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.     

Hepatocyte apoB-containing lipoprotein secretion is decreased by the grapefruit flavonoid,naringenin, via inhibition of MTP-mediated microsomal triglyceride accumulation

Naringenin, the principal flavonoid in grapefruit, reduces plasma lipids in vivo and inhibits apoB secretion, cholesterol esterification, and MTP activity in HepG2 human hepatoma cells. Although naringenin inhibits ACAT, we recently demonstrated that CE availability in the microsomal lumen does not regulate apoB secretion in HepG2 cells. We therefore hypothesized that inhibition of TG accumulation in the ER lumen, secondary to MTP inhibition, is the primary mechanism whereby naringenin blocks lipidation and subsequent secretion of apoB. Multicompartmental modeling of pulse-chase studies was used to compare cellular apoB kinetics in the presence of either naringenin or the specific MTP inhibitor, BMS-197636. At concentrations that reduced apoB secretion by 50%, both compounds selectively enhanced degradation via a kinetically defined, rapid, proteasomal pathway to the same extent. Subcellular fractionation experiments revealed that naringenin and BMS-197636 reduced accumulation of newly synthesized TG in the microsomal lumen by 48% and 54%, respectively. Newly synthesized CE accumulation in the lumen was reduced by 80% and 33% with naringenin and BMS-197636, respectively, demonstrating for the first time that MTP is involved in CE accumulation in the microsomal lumen. Reduced TG availability at this initial site of lipoprotein assembly was associated with significant reductions in the secretion of apoB-containing lipoproteins. Both naringenin and BMS-197636 were most effective in reducing secretion of IDL and LDL, but also inhibited secretion of apoB-containing HDL-sized particles. Furthermore, in McA-RH7777-derived cell lines, naringenin reduced secretion of hapoB72 and hapoB100, which require significant assembly with lipid to be secreted, but did not reduce secretion of hapoB17, hapoB23, and hapoB48, which require only minimal lipidation. Taken together, our results indicate that naringenin inhibits the lipidation and subsequent secretion of apoB-containing lipoproteins primarily by limiting the accumulation of TG in the ER lumen, secondary to MTP inhibition.     

Microsomal triglyceride transfer protein activity is not required for the initiation of apolipoprotein B-containing lipoprotein assembly in McA-RH7777 cells

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. (2004) J. Biol. Chem. 279, 39757-39766). In this study we have investigated the putative functional role of MTP in the initial lipidation of apoB:1000 in stable transformants of McA-RH7777 cells. Inhibition of MTP lipid transfer activity by 0.1 microm BMS-197636 and 5, 10, and 20 microm of BMS-200150 had no detectable effect on the synthesis, lipidation, and secretion of apoB:1000-containing particles. Under identical experimental conditions, the synthesis, lipidation, and secretion of endogenous apoB100-containing particles in HepG2 and parental untransfected McA-RH7777 cells were inhibited by 86-94%. BMS-200150 at 40 microm nearly abolished the secretion of endogenous apoB100-containing particles in HepG2 and parental McA-RH cells but caused only 15-20% inhibition in the secretion of apoB: 1000-containing particles. This modest decrease was attributable to the nonspecific effect of a high concentration of this compound on hepatic protein synthesis, as reflected in a similar (20-25%) reduction in albumin secretion. Suppression of MTP gene expression in stable transformants of McA-RH7777 cells by micro-interfering RNA led to 60-70% decrease in MTP mRNA and protein levels, but it had no detectable effect on the secretion of apoB:1000. Our results provide a compelling argument that the initial addition of phospholipids to apoB:1000 and initiation of apoB-containing lipoprotein assembly occur independently of MTP lipid transfer activity.     

The late addition of core lipids to nascent apolipoprotein B100, resulting in the assembly and secretion of triglyceride-rich lipoproteins, is independent of both microsomal triglyceride transfer protein activity and new triglyceride synthesis.

Although microsomal triglyceride transfer protein (MTP) and newly synthesized triglyceride (TG) are critical for co-translational targeting of apolipoprotein B (apoB100) to lipoprotein assembly in hepatoma cell lines, their roles in the later stages of lipoprotein assembly remain unclear. Using N-acetyl-Leu-Leu-norleucinal to prevent proteasomal degradation, HepG2 cells were radiolabeled and chased for 0-90 min (chase I). The medium was changed and cells chased for another 150 min (chase II) in the absence (control) or presence of Pfizer MTP inhibitor CP-10447 (CP). As chase I was extended, inhibition of apoB100 secretion by CP during chase II decreased from 75.9% to only 15% of control (no CP during chase II). Additional studies were conducted in which chase I was either 0 or 90 min, and chase II was in the presence of [(3)H]glycerol and either BSA (control), CP (inhibits both MTP activity and TG synthesis),BMS-1976360-1) (BMS) (inhibits only MTP activity), or triacsin C (TC) (inhibits only TG synthesis). When chase I was 0 min, CP, BMS, and TC reduced apoB100 secretion during chase II by 75.3, 73.9, and 53.9%. However, when chase I was 90 min, those agents reduced apoB100 secretion during chase II by only 16.0, 19.2, and 13.9%. Of note, all three inhibited secretion of newly synthesized TG during chase II by 80, 80, and 40%, whether chase I was 0 or 90 min. In both HepG2 cells and McA-RH7777 cells, if chase I was at least 60 min, inhibition of TG synthesis and/or MTP activity did not affect the density of secreted apoB100-lipoproteins under basal conditions. Oleic acid increased secretion of TG-enriched apoB100-lipoproteins similarly in the absence or presence of either of CP, BMS, or TC. We conclude that neither MTP nor newly synthesized TG is necessary for the later stages of apoB100-lipoprotein assembly and secretion in either HepG2 or McA-RH7777 cells.     

Microsomal triacylglycerol transfer protein is required for lumenal accretion of triacylglycerol not associated with ApoB,as well as for ApoB lipidation

The assembly of very low density lipoproteins in hepatocytes requires the microsomal triacylglycerol transfer protein (MTP). This microsomal lumenal protein transfers lipids, particularly triacylglycerols (TG), between membranes in vitro and has been proposed to transfer TG to nascent apolipoprotein (apo) B in vivo. We examined the role of MTP in the assembly of apoB-containing lipoproteins in cultured murine primary hepatocytes using an inhibitor of MTP. The MTP inhibitor reduced TG secretion from hepatocytes by 85% and decreased the amount of apoB100 in the microsomal lumen, as well as that secreted into the medium, by 70 and 90%, respectively, whereas the secretion of apoB48 was only slightly decreased and the amount of lumenal apoB48 was unaffected. However, apoB48-containing particles formed in the presence of inhibitor were lipid-poor compared with those produced in the absence of inhibitor. We also isolated a pool of apoB-free TG from the microsomal lumen and showed that inhibition of MTP decreased the amount of TG in this pool by approximately 45%. The pool of TG associated with apoB was similarly reduced. However, inhibition of MTP did not directly block TG transfer from the apoB-independent TG pool to partially lipidated apoB in the microsomal lumen. We conclude that MTP is required for TG accumulation in the microsomal lumen and as a source of TG for assembly with apoB, but normal levels of MTP are not required for transferring the bulk of TG to apoB during VLDL assembly in murine hepatocytes.     

The assembly of very low density lipoproteins in rat hepatoma McA-RH7777 cells is inhibited by phospholipase A2 antagonists

In McA-RH7777 cells, the oleate-stimulated assembly and secretion of very low density lipoproteins (VLDL) was associated with enhanced deacylation of phospholipids, which was markedly decreased by inactivation of the cellular phospholipase A(2). Treatment of the cells with antagonists or antisense oligonucleotide of the Ca(2+)-independent phospholipase A(2) (iPLA(2)) significantly inhibited secretion of apoB100-VLDL and triglyceride. Similar inhibitory effect of the iPLA(2) antagonists was observed on apoB48-VLDL secretion, but secretion of high density lipoprotein particles (such as apoAI- and apoB48-high density lipoprotein) or proteins in general was unaffected. The iPLA(2) antagonist did not affect the synthesis of apoB100 or triglyceride, nor did it affect the activities of phospholipase D, phosphatidate phosphohydrolase, or microsomal triglyceride transfer protein. Inactivation of iPLA(2) resulted in impaired apoB100-VLDL assembly as shown by decreased apoB100-VLDL and triglyceride within the microsomal lumen, with concomitant increase in apoB100 association with the microsomal membranes. The inhibitory effect of iPLA(2) antagonists on apoB100-VLDL assembly/secretion could be abated by pretreatment of cells with oleate. Analysis of molecular species of microsomal phosphatidylcholine and phosphatidylethanolamine by electron spray tandem mass spectrometry revealed that the enrichment of oleoyl moieties was altered by the treatment of iPLA(2) antagonist. These results suggest that the oleate-induced VLDL assembly/secretion may depend upon the establishment of membrane glycerolipids enriched in oleoyl chain, a process mediated by the iPLA(2) activity.     

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.     

Translocational status of ApoB in the presence of an inhibitor of microsomal triglyceride transfer protein

Despite numerous studies demonstrating that microsomal triglyceride transfer protein (MTP) activity is critical to apoB secretion, there is still controversy as to whether MTP directly facilitates the translocation of apoB across the membrane of the endoplasmic reticulum (ER) through either the recruitment of lipids and/or chaperone activity. In the present study, a specific inhibitor of MTP (BMS 197636) was utilized in HepG2 cells to investigate whether a direct relationship exists between the translocation of apoB across the ER membrane and the lipid-transferring activity of MTP. Inhibition of MTP (with 10 and 50 nmol/L of the inhibitor) did not significantly affect the translocation of newly synthesized apoB (P = 0.77) or the translocational efficiency of the steady-state apoB mass (P = 0.45), despite a 49% decrease in apoB secretion and increased proteosomal degradation. These results compared well with subcellular fractionation experiments which showed no significant change in the fraction of apoB accumulated in the lumen of isolated microsomes in MTP-treated cells (P = 0.35). In summary, MTP lipid transfer activity does not appear to influence translocational status of apoB, but its inhibition is associated with an increased susceptibility to proteasome-mediated degradation and reduced assembly and secretion of apoB lipoprotein particles.     

Very-low-density lipoprotein assembly and secretion

The assembly of apolipoprotein B (apoB) into VLDL is broadly divided into two steps. The first involves transfer of lipid by the microsomal triglyceride transfer protein (MTP) to apoB during translation. The second involves fusion of apoB-containing precursor particles with triglyceride droplets to form mature VLDL. ApoB and MTP are homologs of the egg yolk storage protein, lipovitellin. Homodimerization surfaces in lipovitellin are reutilized in apoB and MTP to achieve apoB-MTP interactions necessary for first step assembly. Structural modeling predicts a small lipovitellin-like lipid binding cavity in MTP and a transient lipovitellin-like cavity in apoB important for nucleation of lipid sequestration. The formation of triglyceride droplets in the endoplasmic reticulum requires MTP however, their fusion with apoB may be MTP-independent. Second step assembly is modulated by phospholipase D and A2. Phospholipases may prime membrane transport steps required for second step fusion and/or channel phospholipids into a pathway for VLDL triglyceride production. The enzymology of VLDL triglyceride synthesis is still poorly understood; however, it appears that ACAT2 is the sole source of cholesterol esters for VLDL and chylomicron assembly. VLDL production is controlled primarily at the level of presecretory degradation. Recently, it was discovered that the LDL receptor modulates VLDL production through its interactions with nascent VLDL in the secretory pathway.     

微粒体甘油三脂转运蛋白MTP的结构和功能研究概况

微粒体甘油三酯转运蛋白MTP(microsomal triglyceride transfer protein,MTP)首先是从牛的肝细胞微粒体碎片中分离获得的,其作用是加速甘油三脂(triglyceride,TG)、胆固醇(cholesteryl ester,CE)和磷脂酰胆碱(phosphatidylcholine,PC)的转运和细胞或亚细胞膜的生物合成。它后来在肝细胞和小肠的微粒体膜中发现[1],由于它的位置及其转运TG可以推测与血浆脂蛋白中极低密度脂蛋白(very low density lipoprotein,VLDL)和乳糜微粒(chylomicrons,CM)的组装过程有关。     

ApoB100 is required for increased VLDL-triglyceride secretion by microsomal triglyceride transfer protein in ob/ob mice

Microsomal triglyceride transfer protein (Mttp) is a key player in the assembly and secretion of hepatic very low density lipoproteins (VLDL). Here we determined the effects of Mttp overexpression on hepatic triglyceride (TG) and VLDL secretion in leptin-deficient (ob/ob) mice, specifically in relation to apolipoproteinB (apoB) isoforms. We crossed Apobec1(-/-) mice with congenic ob/ob mice to generate apoB100-only ob/ob mice (A-ob/ob). The obesity phenotype in both genotypes was similar, but A-ob/ob mice had greater hepatic TG content. Administration of recombinant adenovirus expressing murine Mttp cDNA (Ad-mMTP) increased hepatic Mttp content and activity and increased hepatic VLDL-TG secretion in A-ob/ob mice. However, despite equivalent overexpression of Mttp, there was no change in VLDL-TG secretion in ob/ob mice in a wild-type Apobec1 background. Metabolic labeling studies in primary hepatocytes from A-ob/ob mice demonstrated that Ad-mMTP increased triglyceride secretion without changing the synthesis and secretion of apoB100, suggesting greater incorporation of TG into existing VLDL particles rather than increased particle number. Ad-mMTP administration failed to increase hepatic VLDL secretion in lean Apobec1(-/-) mice or controls. By contrast, VLDL secretion increased and hepatic TG content decreased following Ad-mMTP administration to human APOB transgenic mice crossed into the Apobec1(-/-) line. These findings demonstrate that Ad-mMTP increases murine hepatic VLDL-TG secretion only in the apoB100 background, and even then only in situations with either increased hepatic TG accumulation or increased apoB100 expression.     

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.     

Intracellular assembly of very low density lipoproteins containing apolipoprotein B100 in rat hepatoma McA-RH7777 cells

Previous studies with McA-RH7777 cells showed a 15-20-min temporal delay in the oleate treatment-induced assembly of very low density lipoproteins (VLDL) after apolipoprotein (apo) B100 translation, suggesting a post-translational process. Here, we determined whether the post-translational assembly of apoB100-VLDL occurred within the endoplasmic reticulum (ER) or in post-ER compartments using biochemical and microscopic techniques. At steady state, apoB100 distributed throughout ER and Golgi, which were fractionated by Nycodenz gradient centrifugation. Pulse-chase experiments showed that it took about 20 min for newly synthesized apoB100 to exit the ER and to accumulate in the cis/medial Golgi. At the end of a subsequent 20-min chase, a small fraction of apoB100 accumulated in the distal Golgi, and a large amount of apoB100 was secreted into the medium as VLDL. VLDL was not detected either in the lumen of ER or in that of cis/medial Golgi where apoB100 was membrane-associated and sensitive to endoglycosidase H treatment. In contrast, VLDL particles were found in the lumen of the distal Golgi where apoB100 was resistant to endoglycosidase H. Formation of lumenal VLDL almost coincided with the appearance of VLDL in the medium, suggesting that the site of VLDL assembly is proximal to the site of secretion. When microsomal triglyceride transfer protein activity was inactivated after apoB had exited the ER, VLDL formation in the distal Golgi and its subsequent secretion was unaffected. Lipid analysis by tandem mass spectrometry showed that oleate treatment increased the masses of membrane phosphatidylcholine (by 68%) and phosphatidylethanolamine (by 27%) and altered the membrane phospholipid profiles of ER and Golgi. Taken together, these results suggest that VLDL assembly in McA-RH7777 cells takes place in compartments at the distal end of the secretory pathway.     

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.     

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.     

Binding of microsomal triglyceride transfer protein to lipids results in increased affinity for apolipoprotein B: evidence for stable microsomal MTP-lipid complexes.

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are known to interact with each other. We evaluated the effect of different lipids on the protein-protein interactions between MTP and apoB100 or its C-terminally truncated forms. Negatively charged lipids decreased protein-protein interactions between apoB and MTP. In contrast, zwitterionic phospholipids enhanced (2-4-fold) the binding of apoB100 to MTP by increasing affinity (1.5-3-fold) between these proteins without affecting the number of binding sites. Similarly, phospholipids augmented (1.5-4-fold) the binding of various C-terminally truncated apoB peptides to MTP. The increased binding was greater for apoB peptides containing lipid-binding domains, such as apoB28 and apoB42. Surprisingly, preincubation of apoB28 with lipid vesicles had no effect on MTP binding. In contrast, incubation of MTP with lipid vesicles resulted in a stable association of MTP with vesicles, and MTP-lipid vesicles bound better (5-fold increase) to LDL than did lipid-free MTP. To determine whether MTP exists stably associated with lipids in cells, microsomal contents from COS cells expressing MTP, HepG2 cells, and mouse liver were ultracentrifuged, and MTP was visualized in different density fractions. MTP was found associated and unassociated with lipids. In contrast, apoB17 and apoB:270-570 were present unassociated with lipids in COS cells. These studies show that the binding of MTP to lipids results in increased affinity for apoB and that stable MTP-lipid complexes exist in the lumen of the endoplasmic reticulum. Protein-protein interactions between apoB and MTP may juxtapose lipids associated with MTP to lipid-binding domains of apoB and facilitate hydrophobic interactions leading to enhance affinity. We speculate that MTP-lipid complexes may serve as nuclei to form "primordial lipoproteins" and may also play a role in the bulk addition of lipids during the "core expansion" of these lipoproteins.     

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.     

Mechanisms of hepatic very low density lipoprotein overproduction in insulin resistance. Evidence for enhanced lipoprotein assembly, reduced intracellular ApoB degradation, and increased microsomal triglyceride transfer protein in a fructose-fed hamster model

A novel animal model of insulin resistance, the fructose-fed Syrian golden hamster, was employed to investigate the mechanisms mediating the overproduction of very low density lipoprotein (VLDL) in the insulin resistant state. Fructose feeding for a 2-week period induced significant hypertriglyceridemia and hyperinsulinemia, and the development of whole body insulin resistance was documented using the euglycemic-hyperinsulinemic clamp technique. In vivo Triton WR-1339 studies showed evidence of VLDL-apoB overproduction in the fructose-fed hamster. Fructose feeding induced a significant increase in cellular synthesis and secretion of total triglyceride (TG) as well as VLDL-TG by primary hamster hepatocytes. Increased TG secretion was accompanied by a 4.6-fold increase in VLDL-apoB secretion. Enhanced stability of nascent apoB in fructose-fed hepatocytes was evident in intact cells as well as in a permeabilized cell system. Analysis of newly formed lipoprotein particles in hepatic microsomes revealed significant differences in the pattern and density of lipoproteins, with hepatocytes derived from fructose-fed hamsters having higher levels of luminal lipoproteins at a density of VLDL versus controls. Immunoblot analysis of the intracellular mass of microsomal triglyceride transfer protein, a key enzyme involved in VLDL assembly, showed a striking 2.1-fold elevation in hepatocytes derived from fructose-fed versus control hamsters. Direct incubation of hamster hepatocytes with various concentrations of fructose failed to show any direct stimulation of its intracellular stability or extracellular secretion, further supporting the notion that the apoB overproduction in the fructose-fed hamster may be related to the fructose-induced insulin resistance in this animal model. In summary, hepatic VLDL-apoB overproduction in fructose-fed hamsters appears to result from increased intracellular stability of nascent apoB and an enhanced expression of MTP, which act to facilitate the assembly and secretion of apoB-containing lipoprotein particles.     

Comparison of the pharmacological profiles of murine antisense oligonucleotides targeting apolipoprotein B and microsomal triglyceride transfer protein

Therapeutic agents that suppress apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) levels/activity are being developed in the clinic to benefit patients who are unable to reach target LDL-C levels with maximally tolerated lipid-lowering drugs. To compare and contrast the metabolic consequences of reducing these targets, murine-specific apoB or MTP antisense oligonucleotides (ASOs) were administered to chow-fed and high fat-fed C57BL/6 or to chow-fed and Western diet-fed LDLr^−/− mice for periods ranging from 2 to 12 weeks, and detailed analyses of various factors affecting fatty acid metabolism were performed. Administration of these drugs significantly reduced target hepatic mRNA and protein, leading to similar reductions in hepatic VLDL/triglyceride secretion. MTP ASO treatment consistently led to increases in hepatic triglyceride accumulation and biomarkers of hepatotoxicity relative to apoB ASO due in part to enhanced expression of peroxisome proliferator activated receptor γ target genes and the inability to reduce hepatic fatty acid synthesis. Thus, although both drugs effectively lowered LDL-C levels in mice, the apoB ASO produced a more positive liver safety profile.