously with rat (37) and rabbit hepatocytes (38).) Treatment with BMS-197636 entirely abolished the oleate-induced accumulation of TG within microsomal lumen, and also decreased the amount of TG associated with microsomal membrane by 30%.…”

Section: Resultsmentioning

confidence: 55%

The Activity of Microsomal Triglyceride Transfer Protein Is Essential for Accumulation of Triglyceride within Microsomes in McA-RH7777 Cells

Wang¹,

Tran²,

Yao³

1999

Journal of Biological Chemistry

128

140

View full text Add to dashboard Cite

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 M) 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 M) 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 g/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.

show abstract

Section: Resultsmentioning

confidence: 55%

The Activity of Microsomal Triglyceride Transfer Protein Is Essential for Accumulation of Triglyceride within Microsomes in McA-RH7777 Cells

Wang¹,

Tran²,

Yao³

1999

Journal of Biological Chemistry

128

140

View full text Add to dashboard Cite

show abstract

“…This, however, is an unlikely scenario occurring in HepG2 cells under the conditions used in this study, because the cells were incubated under conditions that support optimal folding of apoB resulting in a very efficient secretion. A more likely explanation for the prolonged interaction between ER-resident chaperones and apoB is that the maturation of apoB to form VLDL/IDL is a multistep process that begins in the ER and continues throughout the secretory pathway including distal compartments of the Golgi, as demonstrated by a number of investigators (56,57,63,64,70). These include modifications of apoB such as glycosylation and phosphorylation, remodeling of the phospholipid on the surface of the particles, and possibly continued lipidation (71,72).…”

Section: Fig 5 Molecular Chaperones Co-immunoprecipitate With Apob mentioning

confidence: 99%

Nascent Lipidated Apolipoprotein B Is Transported to the Golgi as an Incompletely Folded Intermediate as Probed by Its Association with Network of Endoplasmic Reticulum Molecular Chaperones, GRP94, ERp72, BiP, Calreticulin, and Cyclophilin B

Zhang

Herscovitz

2003

Journal of Biological Chemistry

View full text Add to dashboard Cite

We have previously demonstrated that endoplasmic reticulum (ER)-resident molecular chaperones interact with apolipoprotein B-100 (apoB) during its maturation. The initial stages of apoB folding occur while it is bound to the ER membrane, where it becomes partially lipidated to form a primordial intermediate. We determined whether this intermediate is dependent on the assistance of molecular chaperones for its subsequent folding steps. To that end, microsomes were prepared from HepG2 cells and luminal contents were subjected to KBr density gradient centrifugation. Immunoprecipitation of apoB followed by Western blotting showed that the luminal pool floated at a density of 1.12 g/ml and, like the membrane-bound pool, was associated with GRP94, ERp72, BiP, calreticulin, and cyclophilin B. Except for calreticulin, chaperone/apoB ratio in the lumen was severalfold higher than that in the membrane, suggesting a role for these chaperones both in facilitating the release of the primordial intermediate into the ER lumen and in providing stability. Subcellular fractionation on sucrose gradients showed that apoB in the Golgi was associated with the same array of chaperones as the pool of apoB recovered from heavy microsomes containing the ER, except that chaperone/apoB ratio was lower. KBr density gradient fractionation showed that the major pool of luminal apoB in the Golgi was recovered from 1.02 < d < 1.08 g/ml, whereas apoB in ER was recovered primarily from 1.08 < d < 1.2 g/ml. Both fractions were associated with the same spectrum of chaperones. Together with the finding that GRP94 was found associated with sialylated apoB, we conclude that correct folding of apoB is dependent on the assistance of molecular chaperone, which play multiple roles in its maturation throughout the secretory pathway including distal compartments such as the trans-Golgi network.The correct folding of apolipoprotein B (apoB) 1 into its mature, secretion-competent form is a complex process that leads to the formation and secretion of triacylglycerol (TAG)-rich lipoproteins, chylomicrons, and the atherogenic lipoproteins, very low density lipoproteins (VLDL) (1, 2). A two-step model for the formation of VLDL was originally proposed on the basis of electron microscopic studies in rat liver (3). This model was further supported by latter studies in rat liver (4), in rat hepatoma cells (McA-RH7777; Refs. 5-8), and in transgenic mice lacking the gene for apoB in the intestine (9). According to this model, the first step involves partial lipidation of apoB to form a primordial intermediate with HDL/LDL-like density. In the second step this intermediate fuses with a large apoB-free lipid droplet composed primarily of TAG to form nascent VLDL or chylomicrons. In HepG2 cells, however, these distinct steps were not clearly demonstrated. Nonetheless, the initial steps of lipidation are thought to be similar to other systems in that they are mediated by microsomal triglyceride transfer protein (MTP) (reviewed in Refs. 10 -12) and occur co-translationally,...

show abstract

“…Whether this full capacity is actually realized in practice depends on (1) the extent of subsequent lipidation of each secretory competent molecule of apoB at the so-called second stage of VLDL assembly, which involves bulk lipid transfer 18 -22 and (2) the extent to which particles unable to complete this step are degraded or secreted. 22,60,61 Although MTP is essential for neutral lipid transfer to apoB, 62,63 it is not yet clear as to whether MTP is required at only 1 or at both of the above lipid acquisition steps. 23,24,64 Nevertheless, the presence of at least 2 such lipid interaction steps, each at a critical phase in VLDL assembly, implies that they may have some regulatory significance as possible foci for hormones or regulatory metabolites.…”

Section: Glucose Enhances Predominantly the Bulk Lipidation Step Of Vmentioning

confidence: 99%

Glucose Phosphorylation Is Essential for the Turnover of Neutral Lipid and the Second Stage Assembly of Triacylglycerol-Rich ApoB-Containing Lipoproteins in Primary Hepatocyte Cultures

Brown

Wiggins

Gibbons

1999

ATVB

View full text Add to dashboard Cite

Abstract-Primary hepatocytes cultured in a medium supplemented with amino acids and lipogenic substrates responded to increased extracellular glucose by increasing the secretion of VLDL apoB. This effect was accompanied by an increased secretion of VLDL triacylglycerol (TAG) derived from endogenous stores. Glucose also stimulated intracellular TAG mobilization via the TAG lipolysis/esterification cycle. All these effects were abolished in the presence of mannoheptulose (MH), an inhibitor of glucose phosphorylation. Glucose also gave rise to a modest (50% to 60%) increase in the incorporation of 35 S methionine into newly synthesized apoB (PϽ0.05) and to a doubling of newly-synthesized apoB secretion as VLDL (PϽ0.05). The magnitude of these effects was similar for apoB-48 and for apoB-100. MH inhibited apoB-48 and apoB-100 synthesis and VLDL secretion at all glucose concentrations. The effects of glucose and MH on the secretion of newly-synthesized apoB-48 or apoB-100 as small dense particles were less pronounced. Glucose had no effects on the posttranslational degradation of newly-synthesized apoB-100 or apoB-48. However, this process was significantly enhanced by MH. The results suggest that glucose stimulates TAG synthesis, turnover, and output as VLDL. These effects are associated with an increased VLDL output of apoB mediated mainly by an increase in the net synthesis of both apoB-48 and apoB-100. All these changes are prevented by interference with glucose phosphorylation. Output of small, dense, apoB-containing particles is relatively unaffected by the glucose and MH-induced changes in TAG synthesis and lipolysis, an observation which suggests that only the bulk lipid addition step of VLDL assembly is affected by changes in glucose metabolism. Key Words: primary hepatocytes Ⅲ apoB Ⅲ glucose Ⅲ lipid recruitment Ⅲ phosphorylation E levated plasma glucose is a characteristic feature of the hypertriglyceridemia that frequently accompanies conditions such as non-insulin-dependent diabetes mellitus (NIDDM) and obesity. [1][2][3][4] The role of glucose in this relationship has been studied for several years and there now seems little doubt that 1 of the direct effects of glucose at the hepatic level is to increase the secretion of VLDL TAG. 5-8 Controversy exists, however, as to whether this effect of glucose is accompanied by a similar increase in the secretion of apoB, and studies with primary cultures of rat hepatocytes 7 and the human hepatoma cell line HepG2 9 showed little effect of glucose in this regard. Recent studies have produced evidence for a glucose-mediated stimulation of apoB output in HepG2, 10,11 and it has been suggested that the precise effect of glucose is dependent on the culture conditions in vitro. 11 We have previously shown that high rates of VLDL output from primary hepatocytes can be maintained only when the culture medium is supplemented with certain amino acids and fatty acid precursors, which provide an environment conducive to vigorous de novo lipogenesis. [12][13][14] It has also ...

show abstract

Intracellular events in the assembly of very-low-density-lipoprotein lipids with apolipoprotein B in isolated rabbit hepatocytes

Cited by 42 publications

References 48 publications

The Activity of Microsomal Triglyceride Transfer Protein Is Essential for Accumulation of Triglyceride within Microsomes in McA-RH7777 Cells

The Activity of Microsomal Triglyceride Transfer Protein Is Essential for Accumulation of Triglyceride within Microsomes in McA-RH7777 Cells

Nascent Lipidated Apolipoprotein B Is Transported to the Golgi as an Incompletely Folded Intermediate as Probed by Its Association with Network of Endoplasmic Reticulum Molecular Chaperones, GRP94, ERp72, BiP, Calreticulin, and Cyclophilin B

Glucose Phosphorylation Is Essential for the Turnover of Neutral Lipid and the Second Stage Assembly of Triacylglycerol-Rich ApoB-Containing Lipoproteins in Primary Hepatocyte Cultures

Contact Info

Product

Resources

About