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.     

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.     

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.     

The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions

Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.     

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

This study investigates the importance of peroxisome proliferator activated receptor alpha (PPARalpha) for serum apolipoprotein B (apoB) levels and hepatic secretion of apoB-containing lipoproteins. Total serum apoB and VLDL-apoB levels were higher in female PPARalpha-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 PPARalpha-null mice compared with female wild-type mice, but no difference was observed in male mice. However, when fed a high fat diet, male PPARalpha-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 PPARalpha deficiency, gender, or the fat diet. Hepatocyte cultures from female PPARalpha-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, PPARalpha-deficient female mice, but not males, display high serum apoB associated with VLDL and increased hepatic triglyceride secretion. Moreover, male PPARalpha-null mice show increased susceptibility to high fat diet in terms of serum apoB levels.     

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.     

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.     

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.     

Human placenta secretes apolipoprotein B-100-containing lipoproteins

Supply of lipids from the mother is essential for fetal growth and development. In mice, disruption of yolk sac cell secretion of apolipoprotein (apo) B-containing lipoproteins results in embryonic lethality. In humans, the yolk sac is vestigial. Nutritional functions are instead established very early during pregnancy in the placenta. To examine whether the human placenta produces lipoproteins, we examined apoB and microsomal triglyceride transfer protein (MTP) mRNA expression in placental biopsies. ApoB and MTP are mandatory for assembly and secretion of apoB-containing lipoproteins. Both genes were expressed in placenta and microsomal extracts from human placenta contained triglyceride transfer activity, indicating expression of bioactive MTP. To detect lipoprotein secretion, biopsies from term placentas were placed in medium with [(35)S]methionine and [(35)S]cysteine for 3-24 h. Upon sucrose gradient ultracentrifugation of the labeled medium, fractions were analyzed by apoB-immunoprecipitation. (35)S-labeled apoB-100 was recovered in d approximately 1.02-1.04 g/ml particles (i.e. similar to the density of plasma low density lipoproteins). Electron microscopy of negatively stained lipoproteins secreted from placental tissue showed spherical particles with a diameter of 47 +/- 10 nm. These results demonstrate that human placenta expresses both apoB and MTP and consequently synthesize and secrete apoB-100-containing lipoproteins. Placental lipoprotein formation constitutes a novel pathway of lipid transfer from the mother to the developing fetus.     

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.     

Microsomal triglyceride transfer protein enhances cellular cholesteryl esterification by relieving product inhibition

Cholesteryl ester synthesis by the acyl-CoA:cholesterol acyltransferase enzymes ACAT1 and ACAT2 is, in part, a cellular homeostatic mechanism to avoid toxicity associated with high free cholesterol levels. In hepatocytes and enterocytes, cholesteryl esters are secreted as part of apoB lipoproteins, the assembly of which is critically dependent on microsomal triglyceride transfer protein (MTP). Conditional genetic ablation of MTP reduces cholesteryl esters and enhances free cholesterol in the liver and intestine without diminishing ACAT1 and ACAT2 mRNA levels. As expected, increases in hepatic free cholesterol are associated with decreases in 3-hydroxy-3-methylglutaryl-CoA reductase and increases in ATP-binding cassette transporter 1 mRNA levels. Chemical inhibition of MTP also decreases esterification of cholesterol in Caco-2 and HepG2 cells. Conversely, coexpression of MTP and apoB in AC29 cells stably transfected with ACAT1 and ACAT2 increases cholesteryl ester synthesis. Liver and enterocyte microsomes from MTP-deficient animals synthesize lesser amounts of cholesteryl esters in vitro, but addition of purified MTP and low density lipoprotein corrects this deficiency. Enrichment of microsomes with cholesteryl esters also inhibits cholesterol ester synthesis. Thus, MTP enhances cellular cholesterol esterification by removing cholesteryl esters from their site of synthesis and depositing them into nascent apoB lipoproteins. Therefore, MTP plays a novel role in regulating cholesteryl ester biosynthesis in cells that produce lipoproteins. We speculate that non-lipoprotein-producing cells may use different mechanisms to alleviate product inhibition and modulate cholesteryl ester biosynthesis.     

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.     

Lysophosphatidylcholine acyltransferase 3 knockdown-mediated liver lysophosphatidylcholine accumulation promotes very low density lipoprotein production by enhancing microsomal triglyceride transfer protein expression

After de novo biosynthesis phospholipids undergo extensive remodeling by the Lands' cycle. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in reacylation of lysophosphopholipids. One key enzyme in the Lands' cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT), which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. In this study we found that LPCAT3 is the major hepatic isoform, and its knockdown significantly reduces hepatic LPCAT activity. Moreover, we report that hepatic LPCAT3 knockdown increases certain species of LysoPCs and decreases certain species of PC. A surprising observation was that LPCAT3 knockdown significantly reduces hepatic triglycerides. Despite this, these mice had higher plasma triglyceride and apoB levels. Lipoprotein production studies indicated that reductions in LPCAT3 enhanced assembly and secretion of triglyceride-rich apoB-containing lipoproteins. Furthermore, these mice had higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration-dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.     

Liver-specific inactivation of the abetalipoproteinemia gene completely abrogates very low density lipoprotein/low density lipoprotein production in a viable conditional knockout mouse

Conventional knockout of the microsomal triglyceride transfer protein large subunit (lMTP) gene is embryonic lethal in the homozygous state in mice. We have produced a conditional lMTP knockout mouse by inserting loxP sequences flanking exons 5 and 6 by gene targeting. Homozygous floxed mice were born live with normal plasma lipids. Intravenous injection of an adenovirus harboring Cre recombinase (AdCre1) produced deletion of exons 5 and 6 and disappearance of lMTP mRNA and immunoreactive protein in a liver-specific manner. There was also disappearance of plasma apolipoprotein (apo) B-100 and marked reduction in apoB-48 levels. Wild-type mice showed no response, and heterozygous mice, an intermediate response, to AdCre1. Wild-type mice doubled their plasma cholesterol level following a high cholesterol diet. This hypercholesterolemia was abolished in AdCre1-treated lMTP-/- mice, the result of a complete absence of very low/intermediate/low density lipoproteins and a slight reduction in high density lipoprotein. Heterozygous mice showed an intermediate lipoprotein phenotype. The rate of accumulation of plasma triglyceride following Triton WR1339 treatment in lMTP-/- mice was <10% that in wild-type animals, indicating a failure of triglyceride-rich lipoprotein production. Pulse-chase experiments using hepatocytes isolated from wild-type and lMTP-/- mice revealed a failure of apoB secretion in lMTP-/- animals. Therefore, the liver-specific inactivation of the lMTP gene completely abrogates apoB-100 and very low/intermediate/low density lipoprotein production. These conditional knockout mice are a useful in vivo model for studying the role of MTP in apoB biosynthesis and the biogenesis of apoB-containing lipoproteins.     

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.     

An apolipoprotein B antisense oligonucleotide lowers LDL cholesterol in hyperlipidemic mice without causing hepatic steatosis

High levels of plasma apolipoprotein B-100 (apoB-100), the principal apolipoprotein of LDL, are associated with cardiovascular disease. We hypothesized that suppression of apoB-100 mRNA by an antisense oligonucleotide (ASO) would reduce LDL cholesterol (LDL-C). Because most of the plasma apoB is made in the liver, and antisense drugs distribute to that organ, we tested the effects of a mouse-specific apoB-100 ASO in several mouse models of hyperlipidemia, including C57BL/6 mice fed a high-fat diet, Apoe-deficient mice, and Ldlr-deficient mice. The lead apoB-100 antisense compound, ISIS 147764, reduced apoB-100 mRNA levels in the liver and serum apoB-100 levels in a dose- and time-dependent manner. Consistent with those findings, total cholesterol and LDL-C decreased by 25-55% and 40-88%, respectively. Unlike small-molecule inhibitors of microsomal triglyceride transfer protein, ISIS 147764 did not produce hepatic or intestinal steatosis and did not affect dietary fat absorption or elevate plasma transaminase levels. These findings, as well as those derived from interim phase I data with a human apoB-100 antisense drug, suggest that antisense inhibition of this target may be a safe and effective approach for the treatment of humans with hyperlipidemia.     

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.     

The assembly and secretion of apolipoprotein B-48-containing very low density lipoproteins in McA-RH7777 cells

We have used an extraction procedure, which released membrane-bound apoB-100, to study the assembly of apoB-48 VLDL (very low density lipoproteins). This procedure released apoB-48, but not integral membrane proteins, from microsomes of McA-RH7777 cells. Upon gradient ultracentrifugation, the extracted apoB-48 migrated in the same position as the dense apoB-48-containing lipoprotein (apoB-48 HDL (high density lipoprotein)) secreted into the medium. Labeling studies with [(3)H]glycerol demonstrated that the HDL-like particle extracted from the microsomes contains both triglycerides and phosphatidylcholine. The estimated molar ratio between triglyceride and phosphatidylcholine was 0.70 +/- 0.09, supporting the possibility that the particle has a neutral lipid core. Pulse-chase experiments indicated that microsomal apoB-48 HDL can either be secreted as apoB-48 HDL or converted to apoB-48 VLDL. These results support the two-step model of VLDL assembly. To determine the size of apoB required to assemble HDL and VLDL, we produced apoB polypeptides of various lengths and followed their ability to assemble VLDL. Small amounts of apoB-40 were associated with VLDL, but most of the nascent chains associated with VLDL ranged from apoB-48 to apoB-100. Thus, efficient VLDL assembly requires apoB chains of at least apoB-48 size. Nascent polypeptides as small as apoB-20 were associated with particles in the HDL density range. Thus, the structural requirements of apoB to form HDL-like first-step particles differ from those to form second-step VLDL. Analysis of proteins in the d < 1.006 g/ml fraction after ultracentrifugation of the luminal content of the cells identified five chaperone proteins: binding protein, protein disulfide isomerase, calcium-binding protein 2, calreticulin, and glucose regulatory protein 94. Thus, intracellular VLDL is associated with a network of chaperones involved in protein folding. Pulse-chase and subcellular fractionation studies showed that apoB-48 VLDL did not accumulate in the rough endoplasmic reticulum. This finding indicates either that the two steps of apoB lipoprotein assembly occur in different compartment or that the assembled VLDL is transferred rapidly out of the rough endoplasmic reticulum.