Secretion Of apoB-containing Lipoproteins Research Articles

Intestinal apolipoprotein B secretion is inhibited by the flavonoid quercetin: potential role of microsomal triglyceride transfer protein and diacylglycerol acyltransferase.

Recent studies have yielded evidence that plant flavonoids reduce hepatic lipid and apolipoprotein B (apoB) secretion. However, the possible role of flavonoids in regulating lipid and apoB secretion by the intestine has not been studied. The purpose of our study was to examine the effects of quercetin, a common dietary flavonoid, on TAG and apoB secretion in a human intestinal cell-line, CaCo-2. Differentiated postconfluent CaCo-2 cells grown on filters and pretreated with quercetin for 8 h were shown by ELISA to inhibit basolateral apoB secretion in a dose-dependent manner. At 15 microM, the secretion of both apoB-100 and apoB-48 were inhibited similarly. This effect was shown to be specific, as quercetin did not affect the incorporation of [35S]methionine/cysteine into secreted TCA-precipitable proteins. To determine the mechanism underlying this inhibitory effect, we examined two regulatory points: TAG availability and lipid transfer to the lipoprotein particle. Quercetin inhibited TAG synthesis under both basal and lipid-rich conditions, indicating that lipid availability is a determining factor in the regulation of apoB secretion by quercetin. The reduction was due at least in part to a decrease in diacylglycerol acyltransferase activity. We next examined lipid transfer or lipidation of the lipoprotein particle by analyzing microsomal TAG transfer protein (MTP) activity. Quercetin decreased MTP activity moderately. In summary, the data demonstrated that pharmacological concentrations of quercetin are a potent inhibitor of intestinal apoB secretion and that reduced lipid availability and lipidation in the lipoprotein assembly step are the mechanism for the suppression of apoB-containing lipoprotein secretion by quercetin in CaCo-2 cells.

Hepatic expression of microsomal triglyceride transfer protein and in vivo secretion of triglyceride-rich lipoproteins are increased in obese diabetic mice.

Secondary hyperlipidemia is a major cardiovascular risk factor in individuals with type 2 diabetes. Increased hepatic production of apolipoprotein B (apoB)-containing lipoproteins contributes to the elevated plasma levels, but the mechanism is poorly understood. Recent results have established that microsomal triglyceride transfer protein (MTP) is rate limiting for the assembly and secretion of apoB-containing lipoproteins. To better understand the mechanism of type 2 diabetes-associated hyperlipidemia, we quantified hepatic MTP mRNA levels, hepatic microsomal triglyceride transfer activity, and in vivo triglyceride secretion from the liver in two diabetic mouse models. Obese diabetic (ob/ob) mice had 45% higher (P = 0.006) hepatic MTP mRNA levels, 54% higher (P < 0.0001) microsomal triglyceride transfer activity, and 70% higher (P < 0.0001) in vivo triglyceride secretion rates compared with ob/+ control mice. In contrast, in lean streptozotocin-treated diabetic mice, hepatic MTP mRNA levels were unchanged, whereas microsomal triglyceride transfer activity and in vivo triglyceride secretion rates were marginally decreased. These studies suggest that obesity-induced type 2 diabetes in mice confers increases in hepatic MTP expression and secretion of triglyceride-rich lipoproteins. High blood glucose and altered hepatic expression of sterol regulatory element binding protein genes play a minor role in this diabetic response.

Apolipoprotein B Metabolism: Tracer Kinetics, Models, and Metabolic Studies

ABSTRACTThe study of apolipoprotein (apo) B metabolism is central to our understanding of lipoprotein metabolism. However, the assembly and secretion of apoB-containing lipoproteins is a complex process. Specialized techniques, developed and applied to in vitro and in vivo studies of apoB metabolism, have provided insights into the mechanisms involved in the regulation of this process. Moreover, these studies have important implications for understanding both the pathophysiology as well as the therapeutic options for the dyslipidemias. The purpose of this review is to examine the role of apoB in lipoprotein metabolism and to explore the applications of kinetic analysis and multicompartmental modeling to the study of apoB metabolism. New developments and significant advances over the last decade are discussed.

PPARα deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins

This study investigates the importance of peroxisome proliferator activated receptor α (PPARα) for serum apolipoprotein B (apoB) levels and hepatic secretion of apoB-containing lipoproteins. Total serum apoB and VLDL-apoB levels were higher in female PPARα-null mice compared with female wild-type mice, but no difference was seen in male mice. Furthermore, hepatic triglyceride secretion rate, determined in vivo after Triton WR1339 injection, was 2.4-fold higher in female PPARα-null mice compared with female wild-type mice, but no difference was observed in male mice. However, when fed a high fat diet, male PPARα-null mice displayed 2-fold higher serum levels of apoB and LDL cholesterol compared with male wild-type mice, but triglyceride levels were not affected. Hepatic LDL receptor protein levels were not influenced by PPARα deficiency, gender, or the fat diet. Hepatocyte cultures from female PPARα-null mice (cultured for 4 days in serum free medium) showed 2-fold higher total apoB secretion and increased secretion of apoB-48 VLDL, as well as 2.7-fold larger accumulation of VLDL-triglycerides in the medium compared with wild-type cultures. In conclusion, PPARα-deficient female mice, but not males, display high serum apoB associated with VLDL and increased hepatic triglyceride secretion. Moreover, male PPARα-null mice show increased susceptibility to high fat diet in terms of serum apoB levels.—Lindén, D., M. Alsterholm, H. Wennbo, and J. Oscarsson. PPARα deficiency increases secretion and serum levels of apolipoprotein B-containing lipoproteins.

Increased Production of Apolipoprotein B-containing Lipoproteins in the Absence of Hyperlipidemia in Transgenic Mice Expressing Cholesterol 7α-Hydroxylase

The finding that expression of a cholesterol 7alpha-hydroxylase (CYP7A1) transgene in cultured rat hepatoma cells caused a coordinate increase in lipogenesis and secretion of apoB-containing lipoproteins led to the hypothesis that hepatic production of apoB-containing lipoproteins may be linked to the expression of CYP7A1 (Wang, S.-L., Du, E., Martin, T. D., and Davis, R. A. (1997) J. Biol. Chem. 272, 19351-19358). To examine this hypothesis in vivo, a transgene encoding CYP7A1 driven by the constitutive liver-specific enhancer of the human apoE gene was expressed in C56BL/6 mice. The expression of CYP7A1 mRNA (20-fold), protein ( approximately 10-fold), and enzyme activity (5-fold) was markedly increased in transgenic mice compared with non-transgenic littermates. The bile acid pool of CYP7A1 transgenic mice was doubled mainly due to increased hydrophobic dihydroxy bile acids. In CYP7A1 transgenic mice, livers contained approximately 3-fold more sterol response element-binding protein-2 mRNA. Hepatic expression of mRNAs encoding lipogenic enzymes (i.e. fatty-acid synthase, acetyl-CoA carboxylase, stearoyl-CoA desaturase, squalene synthase, farnesyl-pyrophosphate synthase, 3-hydroxy-3-methylglutaryl-CoA reductase, and low density lipoprotein receptor) as well as microsomal triglyceride transfer protein were elevated approximately 3-5-fold in transgenic mice. CYP7A1 transgenic mice also displayed a >2-fold increase in hepatic production and secretion of triglyceride-rich apoB-containing lipoproteins. Despite the increased hepatic secretion of apoB-containing lipoproteins in CYP7A1 mice, plasma levels of triglycerides and cholesterol were not significantly increased. These data suggest that the 5-fold increased expression of the low density lipoprotein receptor displayed by the livers of CYP7A1 transgenic mice was sufficient to compensate for the 2-fold increase production of apoB-containing lipoproteins. These findings emphasize the important homeostatic role that CYP7A1 plays in balancing the anabolic lipoprotein assembly/secretion pathway with the cholesterol catabolic bile acid synthetic pathway.

2000 George Lyman Duff Memorial Lecture: atherosclerosis is a liver disease of the heart.

The production of apolipoprotein B (apoB)-containing lipoproteins by the liver is regulated by a complex series of processes involving apoB being cotranslationally translocated across the endoplasmic reticulum and assembled into a lipoprotein particle. The translocation of apoB across the endoplasmic reticulum is facilitated by the intraluminal chaperone, microsomal triglyceride transfer protein (MTP). MTP facilitates the translocation and folding of apoB, as well as the addition of lipid to lipid-binding domains (which consist of amphipathic beta sheets and alpha helices). In the absence of MTP or sufficient lipid, apoB exhibits translocation arrest. Thus, apoB translation, translocation, and assembly with lipids to form a core-containing lipoprotein particle occur as concerted processes. Abrogation of >/=1 of these processes diverts apoB into a degradation pathway that is dependent on conjugation with ubiquitin and proteolysis by the proteasome. The nascent core-containing lipoprotein particle that forms within the lumen of the endoplasmic reticulum can be "enlarged" to form a mature very low density lipoprotein particle. Additional studies show that the assembly and secretion of apoB-containing lipoproteins are linked to the cholesterol/bile acid synthetic pathway controlled by cholesterol 7alpha-hydroxylase. Studies in cultured cells and transgenic mice indicate that the expression of cholesterol 7alpha-hydroxylase indirectly regulates the expression of lipogenic enzymes through changes in the cellular content of mature sterol response element binding proteins. Oxysterols and bile acids may also act via the ligand-activated nuclear receptors LXR and FXR to link the metabolic pathways controlling energy balance and lipid metabolism to nutritional state.

A Mechanism of Membrane Neutral Lipid Acquisition by the Microsomal Triglyceride Transfer Protein

The microsomal triglyceride transfer protein (MTP) and apolipoprotein B (apoB) belong to the vitellogenin (VTG) family of lipid transfer proteins. MTP is essential for the intracellular assembly and secretion of apoB-containing lipoproteins, the key intravascular lipid transport proteins in vertebrates. We report the predicted three-dimensional structure of the C-terminal lipid binding cavity of MTP, modeled on the crystal structure of the lamprey VTG gene product, lipovitellin. The cavity in MTP resembles those found in the intracellular lipid-binding proteins and bactericidal/permeability-increasing protein. Two conserved helices, designated A and B, at the entrance to the MTP cavity mediate lipid acquisition and binding. Helix A (amino acids 725-736) interacts with membranes in a manner similar to viral fusion peptides. Mutation of helix A blocks the interaction of MTP with phospholipid vesicles containing triglyceride and impairs triglyceride binding. Mutations of helix B (amino acids 781-786) and of N780Y, which causes abetalipoproteinemia, have no impact on the interaction of MTP with phospholipid vesicles but impair triglyceride binding. We propose that insertion of helix A into lipid membranes is necessary for the acquisition of neutral lipids and that helix B is required for their transfer to the lipid binding cavity of MTP.

APOLIPOPROTEIN B: mRNA editing, lipoprotein assembly, and presecretory degradation.

Apolipoprotein (apo)B circulates in two distinct forms, apoB100 and apoB48. Human liver secretes apoB100, the product of a large mRNA encoding 4536 residues. The small intestine of all mammals secretes apoB48, which arises following C-to-U deamination of a single cytidine base in the nuclear apoB transcript, introducing a translational stop codon. This process, referred to as apoB RNA editing, operates through a multicomponent enzyme complex that contains a single catalytic subunit, apobec-1, in addition to other protein factors that have yet to be cloned. ApoB RNA editing also exhibits stringent cis-acting requirements that include both structural and sequence-specific elements-specifically efficiency elements that flank the minimal cassette, an AU-rich RNA context, and an 11-nucleotide mooring sequence-located in proximity to a suitably positioned (usually upstream) cytidine. C-to-U RNA editing may become unconstrained under circumstances where apobec-1 is overexpressed, in which case multiple cytidines in apoB RNA, as well as in other transcripts, undergo C-to-U editing. ApoB RNA editing is eliminated following targeting of apobec-1, establishing that there is no genetic redundancy in this function. Under physiological circumstances, apoB RNA editing exhibits developmental, hormonal, and nutritional regulation, in some cases related to transcriptional regulation of apobec-1 mRNA. ApoB and the microsomal triglyceride transfer protein (MTP) are essential for the assembly and secretion of apoB-containing lipoproteins. MTP functions by transferring lipid to apoB during its translation and by transporting triglycerides into the endoplasmic reticulum to form apoB-free lipid droplets. These droplets fuse with nascent apoB-containing particles to form mature, very low-density lipoproteins or chylomicrons. In cultured hepatic cells, lipid availability dictates the rate of apoB production. Unlipidated or underlipidated forms of apoB are subjected to presecretory degradation, a process mediated by retrograde transport from the lumen of the endoplasmic reticulum to the cytosol, coupled with multiubquitination and proteasomal degradation. Although control of lipid secretion in vivo is primarily achieved at the level of lipoprotein particle size, regulation of apoB production by presecretory degradation may be relevant in some dyslipidemic states.

Role of acyl-coenzyme A:cholesterol acyltransferase-1 in the control of hepatic very low density lipoprotein secretion and low density lipoprotein receptor expression in the mouse and hamster.

Cholesteryl esters present in nascent very low density lipoproteins are generated in a reaction catalyzed by acyl CoA:cholesterol acyltransferase (ACAT). To examine the effect of cholesteryl esters on the secretion of apoB-containing lipoproteins, we transiently overexpressed human (h) ACAT-1 in the livers of low density lipoprotein (LDL) receptor(-/-) mice using adenovirus-mediated gene transfer. Overexpression of hACAT-1 increased hepatic total and esterified cholesterol but did not reduce hepatic free cholesterol due to a compensatory increase in the rate of de novo cholesterol synthesis. Overexpression of hACAT-1 markedly increased the plasma concentration and hepatic secretion of apoB-containing lipoproteins but had no effect on the clearance of very low density lipoprotein-apoB from plasma indicating that cholesteryl esters play an important role in regulating the assembly and secretion of apoB-containing lipoproteins. ACAT activity has been implicated in the regulation of the LDL receptor pathway by dietary fatty acids. It has been hypothesized that unsaturated fatty acids, by enhancing ACAT activity, reduce the amount of free cholesterol in a putative regulatory pool that feeds back on LDL receptor expression. We directly tested this hypothesis in hamsters by transiently overexpressing hACAT-1 in the liver. Enhanced cholesterol esterification in the liver resulted in a compensatory increase in de novo cholesterol synthesis but no induction of LDL receptor expression suggesting that fatty acids regulate LDL receptor expression via a mechanism independent of ACAT.

The effects of fatty acids on apolipoprotein B secretion by human hepatoma cells (HEP G2)

We have investigated the effect of fatty acids on the rate of apolipoprotein B (apo B) secretion by human hepatoma cells (Hep G2). When Hep G2 cells were maintained in tissue culture flasks oleic acid up to 0.4 mM increased apo B secretion in a dose-dependent manner, whereas increases in triacylglycerol (TG) were smaller and dose dependency was less evident. In the absence of oleic acid, apo B accumulating in the tissue culture medium was predominantly in lipoproteins of higher density than very low density lipoproteins (VLDL). However, when the rate of secretion was stimulated with oleic acid the apo B-containing lipoproteins became lower in density. We postulated that there was a high rate of lipolysis of newly secreted VLDL by Hep G2 cells, which would account both for the relatively smaller effect of oleic acid on TG as opposed to apo B accumulating in the culture medium and the predominance of apo B in lipoproteins of a higher density than VLDL, which became less evident when VLDL secretory rates were stimulated by oleic acid. To test this hypothesis, cultured Hep G2 cells were transferred to columns containing Cytodex beads, permitting their continuous perfusion with culture medium so that newly secreted VLDL did not remain in contact with the cells. Apo B recovered from the perfusate was largely in VLDL range lipoproteins and the TG measured in the perfusate indicated that the true secretory rate of TG-rich lipoproteins was substantially higher than had been reflected by TG accumulating in culture medium left in contact with cells. Apo B measured in the culture medium of Hep G2 cells may thus be a better reflection of VLDL secretion, even though it is contained in higher density lipoproteins due to removal of TG by lipolysis. The effects of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) on apo B (apo B) secretion by Hep G2 cells maintained in tissue culture flasks were next investigated. SFA (0.4 mM), with the exception of stearic acid (C18:0), increased apo B secretion. Lauric acid (C12:0) increased apo B secretion by 32%, myristic acid (C14:0) by 41% ( P<0.005), palmitic acid (C16:0) by 154% ( P<0.025), and arachidic acid (C20:0) by 186% ( P<0.005). The effect of MUFA (0.4 mM) was to increase apo B secretion, oleic acid (C18:1) by 239% (( P<0.0005) and palmitoleic acid (C16: 1) by 125% ( P<0.005). Of the PUFA investigated, linolenic acid (C18:3) (0.4 mM) did not have any significant effect on apo B secretion, whereas linoleic acid (C18:2) (0.4mM) arachidonic acid (C20:4) (0.1 mM) and eicosapentaenoic acid (C20:5) (0.1 mM) caused significant increases of 164, 171 and 171%, respectively ( P<0.005). The fatty acids studied increased intracellular TG and cholesteryl ester concentrations to varying extents. The increase in intracellular TG produced by the different fatty acids correlated with the rate of apo B secretion ( r=0.6; P<0.05). In this human hepatoma cell line, with the exception of the saturated fatty acids, the rate of secretion of apo B-containing lipoproteins does not follow the same pattern as changes in circulating low density lipoprotein (LDL) concentrations reported with dietary manipulation in man. If our findings reflect the in vivo situation, we suggest that whilst the dietary effects of SFA on serum LDL may in part be determined by the hepatic apo B secretory rate, the effects of MUFA and PUFA must be largely mediated through a catabolic effect rather than an effect on hepatic secretion. The marked increase in apo B secretion with the more highly polyunsaturated fatty acids, such as eicosapentaenoic acid, may also explain why they do not lower circulating LDL, despite reports of their apparently favourable effect on LDL-receptor mediated clearance.

In vivo metabolism of ApoB, ApoA-I, and VLDL triglycerides in a form of hypobetalipoproteinemia not linked to the ApoB gene.

Familial hypobetalipoproteinemia (FHBL) is an autosomal codominant disorder that may result from different mutations in the apolipoprotein B (apoB) gene or chromosome 2. However, linkage of FHBL to the apoB gene was ruled out in 2 kindreds reported to date, and the genetic and metabolic bases for FHBL remain unknown. One of the reported kindreds is our 40-member F kindred, in which we found linkage of FHBL to a novel susceptibility region on chromosome 3p21. 1-2. In addition to having low apoB levels, some, but not all, of the affected subjects in the F kindred also had low levels of high density lipoprotein (HDL) cholesterol and apoA-I. Our aim was to define the metabolic bases of the disorder in the F kindred. Therefore, we studied the in vivo kinetics of apoB and apoA-I and very low density lipoprotein (VLDL) triglycerides in 4 affected subjects and 5 normolipidemic relatives. Deuterated leucine and deuterated glycerol were used to label the apolipoproteins and triglycerides, respectively. Compartmental modeling was used to obtain the kinetic parameters. Affected subjects had (1) normal fractional catabolic rates (FCRs) for VLDL apoB, (2) increased FCRs for low density lipoprotein (LDL) apoB (0.050+/-0.009 versus 0. 030+/-0.006 pools per hour for normal subjects, P=0.005), and (3) decreased production rates of VLDL apoB (11.4+/-1.7 versus 25.6+/-4. 9 mg. kg(-1). d(-1), P=0.003), LDL apoB (7.8+/-1.3 versus 12.7+/-3.7 mg. kg(-1). d(-1), P=0.04), and VLDL triglycerides (8.2+/-4.5 versus 19.6+/-10.8 58 micromol. kg(-1). h(-1), P=0.09). These data differ from those obtained in previously studied FHBL heterozygotes bearing apoB-2 and apoB-9, 2 very short truncations of apoB. Low HDL cholesterol and apoA-I levels were caused by higher apoA-I FCRs (0. 035+/-0.005 versus 0.018+/-0.005 pools per hour in controls, P<0.01) without significant decrease in apoA-I production rates (18.7+/-2.7 versus 22.8+/-5.6 mg. kg(-1). d(-1)). In conclusion, decreased secretion of apoB-containing lipoproteins and hypercatabolism of LDL account for low apoB and cholesterol levels in this novel form of FHBL.

Decreased secretion of ApoB follows inhibition of ApoB-MTP binding by a novel antagonist.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly of triglyceride-rich lipoproteins. Evidence has been presented for physical interactions between these proteins. To study the importance of apoB-MTP binding in apoB secretion, we have identified a compound, AGI-S17, that inhibited (60-70% at 40 microM) the binding of various apoB peptides to MTP but not to an anti-apoB monoclonal antibody, 1D1, whose epitope overlaps with an MTP binding site in apoB. AGI-S17 had no significant effect on the lipid transfer activity of the purified MTP. In contrast, another antagonist, BMS-200150, did not affect apoB-MTP binding but inhibited MTP's lipid transfer activity. The differential effects of these inhibitors suggest two functionally independent, apoB binding and lipid transfer, domains in MTP. AGI-S17 was then used to study its effect on the lipid transfer and apoB binding activities of MTP in HepG2 cells. AGI-S17 had no effect on cellular lipid transfer activities, but it inhibited coimmunoprecipitation of apoB with MTP. These studies indicate that AGI-S17 inhibits apoB-MTP binding but has no effect on MTP's lipid transfer activity. Experiments were then performed to study the effect of inhibition of apoB-MTP binding on apoB secretion in HepG2 cells. AGI-S17 (40 microM) did not affect cell protein levels but decreased the total mass of apoB secreted by 70-85%. Similarly, AGI-S17 inhibited the secretion of nascent apoB by 60-80%, but did not affect albumin secretion. These studies indicate that AGI-S17 decreases apoB secretion most likely by inhibiting apoB-MTP interactions. Thus, the binding of MTP to apoB may be important for the assembly and secretion of apoB-containing lipoproteins and can be a potential target for the development of lipid-lowering drugs. It is proposed that the apoB binding may represent MTP's chaperone activity that assists in the transfer from the membrane to the lumen of the endoplasmic reticulum and in the net lipidation of nascent apoB, and may be essential for lipoprotein assembly and secretion.

Efficient glycosylation site utilization by intracellular apolipoprotein B: implications for proteasomal degradation

The balance between the hepatic assembly of apolipoprotein B (apoB) and its presecretory degradation at the level of the endoplasmic reticulum (ER) may control the secretion of apoB-containing lipoproteins. In one model, apoB that fails to assemble with lipid undergoes translocation arrest, exposing the protein to the cytosolic proteasome. To examine apoB's translocation behavior under various metabolic conditions, glycosylation site utilization studies were performed. A 70-amino acid peptide containing three sites for N-linked glycosylation was appended to the C-terminus of apoB-50 (amino-terminal 50% of apoB) and expressed in both hepatic and nonhepatic cell lines. When the C-terminal reporter peptide was released by cyanogen bromide cleavage, all of the sites were glycosylated irrespective of cell type, labeling time, or assembly status. Similar peptide mapping of endogenous apoB-100 expressed in HepG2 cells was performed to monitor glycosylation at Asn residues 2752 (apoB-61), 2955 (apoB-65), and 3074 (apoB-68). N-linked glycosylation occurred at a minimum of two of the three sites, a frequency identical to that observed in apoB-100 recovered from cell media. Treatment of cells with proteasome inhibitors produced a 2.5-fold increase in intracellular apoB but failed to cause accumulation of an unglycosylated form.▪ These results indicate that 1) the efficient translocation of apoB into the ER occurs independently of microsomal triglyceride transfer protein and its assembly with lipid and 2) despite its large size and affinity for lipid, delivery of misassembled apoB to the proteasome requires retrograde translocation from the ER lumen to cytosol.—Huang, X. F., and G. S. Shelness. Efficient glycosylation site utilization by intracellular apolipoprotein B: implications for proteasomal degradation.

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.

Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase

The cDNA encoding rat betaine-homocysteine S-methyltransferase (BHMT) was isolated through production of monoclonal antibodies against protein fractions enriched with apolipoprotein B (apo B)-mRNA-editing complexes. BHMT mRNA was expressed predominantly in liver, and also in kidney, but not in small intestine. In stable McArdle RH-7777 (McA) cell lines expressing differing levels of BHMT, the editing efficiency of apo B mRNA was unchanged. Evaluation of apo B-mRNA expression revealed that steady-state levels were increased significantly and in parallel with BHMT protein expression. The highest levels of BHMT mRNA and BHMT enzyme activity expressed in stably transfected McA cells were comparable with those found in rat hepatocytes. In contrast to the changes in apo B-mRNA abundance, levels of other apolipoprotein-encoding mRNAs and several liver-specific and ubiquitously expressed mRNAs were unchanged by BHMT expression. In the cell line expressing the highest level of BHMT, apo B-containing lipoprotein secretion was increased, indicating utilization of increased endogenous message. Results suggest that apo B-mRNA abundance in McA cells is related to the expression of BHMT, an enzyme important in homocysteine metabolism.

Apolipoprotein B mRNA and lipoprotein secretion are increased in McArdle RH-7777 cells by expression of betaine-homocysteine S-methyltransferase

The cDNA encoding rat betaine-homocysteine S-methyltransferase (BHMT) was isolated through production of monoclonal antibodies against protein fractions enriched with apolipoprotein B (apo B)-mRNA-editing complexes. BHMT mRNA was expressed predominantly in liver, and also in kidney, but not in small intestine. In stable McArdle RH-7777 (McA) cell lines expressing differing levels of BHMT, the editing efficiency of apo B mRNA was unchanged. Evaluation of apo B-mRNA expression revealed that steady-state levels were increased significantly and in parallel with BHMT protein expression. The highest levels of BHMT mRNA and BHMT enzyme activity expressed in stably transfected McA cells were comparable with those found in rat hepatocytes. In contrast to the changes in apo B-mRNA abundance, levels of other apolipoprotein-encoding mRNAs and several liver-specific and ubiquitously expressed mRNAs were unchanged by BHMT expression. In the cell line expressing the highest level of BHMT, apo B-containing lipoprotein secretion was increased, indicating utilization of increased endogenous message. Results suggest that apo B-mRNA abundance in McA cells is related to the expression of BHMT, an enzyme important in homocysteine metabolism.

Chinese Hamster Ovary Cells Require the Coexpression of Microsomal Triglyceride Transfer Protein and Cholesterol 7α-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.

ApoB100 secretion from HepG2 cells is decreased by the ACAT inhibitor CI-1011: an effect associated with enhanced intracellular degradation of ApoB.

The concept that hepatic cholesteryl ester (CE) mass and the rate of cholesterol esterification regulate hepatocyte assembly and secretion of apoB-containing lipoproteins remains controversial. The present study was carried out in HepG2 cells to correlate the rate of cholesterol esterification and CE mass with apoB secretion by CI-1011, an acyl CoA:cholesterol acyltransferase (ACAT) inhibitor that is known to decrease apoB secretion, in vivo, in miniature pigs. HepG2 cells were incubated with CI-1011 (10 nmol/L, 1 micromol/L, and 10 micromol/L) for 24 hours. ApoB secretion into media was decreased by 25%, 27%, and 43%, respectively (P<0.0012). CI-1011 (10 micromol/L) inhibited HepG2 cell ACAT activity by 79% (P<0.002) and cellular CE mass by 32% (P<0.05). In contrast, another ACAT inhibitor, DuP 128 (10 micromol/L), decreased cellular ACAT activity and CE mass by 85% (P<0.002) and 42% (P=0.01), respectively, but had no effect on apoB secretion into media. To characterize the reduction in apoB secretion by CI-1011, pulse-chase experiments were performed and analyzed by multicompartmental modelling using SAAM II. CI-1011 did not affect the synthesis of apoB or albumin. However, apoB secretion into the media was decreased by 42% (P=0.019). Intracellular apoB degradation increased proportionately (P=0.019). The secretion of albumin and cellular reuptake of labeled lipoproteins were unchanged. CI-1011 and DuP 128 did not affect apoB mRNA concentrations. These results show that CI-1011 decreases apoB secretion by a mechanism that involves an enhanced intracellular degradation of apoB. This study demonstrates that ACAT inhibitors can exert differential effects on apoB secretion from HepG2 cells that do not reflect their efficacy in inhibiting cholesterol esterification.

Identification of Domains in Apolipoprotein B100 That Confer a High Requirement for the Microsomal Triglyceride Transfer Protein

The microsomal triglyceride transfer protein (MTP) is required for the assembly and secretion of apoB-containing lipoproteins. To investigate the role of MTP in lipoprotein assembly, we determined the ability of carboxyl-terminally truncated forms of apoB to be secreted from cells treated with the MTP inhibitor 4'-bromo-3'-methylmetaqualone (Benoist, F., Nicodeme, E., and Grand-Perret, T. (1996) Eur. J. Biochem. 240, 713-720). In Caco-2 and mhAT3F cells that produce apoB100 and apoB48, the inhibitor preferentially blocked apoB100 secretion. When the inhibitor was tested on McA-RH7777 cells stably transfected with cDNAs encoding human apoB100, apoB72, apoB53, apoB29, and apoB18, the secretion of apoB100, apoB72, and apoB53 was preferentially impaired relative to apoB48 and shorter forms. To delineate the region between apoB48 and apoB53 that has a high requirement for MTP, we used puromycin to generate a range of truncated forms of apoB in HepG2 cells. The secretion of apoB53 and longer forms of apoB was markedly affected by low concentrations of the MTP inhibitor (approximately 1 microM), whereas apoB51 and smaller forms of apoB were only affected at higher concentrations (> 10 microM). The size-related sensitivity to MTP inhibitor was not due to late processing or retention, since the same result was observed when nascent lipoproteins were isolated from the endoplasmic reticulum. The MTP inhibitor did not alter the density of the secreted lipoproteins, indicating that each apoB polypeptide requires a minimally defined amount of lipid to attain a secretable conformation. Our results suggest that the folding of the domain between apoB51 and apoB53 has a high requirement for lipid. This domain is predicted to form amphipathic alpha-helices and to bind lipid reversibly. It proceeds and is followed by rigid amphipathic beta-sheets that are predicted to associate with lipid irreversibly. We speculate that these domains enable apoB to switch from a stable lipid-poor conformation in apoB48 to another lipid-rich conformation in apoB100 during lipoprotein assembly.

A Common Binding Site on the Microsomal Triglyceride Transfer Protein for Apolipoprotein B and Protein Disulfide Isomerase

The assembly of triglyceride-rich lipoproteins requires the formation in the endoplasmic reticulum of a complex between apolipoprotein B (apoB), a microsomal triglyceride transfer protein (MTP), and protein disulfide isomerase (PDI). In the MTP complex, the amino-terminal region of MTP (residues 22-303) interacts with the amino-terminal region of apoB (residues 1-264). Here, we report the identification and characterization of a site on apoB between residues 512 and 721, which interacts with residues 517-603 of MTP. PDI binds in close proximity to this apoB binding site on MTP. The proximity of these binding sites on MTP for PDI and amino acids 512-721 of apoB was evident from studies carried out in a yeast two-hybrid system and by co-immunoprecipitation. The expression of PDI with MTP and apoB16 (residues 1-721) in the baculovirus expression system reduced the amount of MTP co-immunoprecipitated with apoB by 73%. The interaction of residues 512-721 of apoB with MTP facilitates lipoprotein production. Mutations of apoB that markedly reduced this interaction also reduced the level of apoB-containing lipoprotein secretion.