Microsomal triglyceride transfer protein is essential for hepatic secretion of apoB-100 and apoB-48 but not triglyceride

Despite a complete lack of microsomal triglyceride transfer protein (MTP), L35 rat hepatoma cells secrete triglyceride-containing lipoproteins, albeit at a rate 25% of that of parental FAO hepatoma cells, which express high levels of MTP. The inability to express MTP was associated with a complete block in the secretion of both apolipoprotein (apo)B-100 and apoB-48. Stable expression of a MTP transgene restored the secretion of both apoB-100 and apoB-48 in L35 cells, indicating that MTP is essential for the secretion of both forms of apoB. Treatment with the MTP inhibitor BMS-200150 reduced the secretion of triglyceride by 70% in FAO cells, whereas the inhibitor did not affect the secretion of triglycerides by L35 cells. Thus, in the presence of the MTP inhibitor, both cell types secreted triglycerides at similar rates. Essentially, all of the triglycerides secreted by L35 cells were associated with HDL containing apoA-IV and apoE but devoid of apoB-100 or apoB-48. These results suggest that these triglyceride-containing lipoproteins are assembled and secreted via a pathway that is independent of both apoB and MTP. Our findings support the concept that apoB and MTP co-evolved and provided a means to augment the secretion of triglyceride through the formation of lipoproteins containing large hydrophobic cores enriched with triglycerides.     

Apolipoprotein B-related gene expression and ultrastructural characteristics of lipoprotein secretion in mouse yolk sac during embryonic development.

In mice, the yolk sac appears to play a crucial role in nourishing the developing embryo, especially during embryonic days (E) 7;-10. Lipoprotein synthesis and secretion may be essential for this function: embryos lacking apolipoprotein (apo) B or microsomal triglyceride transfer protein (MTP), both of which participate in the assembly of triglyceride-rich lipoproteins, are apparently defective in their ability to export lipoproteins from yolk sac endoderm cells and die during mid-gestation. We therefore analyzed the embryonic expression of apoB, MTP, and alpha-tocopherol transfer protein (alpha-TTP), which have been associated with the assembly and secretion of apoB-containing lipoproteins in the adult liver, at different developmental time points. MTP expression or activity was found in the yolk sac and fetal liver, and low levels of activity were detected in E18.5 placentas. alpha-TTP mRNA and protein were detectable in the fetal liver, but not in the yolk sac or placenta. Ultrastructural analysis of yolk sac visceral endoderm cells demonstrated nascent VLDL within the luminal spaces of the rough endoplasmic reticulum and Golgi apparatus at E7.5 and E8.5. The particles were reduced in diameter at E13.5 and reduced in number at E18.5;-19.The data support the hypothesis that the yolk sac plays a vital role in providing lipids and lipid-soluble nutrients to embryos during the early phases (E7;-10) of mouse development. secretion in mouse yolk sac during embryonic development.     

Microsomal triglyceride transfer protein -493T variant reduces IDL plus LDL apoB production and the plasma concentration of large LDL particles

The microsomal triglyceride transfer protein (MTP) is essential for the synthesis and secretion of apolipoprotein B (apoB)-containing lipoproteins. We investigated the role the MTP -493G/T gene polymorphism in determining the apoB-100 secretion pattern and LDL heterogeneity in healthy human subjects. Groups of carriers of the T and the G variants (n = 6 each) were recruited from a cohort of healthy 50-yr-old men. Kinetic studies were performed by endogenous [(2)H(3)]leucine labeling of apoB and subsequent quantification of the stable isotope incorporation. apoB production rates, metabolic conversions, and eliminations were calculated by multicompartmental modeling (SAAM-II). LDL subfraction distribution was analyzed in the entire cohort (n = 377). Carriers of the MTP -493T allele had lower plasma LDL apoB and lower concentration of large LDL particles [LDL-I: 136 +/- 57 (TT) vs. 175 +/- 55 (GG) mg/l, P < 0.01]. Kinetic modeling suggested that MTP -493T homozygotes had a 60% lower direct production rate of intermediate-density lipoprotein (IDL) plus LDL compared with homozygotes for the G allele (P < 0.05). No differences were seen in production rates of large and small VLDL, nor were there any differences in metabolic conversion or elimination rates of apoB between the genotype groups. This study shows that a polymorphism in the MTP gene affects the spectrum of endogenous apoB-containing lipoprotein particles produced in humans. Reduced direct production of LDL plus IDL appears to be related to lower plasma concentrations of large LDL particles.     

Hepatic overexpression of microsomal triglyceride transfer protein (MTP) results in increased in vivo secretion of VLDL triglycerides and apolipoprotein B.

The microsomal triglyceride transfer protein (MTP) is essential for the hepatic secretion of apolipoprotein (apo) B-containing lipoproteins. Previous studies have indicated that inhibition of MTP results in decreased apoB plasma levels and decreased hepatic triglyceride secretion. However, the metabolic effects of overexpression of MTP have not been investigated. We constructed a recombinant adenovirus expressing MTP (AdhMTP) and used it to assess the effects of hepatic overexpression of MTP in mice. Injection of AdhMTP into C57BL/6 mice resulted in a 3-fold increase in hepatic microsomal triglyceride transfer activity compared to mice injected with Adnull. On day 4 after virus injection, AdhMTP-injected mice had significantly elevated plasma TG levels as compared to control virus (Adnull)-injected mice. Hepatic TG secretion rates were significantly greater in AdhMTP-injected mice (184 +/- 12 mg/kg/h) compared with Adnull-injected mice (65 +/- 9 mg/kg/h, P < 0.001). In addition, hepatic very low density lipoprotein (VLDL) apoB secretion in the AdhMTP-injected group was 74% higher than in the control virus group. Hepatic secretion of apoB-48 and apoB-100 contributed equally to this increase.These results provide the first data that hepatic overexpression of MTP results in increased secretion of VLDL-triglycerides as well as VLDL-apoB in vivo. These results suggest that MTP is rate-limiting for VLDL apoB secretion in wild-type mice under basal chow-fed conditions.     

Suppression of cytosolic triacylglycerol recruitment for very low density lipoprotein assembly by inactivation of microsomal triglyceride transfer protein results in a delayed removal of apoB-48 and apoB-100 from microsomal and Golgi membranes of primary rat hepatocytes.

Cellular apoB in primary rat hepatocyte cultures was pulse-labeled with [(35)S]methionine for 1 h. Cells were then chased with excess unlabeled methionine for periods of up to 16 h in the presence or absence of BMS-200150, an inhibitor of microsomal triglyceride transfer protein (MTP). The secretion of apoB-48-VLDL was more sensitive to MTP inhibition than was apoB-100-VLDL. Inhibition of MTP had no inhibitory effect on the secretion of denser particles (apoB-48 HDL and apoB-100 HDL). BMS-200150 delayed the net removal of newly synthesized apoB-48 and apoB-100 from the microsomal and Golgi membranes, but not from the corresponding lumenal compartments. Only minor proportions of the microsomal lumen apoB-48 and apoB-100 (12-16% and 17-19%, respectively) were present as VLDL irrespective of whether MTP was inactivated or not. The HDL fraction contained most of the lumenal apoB-48 (67-73%) and a somewhat smaller proportion of apoB-100 (44-47%). The remainder of the lumenal apoB was associated with the IDL/LDL fraction. These proportions were unaffected by MTP inactivation. Excess labeled apoB which accumulated in the membranes in the presence of BMS-200150 was degraded. Inhibition of MTP prevented the removal of pre-synthesized triacylglycerol (TAG) from the hepatocytes as apoB-VLDL. Under these conditions intracellular TAG accumulated mainly in the cell cytosol, but also, to a lesser extent, in the microsomal membranes. The results suggest that inactivation of MTP inhibits a pathway of VLDL assembly which does not involve the bulk lumenal compartments of the microsomes. Suppression of this pathway ultimately prevents the net transfer of cytosolic TAG into mature apoB-VLDL.     

N-terminal domain of apolipoprotein B has structural homology to lipovitellin and microsomal triglyceride transfer protein: a "lipid pocket" model for self-assembly of apob-containing lipoprotein particles.

The process of assembly of apolipoprotein (apo) B-containing lipoprotein particles occurs co-translationally after disulfide-dependent folding of the N-terminal domain of apoB but the mechanism is not understood. During a recent database search for protein sequences that contained similar amphipathic beta strands to apoB-100, four vitellogenins, the precursor form of lipovitellin, an egg yolk lipoprotein, from chicken, frog, lamprey, and C. elegans appeared on the list of candidate proteins. The X-ray crystal structure of lamprey lipovitellin is known to contain a "lipid pocket" lined by antiparallel amphipathic beta sheets. Here we report that the first 1000 residues of human apoB-100 (the alpha(1) domain plus the first 200 residues of the beta(1) domain) have sequence and amphipathic motif homologies to the lipid-binding pocket of lamprey lipovitellin. We also show that most of the alpha(1) domain of human apoB-100 has sequence and amphipathic motif homologies to human microsomal triglyceride transfer protein (MTP), a protein required for assembly of apoB-containing lipoproteins. Based upon these results, we suggest that an LV-like "proteolipid" intermediate containing a "lipid pocket" is formed by the N-terminal portion of apoB alone or, more likely, as a complex with MTP. This intermediate produces a lipid nidus required for assembly of apoB-containing lipoprotein particles; pocket expansion through the addition of amphipathic beta strands from the beta(1) domain of apoB results in the formation of a progressively larger high density lipoprotein (HDL)-like, then very low density lipoprotein (VLDL)-like, spheroidal lipoprotein particle.     

Microsomal triglyceride transfer protein expression during mouse development

Feto-maternal transfer of lipophilic nutrients is an important factor in the normal development of the fetus and may be mediated by lipoproteins as carriers of these nutrients. Two proteins that may be important in this process are apolipoprotein B (apoB, the major structural protein of secreted lipoproteins) and microsomal triglyceride transfer protein (MTP) whose normal activity is required for the secretion of apoB-containing lipoproteins. Although no abnormalities of conception and embryonic lethality are known in humans who inherit genetic deficiencies of either of these proteins, homozygous mice bearing knockouts of either apoB or MTP show early embryonic lethality. To characterize the ontogeny of MTP expression during embryonic mouse development, we have used in situ hybridization to characterize the pattern of expression. By using microwave heating of tissue sections to optimize hybridization, we show that there is robust MTP expression in the yolk sac tissues followed by expression in the primordial liver cell nests as early as day 9 post-coitum (E9.5). Intestinal expression is detected around E12.5 and attains full adult expression patterns by E14.5. No expression in any other tissues was observed, including developing heart, kidney, placenta, and maternal decidua.Thus the pattern of MTP expression is compatible with a role in the transfer of lipophilic nutrients from the yolk sac, prior to hepatic development and to the liver, once the circulatory system has been established.     

Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.     

apoA-IV tagged with the ER retention signal KDEL perturbs the intracellular trafficking and secretion of apoB

To examine the role of apolipoprotein A-IV (apoA-IV) in the intracellular trafficking and secretion of apoB, COS cells were cotransfected with microsomal triglyceride transfer protein (MTP), apoB-41 (amino terminal 41% of apoB), and either native apoA-IV or apoA-IV modified with the carboxy-terminal endoplasmic reticulum (ER) retention signal, KDEL (apoA-IV-KDEL). As expected, apoA-IV-KDEL was inefficiently secreted relative to native apoA-IV. Coexpression of apoB-41 with apoA-IV-KDEL reduced the secretion of apoB-41 by approximately 80%. The apoA-IV-KDEL effect was specific, as neither KDEL-modified forms of human serum albumin or apoA-I affected apoB-41 secretion. Similar results were observed in McA-RH7777 rat hepatoma cells, which express endogenous MTP. The full inhibitory effect of apoA-IV-KDEL on apoB secretion was observed only for forms of apoB containing a minimum of the amino-terminal 25% of the protein (apoB-25). However, apoA-IV-KDEL inhibited the secretion of both lipid-associated and lipid-poor forms of apoB-25. Dual-label immunofluorescence microscopy of cells transfected with native apoA-IV and apoB-25 revealed that both apolipoproteins were localized to the ER and Golgi, as expected. However, when apoA-IV-KDEL was cotransfected with apoB-25, both proteins localized primarily to the ER. These data suggest that apoA-IV may physically interact with apoB in the secretory pathway, perhaps reflecting a role in modulating the process of triglyceride-rich lipoprotein assembly and secretion.     

Apolipoprotein B-containing lipoprotein assembly in microsomal triglyceride transfer protein-deficient McA-RH7777 cells

Microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apolipoprotein (apo) B-containing lipoproteins. Previously, we demonstrated that the N-terminal 1,000 residues of apoB (apoB:1000) are necessary for the initiation of apoB-containing lipoprotein assembly in rat hepatoma McA-RH7777 cells and that these particles are phospholipid (PL) rich. To determine if the PL transfer activity of MTP is sufficient for the assembly and secretion of primordial apoB:1000-containing lipoproteins, we employed microRNA-based short hairpin RNAs (miR-shRNAs) to silence Mttp gene expression in parental and apoB:1000-expressing McA-RH7777 cells. This approach led to 98% reduction in MTP protein levels in both cell types. Metabolic labeling studies demonstrated a drastic 90–95% decrease in the secretion of rat endogenous apoB100-containing lipoproteins in MTP-deficient McA-RH7777 cells compared with cells transfected with negative control miR-shRNA. A similar reduction was observed in the secretion of rat endogenous apoB48 under the experimental conditions employed. In contrast, MTP absence had no significant effect on the synthesis, lipidation, and secretion of human apoB:1000-containing particles. These results provide strong evidence in support of the concept that in McA-RH7777 cells, acquisition of PL by apoB:1000 and initiation of apoB-containing lipoprotein assembly, a process distinct from the conventional first-step assembly of HDL-sized apoB-containing particles, do not require MTP. This study indicates that, in hepatocytes, a factor(s) other than MTP mediates the formation of the PL-rich primordial apoB:1000-containing initiation complex.     

Chinese hamster ovary cells require the coexpression of microsomal triglyceride transfer protein and cholesterol 7alpha-hydroxylase for the assembly and secretion of apolipoprotein B-containing lipoproteins

Due to the absence of microsomal triglyceride transfer protein (MTP), Chinese hamster ovary (CHO) cells lack the ability to translocate apoB into the lumen of the endoplasmic reticulum, causing apoB to be rapidly degraded by an N-acetyl-leucyl-leucyl-norleucinal-inhibitable process. The goal of this study was to examine if expression of MTP, whose genetic deletion is responsible for the human recessive disorder abetalipoproteinemia, would recapitulate the lipoprotein assembly pathway in CHO cells. Unexpectedly, expression of MTP mRNA and protein in CHO cells did not allow apoB-containing lipoproteins to be assembled and secreted by CHO cells expressing apoB53. Although expression of MTP in cells allowed apoB to completely enter the endoplasmic reticulum, it was degraded by a proteolytic process that was inhibited by dithiothreitol (1 mM) and chloroquine (100 microM), but resistant to N-acetyl-leucyl-leucyl-norleucinal. In marked contrast, coexpression of the liver-specific gene product cholesterol 7alpha-hydroxylase with MTP resulted in levels of MTP lipid transfer activity that were similar to those in mouse liver and allowed intact apoB53 to be secreted as a lipoprotein particle. These data suggest that, although MTP-facilitated lipid transport is not required for apoB translocation, it is required for the secretion of apoB-containing lipoproteins. We propose that, in CHO cells, MTP plays two roles in the assembly and secretion of apoB-containing lipoproteins: 1) it acts as a chaperone that facilitates apoB53 translocation, and 2) its lipid transfer activity allows apoB-containing lipoproteins to be assembled and secreted. Our results suggest that the phenotype of the cell (e.g. expression of cholesterol 7alpha-hydroxylase by the liver) may profoundly influence the metabolic relationships determining how apoB is processed into lipoproteins and/or degraded.     

Generation of hepatoma cell lines deficient in microsomal triglyceride transfer protein

The microsomal triglyceride transfer protein (MTP) is essential for the secretion of apolipoprotein B (apoB)48- and apoB100-containing lipoproteins in the intestine and liver, respectively. Loss of function mutations in MTP cause abetalipoproteinemia. Heterologous cells are used to evaluate the function of MTP in apoB secretion to avoid background MTP activity in liver and intestine-derived cells. However, these systems are not suitable to study the role of MTP in the secretion of apoB100-containing lipoproteins, as expression of a large apoB100 peptide using plasmids is difficult. Here, we report a new cell culture model amenable for studying the role of different MTP mutations on apoB100 secretion. The endogenous MTTP gene was ablated in human hepatoma Huh-7 cells using single guide RNA and RNA-guided clustered regularly interspaced short palindromic repeats-associated sequence 9 ribonucleoprotein complexes. We successfully established three different clones that did not express any detectable MTTP mRNA or MTP protein or activity. These cells were defective in secreting apoB-containing lipoproteins and accumulated lipids. Furthermore, we show that transfection of these cells with plasmids expressing human MTTP cDNA resulted in the expression of MTP protein, restoration of triglyceride transfer activity, and secretion of apoB100. Thus, these new cells can be valuable tools for studying structure-function of MTP, roles of different missense mutations in various lipid transfer activities of MTP, and their ability to support apoB100 secretion, compensatory changes associated with loss of MTP, and in the identification of novel proteins that may require MTP for their synthesis and secretion.     

Disulfide bonds are required for folding and secretion of apolipoprotein B regardless of its lipidation state

Apolipoprotein (apo) B-100, an essential protein for the assembly and secretion of very low density lipoproteins depends on lipid binding (lipidation) for its secretion. Seven of its 8 disulfides are clustered within the N-terminal 21%. The role of these disulfides in the secretion of lipidated or unlipidated truncated forms of apoB was studied in C127 cells expressing apoB-17, apoB-29, or apoB-41. These cells do not express microsomal triglyceride transfer protein yet secrete apoB-41 on triacylglycerol-rich lipoproteins while apoB-29 and apoB-17 are secreted with little or no lipid, respectively. Dithiothreitol utilized in pulse-chase studies prevented the cotranslational formation of disulfides and when added posttranslationally reduced native disulfides. As a result, the secretion of reduced apoB forms was blocked and they were retained in the cells. Reduced apoB polypeptides were rescued following removal of dithiothreitol, as they underwent post-translational disulfide bonding, attained their mature form, and were subsequently secreted. Together the data suggest that in C127 cells the formation of native disulfides is critical for the folding and secretion of apoB independent of its length, its requirement for lipidation or microsomal triglyceride transfer protein expression. Therefore, these cells provide an appropriate model to study the folding of apoB in great detail.     

The low density lipoprotein receptor prevents secretion of dense apoB100-containing lipoproteins from the liver

The assembly and secretion of very low density lipoproteins (VLDL) require microsomal triglyceride transfer protein (MTP). Recent evidence also suggests a role for the low density lipoprotein (LDL) receptor in this process. However, the relative importance of MTP in the two steps of VLDL assembly and the specific role of the LDL receptor still remain unclear. To further investigate the role of MTP and the LDL receptor in VLDL assembly, we bred mice harboring "floxed" Mttp alleles (Mttpflox/flox) and a Cre transgene on a low-density lipoprotein receptor-deficient background to generate mice with double deficiency in the liver (Ldlr-/- MttpDelta/Delta). In contrast to the plasma of Ldlr+/+ MttpDelta/Delta mice, the plasma of Ldlr-/- MttpDelta/Delta mice contained apoB100. Accordingly, Ldlr-/- MttpDelta/Delta but not Ldlr+/+ MttpDelta/Delta hepatocytes secreted apoB100-containing lipoprotein particles. The secreted lipoproteins were of LDL and HDL sizes but no VLDL-sized lipoproteins could be detected. These findings indicate that hepatic LDL receptors function as "gatekeepers" targeting dense apoB100-containing lipoproteins for degradation. In addition, these results suggest that very low levels of MTP are insufficient to mediate the second step but sufficient for the first step of VLDL assembly.     

A Drosophila microsomal triglyceride transfer protein homolog promotes the assembly and secretion of human apolipoprotein B. Implications for human and insect transport and metabolism

The assembly and secretion of triglyceride-rich lipoproteins in vertebrates requires apolipoprotein B (apoB) and the endoplasmic reticulum-localized cofactor, microsomal triglyceride transfer protein (MTP). Invertebrates, particularly insects, transport the majority of their neutral and polar lipids in lipophorins; however, the assembly of lipophorin precursor particles was presumed to be MTP-independent. A Drosophila melanogaster expressed gene sequence (CG9342), displaying 23% identity with human MTP, was recently identified. When coexpressed in COS cells, CG9342 promoted the assembly and secretion of apoB34 and apoB41 (N-terminal 34 and 41% of human apoB). The apoB34-containing particles assembled by human MTP and CG9342 displayed similar peak densities of approximately 1.169 g/ml and similar lipid compositions. However, CG9342 displayed differential sensitivities to two inhibitors of human MTP and low vesicle-based lipid transfer activity, in vitro. In addition, important predicted structural distinctions exist between the human and Drosophila proteins suggesting overlapping but not identical functional roles. We conclude that CG9342 and human MTP are orthologs that share only a subset of functions, consistent with known differences in intracellular and extracellular aspects of vertebrate and invertebrate lipid transport and metabolism.     

Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly

The microsomal triglyceride transfer protein (MTP) is necessary for the proper assembly of the apolipoprotein B containing lipoproteins, very low density lipoprotein and chylomicrons. Recent research has significantly advanced our understanding of the role of MTP in these pathways at the molecular and cellular level. Biochemical studies suggest that initiation of lipidation of the nascent apolipoprotein B polypeptide may occur through a direct association with MTP. This early lipidation may be required to allow the nascent polypeptide to fold properly and therefore avoid ubiquitination and degradation. Concerning the addition of core neutral lipids in the later stages of lipoprotein assembly, cell culture studies show that MTP lipid transfer activity is not required for this to occur for apolipoprotein B-100 containing lipoproteins. Likewise, MTP does not appear to directly mediate addition of core neutral lipid to nascent apoB-48 particles. However, new data indicate that MTP is required to produce triglyceride rich droplets in the smooth endoplasmic reticulum which may supply the core lipids for conversion of nascent, dense apoB-48 particles to mature VLDL. In addition, assembly of dense apolipoprotein B-48 containing lipoproteins has been observed in mouse liver in the absence of MTP. As a result of these new data, an updated model for the role of MTP in lipoprotein assembly is proposed.     

Blocking microsomal triglyceride transfer protein interferes with apoB secretion without causing retention or stress in the ER

Microsomal triglyceride transfer protein (MTP) is an intraluminal protein in the endoplasmic reticulum (ER) that is essential for the assembly of apolipoprotein B (apoB)-containing lipoproteins. In this study, we examine how the livers of mice respond to two distinct methods of blocking MTP function: Cre-mediated disruption of the gene for MTP and chemical inhibition of MTP activity. Blocking MTP significantly reduced plasma levels of triglycerides, cholesterol, and apoB-containing lipoproteins in both wild-type C57BL/6 and LDL receptor-deficient mice. While treating LDL receptor-deficient mice with an MTP inhibitor for 7 days lowered plasma lipids to control levels, liver triglyceride levels were increased by only 4-fold. Plasma levels of apoB-100 and apoB-48 fell by >90% and 65%, respectively, but neither apoB isoform accumulated in hepatic microsomes. Surprisingly, loss of MTP expression was associated with a nearly complete absence of apoB-100 in hepatic microsomes. Levels of microsomal luminal chaperone proteins [e.g., protein disulfide isomerase, glucose-regulated protein 78 (GRP78), and GRP94] and cytosolic heat shock proteins (HSPs) (e.g., HSP60, HSC, HSP70, and HSP90) were unaffected by MTP inhibition. These findings show that the liver responds rapidly to inhibition of MTP by degrading apoB and preventing its accumulation in the ER. The rapid degradation of secretion-incompetent apoB in the ER may block the induction of proteins associated with unfolded protein and heat shock responses.     

Effects of overexpression of the amino-terminal fragment of apolipoprotein B on apolipoprotein B and lipoprotein production

In vitro studies have shown that the binding site for microsomal triglyceride transfer protein (MTP) is within the first 17% of apoB (apoB-17). Expression of apoB-48 in McArdle cells decreases endogenous lipoprotein production; however, overexpression of human apoB in transgenic mice does not decrease endogenous mouse apoB expression. To assess this inconsistency, adenoviruses expressing human apoB-17 (AdB17) or apoB-17-beta (which contains apoB-17 plus a small lipid-binding beta-sheet region of apoB, AdB-17beta) were produced. Hepatoma cells were infected with AdB17 or AdB17-beta with AdLacZ, an adenovirus expressing beta-galactosidase, as a control. Overexpression of apoB-17 and apoB-17-beta in hepatoma cells to levels 2- to 3-fold greater than that of endogenous apoB did not alter endogenous apoB production. This was also true in the presence of oleic acid and N-acetyl-leucyl-leucyl-norleucinal. High levels of apoB-17 or beta-galactosidase expression reduced apoB-100 production; however, control protein production was also reduced. To assess the effects of apoB-17 expression in vivo, mice of three different strains were injected with AdB17. Two days after injection, plasma apoB-17 was approximately 24 times the amount of endogenous apoB in the C57BL/6 mice, 2 times the apoB-100 in human apoB transgenic mice, and 4 times the apoB-48 in apoE knockout mice. Overexpression of apoB-17 did not decrease apoB-100 or apoB-48 concentrations in mouse plasma as assessed by Western blot analysis. These results demonstrate that although the apoB-17 binds to MTP in vitro, it does not alter endogenous apoB expression in mice or in hepatoma cells.