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.     

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.     

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.     

Studies on the expression of genes encoding apolipoproteins B100 and B48 and the low density lipoprotein receptor in nonhuman primates. Comparison of dietary fat and cholesterol

African green monkeys were fed diets containing low and moderate cholesterol concentrations with either polyunsaturated or unsaturated fat as 40% of calories. Plasma total cholesterol, low density lipoprotein (LDL) cholesterol, and apoB concentrations generally were higher in animals fed (a) the higher dietary cholesterol concentration and (b) saturated fat. At necropsy, liver and intestine were removed, and measurement of mRNAs for LDL receptors (liver) and for apolipoprotein B (liver and intestine) was done. Monkey small intestine mucosa made exclusively apoB48 while the liver made only apoB100, although apoB mRNA in both tissues was the same size (14 kilobases). No dietary cholesterol or fat effects were found for apoB mRNA abundance in the liver, while the animals fed the higher dietary cholesterol level had 50% lower levels of hepatic LDL receptor mRNA. In a separate group of animals, livers were perfused and the rate of apoB secretion was measured. No dietary fat effect on apoB secretion rate was found, and no relationship between plasma LDL cholesterol concentration and the rate of hepatic apoB production existed. These findings support the idea that the dietary factors that increase LDL concentrations act by reducing clearance of apoB-containing particles rather than by increasing production of these lipoproteins. Hepatic LDL receptor mRNA was similar in abundance in polyunsaturated fat and saturated fat-fed animals, suggesting that the difference in plasma cholesterol concentration between these groups is not mediated via effects on LDL receptor mRNA abundance. The level of intestinal apoB mRNA was about 30% higher in animals fed the moderate dietary cholesterol concentration. Earlier studies have shown that more cholesterol is transported in chylomicrons from the intestine when dietary cholesterol levels are higher, and the increased intestinal apoB mRNA abundance may reflect increased intestinal cholesterol transport and chylomicron apoB48 production.     

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.     

Influence of dietary lipids on hepatic mRNA levels of proteins regulating plasma lipoproteins in baboons with high and low levels of large high density lipoproteins.

Selective breeding of baboons has produced families with increased plasma levels of large high density lipoproteins (HDL1) and very low (VLDL) and low (LDL) density lipoproteins when the animals consume a diet enriched in cholesterol and saturated fat. High HDL1 baboons have a slower cholesteryl ester transfer, which may account for the accumulation of HDL1, but not of VLDL and LDL. To investigate the mechanism of accumulation of VLDL + LDL in plasma of the high HDL1 phenotype, we selected eight half-sib pairs of baboons, one member of each pair with high HDL1, the other member with little or no HDL1 on the same high cholesterol, saturated fat diet. Baboons were fed a chow diet and four experimental diets consisting of high and low cholesterol with corn oil, and high and low cholesterol with lard, each for 6 weeks, in a crossover design. Plasma lipids and lipoproteins and hepatic mRNA levels were measured on each diet. HDL1 phenotype, type of dietary fat, and dietary cholesterol affected plasma cholesterol and apolipoprotein (apo) B concentrations, whereas dietary fat alone affected plasma triglyceride and apoA-I concentrations. HDL1 phenotype and dietary cholesterol alone did not influence hepatic mRNA levels, whereas dietary lard, compared to corn oil, significantly increased hepatic apoE mRNA levels and decreased hepatic LDL receptor and HMG-CoA synthase mRNA levels. Hepatic apoA-I message was associated with cholesterol concentration in HDL fractions as well as with apoA-I concentrations in the plasma or HDL. However, hepatic apoB message level was not associated with plasma or LDL apoB levels. Total plasma cholesterol, including HDL, was negatively associated with hepatic LDL receptor and HMG-CoA synthase mRNA levels. However, compared with low HDL1 baboons, high HDL1 baboons had higher concentrations of LDL and HDL cholesterol at the same hepatic mRNA levels. These studies suggest that neither overproduction of apoB from the liver nor decreased hepatic LDL receptor levels cause the accumulation of VLDL and LDL in the plasma of high HDL1 baboons. These studies also show that, in spite of high levels of VLDL + LDL and HDL1, the high HDL1 baboons had higher levels of mRNA for LDL receptor and HMG-CoA synthase. This paradoxical relationship needs further study to understand the pathophysiology of VLDL and LDL accumulation in the plasma of animals with the high HDL1 phenotype.     

Direct effects of growth hormone on production and secretion of apolipoprotein B from rat hepatocytes

The aim of this study was to investigate the direct effects of growth hormone (GH) on production and secretion of apolipoprotein B (apoB)-containing lipoproteins from hepatocytes. Bovine GH (5-500 ng/ml) was given for 1 or 3 days to rat hepatocytes cultured on laminin-rich matrigel in serum-free medium. The effects of GH were compared with those of 3 nM insulin and 500 microM oleic acid. GH increased the editing of apoB mRNA, and the proportion of newly synthesized apoB-48 (of total apoB) in the cells and secreted into the medium changed in parallel. GH increased total secretion of apoB-48 (+30%) and apoB-48 in very low density lipoproteins (VLDL) more than twofold. Total apoB-100 secretion decreased 63%, but apoB-100-VLDL secretion was unaffected by GH. Pulse-chase studies indicated that GH increased intracellular early degradation of apoB-100 but not apoB-48. GH had no effect on apoB mRNA or LDL receptor mRNA levels. The triglyceride synthesis, the mass of triglycerides in the cells, and the VLDL fraction of the medium increased after GH incubation. Three days of insulin incubation had effects similar to those of GH. Combined incubation with oleic acid and GH had additive effects on apoB mRNA editing and apoB-48-VLDL secretion. In summary, GH has direct effects on production and secretion of apoB-containing lipoproteins, which may add to the effects of hyperinsulinemia and increased flux of fatty acids to the liver during GH treatment in vivo.     

Immunochemical evidence that human apoB differs when expressed in rodent versus human cells

LDL from human apolipoprotein B-100 (apoB-100) transgenic (HuBTg+/+) mice contains more triglyceride than LDL from normolipidemic subjects. To obtain novel monoclonal antibody (MAb) probes of apoB conformation, we generated hybridomas from HuBTg+/+ that had been immunized with LDL isolated from human plasma. One apoE-specific and four anti-apoB-100-specific hybridomas were identified. Two MAbs, 2E1 and 3D11, recognized an epitope in the amino-terminal 689 residues of apoB in native apoB-containing lipoproteins (LpBs) from human plasma or from the supernatant of human hepatoma HepG2 cells, but did not react with LpB from HuBTg+/+ mice or LpB secreted by human apoB-100-transfected rat McArdle 7777 hepatoma cells. 2E1 reacted weakly and 3D11 reacted strongly with apoB from HuBTg+/+ mice after SDS-PAGE. The lack of expression of the 2E1 and 3D11 epitopes on native LpB from HuBTg+/+ mice did not solely reflect the abnormal lipid composition of murine LpB. Both epitopes were detected in all human plasma samples tested and in all human plasma LpB classes. Therefore, human apoB expressed by rodent hepatocytes or hepatoma cells appears to adopt a different conformation or undergoes different posttranslational modification than apoB expressed in human hepatocytes or hepatoma cells.     

Hepatic lipase overexpression lowers remnant and LDL levels by a noncatalytic mechanism in LDL receptor-deficient mice

To address the role of the noncatalytic ligand function of hepatic lipase (HL) in low density lipoprotein (LDL) receptor-mediated lipoprotein metabolism, we characterized transgenic mice lacking the LDL receptor (LDLR) that express either catalytically active (Ldlr(-/-)HL) or inactive (Ldlr(-/-)HL(S145G)) human HL on both chow and high fat diets and compared them with nontransgenic Ldlr(-/-) mice. In mice fed a chow diet, apolipoprotein (apo)B-containing lipoprotein levels were 40-60% lower in Ldlr(-/-)HL and Ldlr(-/-)HL(S145G) mice than in Ldlr(-/-) mice. This decrease was mainly reflected by decreased apoB-48 levels in the Ldlr(-/-)HL mice and by decreased apoB-100 levels in Ldlr(-/-) HL(S145G) mice. These findings indicate that HL can reduce apoB-100-containing lipoproteins through a noncatalytic ligand activity that is independent of the LDLR. Cholesterol enrichment of the apoB-containing lipoproteins induced by feeding Ldlr(-/-)HL and Ldlr(-/-)HL(S145G) mice a cholesterol-enriched high fat (Western) diet resulted in parallel decreases in both apoB-100 and apoB-48 levels, indicating that HL is particularly efficient at reducing cholesterol-enriched apoB-containing lipoproteins through both catalytic and noncatalytic mechanisms. These data suggest that the noncatalytic function of HL provides an alternate clearance pathway for apoB-100- and apoB-48-containing lipoproteins that is independent of the LDLR and that contributes to the clearance of high density lipoproteins.     

Effects of dietary fats and cholesterol on liver lipid content and hepatic apolipoprotein A-I, B, and E and LDL receptor mRNA levels in cebus monkeys.

The effects of the long-term administration of the dietary fats coconut oil and corn oil at 31% of calories with or without 0.1% (wt/wt) dietary cholesterol on plasma lipoproteins, apolipoproteins (apo), hepatic lipid content, and hepatic apoA-I, apoB, apoE, and low density lipoprotein (LDL) receptor mRNA abundance were examined in 27 cebus monkeys. Relative to the corn oil-fed animals, no significant differences were noted in any of the parameters of the corn oil plus cholesterol-fed group. In animals fed coconut oil without cholesterol, significantly higher (P less than 0.05) plasma total cholesterol (145%), very low density lipoprotein (VLDL) + LDL (201%) and high density lipoprotein (HDL) (123%) cholesterol, apoA-I (103%), apoB (61%), and liver cholesteryl ester (263%) and triglyceride (325%) levels were noted, with no significant differences in mRNA levels relative to the corn oil only group. In animals fed coconut oil plus cholesterol, all plasma parameters were significantly higher (P less than 0.05), as were hepatic triglyceride (563%) and liver apoA-I (123%) and apoB (87%) mRNA levels relative to the corn oil only group, while hepatic LDL receptor mRNA (-29%) levels were significantly lower (P less than 0.05). Correlation coefficient analyses performed on pooled data demonstrated that liver triglyceride content was positively associated (P less than 0.05) with liver apoA-I and apoB mRNA levels and negatively associated (P less than 0.01) with hepatic LDL receptor mRNA levels. Liver free and esterified cholesterol levels were positively correlated (P less than 0.05) with liver apoE mRNA levels and negatively correlated (P less than 0.025) with liver LDL receptor mRNA levels. Interestingly, while a significant correlation (P less than 0.01) was noted between hepatic apoA-I mRNA abundance and plasma apoA-I levels, no such relationship was observed between liver apoB mRNA and plasma apoB levels, suggesting that the hepatic mRNA of apoA-I, but not that of apoB, is a major determinant of the circulating levels of the respective apolipoprotein. Our data indicate that a diet high in saturated fat and cholesterol may increase the accumulation of triglyceride and cholesterol in the liver, each resulting in the suppression of hepatic LDL receptor mRNA levels. We hypothesize that such elevations in hepatic lipid content differentially alter hepatic apoprotein mRNA levels, with triglyceride increasing hepatic mRNA concentrations for apoA-I and B and cholesterol elevating hepatic apoE mRNA abundance.     

Lipolysis stimulated lipoprotein receptor: a novel molecular link between hyperlipidemia, weight gain, and atherosclerosis in mice

The lipolysis-stimulated lipoprotein receptor, LSR, is a multimeric protein complex in the liver that undergoes conformational changes upon binding of free fatty acids, thereby revealing a binding site (s) that recognizes both apoB and apoE. Complete inactivation of the LSR gene is embryonic lethal in mice. Here we show that removal of a single LSR allele (LSR(-/+)) caused statistically significant increases in both plasma triglyceride and cholesterol levels, a 2-fold increase in plasma triglyceride changes during the post-prandial phase, and delayed clearance of lipid emulsions or a high fat meal. The longer postprandial lipoprotein clearance time observed in LSR(-/+) mice was further increased in LSR(-/+) mice lacking functional low density lipoprotein (LDL) receptors. LSR(-/+) mice placed on a Western-type diet displayed higher plasma triglycerides and cholesterol levels, increased triglyceride-rich lipoproteins and LDL, and increased aorta lipid content, as compared with control mice on the same diet. Furthermore, a direct correlation was observed between the hyperlipidemia and weight gain but only in the LSR(-/+) mice. Knockdown of LSR expression by small interfering RNA in mouse Hepa1-6 cells led to decreased internalization of both DiI-labeled cyclohexanedione-LDL and very low density lipoprotein in the presence of oleate. These data led us to conclude that LSR contributes to the physiological clearance of atherogenic triglyceride-rich lipoproteins and LDL. We propose that LSR cooperates with the LDL receptor in the final hepatic processing of apoB-containing lipoproteins and represents a novel therapeutic target for the treatment of hyperlipidemia associated with obesity and atherosclerosis.     

Association between 12α-hydroxylated bile acids and hepatic steatosis in rats fed a high-fat diet

High-fat (HF) diet induces hepatic steatosis that is a risk factor for noncommunicable diseases such as obesity, type 2 diabetes and cardiovascular disease. Previously, we found that HF feeding in rats increases the excretion of fecal bile acids (BAs), specifically 12α-hydroxylated (12αOH) BAs. Although the liver is the metabolic center in our body, the association between hepatic steatosis and 12αOH BAs in HF-fed rats is unclear. Thus, we investigated extensively BA composition in HF-fed rats and evaluated the association between hepatic steatosis and 12αOH BAs. Acclimated male inbred WKAH/HkmSlc rats were divided into two groups and fed either control or HF diet for 8 weeks. Feeding HF diet increased hepatic triglyceride and total cholesterol concentrations, which correlated positively with 12αOH BAs concentrations but not with non-12αOH BAs in the feces, portal plasma and liver. Accompanied by the increase in 12αOH BAs, the rats fed HF diet showed increased fat absorption and higher mRNA expression of liver Cidea. The enhancement of 12αOH BA secretion may contribute to hepatic steatosis by the promotion of dietary fat absorption and hepatic Cidea mRNA expression. The increase in 12αOH BAs was associated with enhanced liver cholesterol 7α-hydroxylase (Cyp7a1) and sterol 12α-hydroxylase (Cyp8b1) mRNA expression. There was a significant increase in 7α-hydroxycholesterol, a precursor of BAs, in the liver of HF-fed rats. Altogether, these data suggest that the HF diet increases preferentially 12αOH BAs synthesis by utilizing the accumulated hepatic cholesterol and enhancing mRNA expression of Cyp7a1 and Cyp8b1 in the liver.     

Hepatic secretion of small lipoprotein particles in apobec-1-/- mice is regulated by the LDL receptor

Recent studies have examined the role of the LDL receptor (LDLR) in regulating murine hepatic lipoprotein production and apolipoprotein B (apoB) secretion, with divergent conclusions from in vivo versus in vitro approaches. We have re-examined this question, both in vivo and in vitro, using apobec-1-/- mice to model the pattern of human hepatic apoB-100 secretion. Hepatic triglyceride production in vivo (using Triton WR-1339) was unchanged in wild-type (WT) C57BL/6, apobec-1-/-, ldlr-/-, and [apobec-1-/-, ldlr-/-] mice, while apoB-100 production (using [35S]methionine incorporation) was increased > 2-fold in [apobec-1-/-, ldlr-/-] mice. Although > 90% of newly synthesized apoB floated within the d < 1.006 fraction of serum from all genotypes, fast-performance liquid chromatography separation revealed that nascent triglyceride-rich particles from [apobec-1-/-, ldlr-/-] mice, but not WT, apobec-1-/-, or ldlr-/- mice, distributed into smaller (intermediate and LDL-sized) particles. Studies in isolated hepatocytes from these different genotypes confirmed secretion of smaller particles exclusively from [apobec-1-/-, ldlr-/-] mice, and pulse-chase analysis demonstrated increased secretion of apoB-100 with virtual elimination of posttranslational degradation. These results directly support the suggestion that the LDLR regulates hepatic apoB-100 production and modulates secretion of small, triglyceride-rich particles, both in vivo and in vitro.     

Cholestasis and hypercholesterolemia in SCD1-deficient mice fed a low-fat, high-carbohydrate diet

Stearoyl-coenzyme A desaturase 1-deficient (SCD1(-/-)) mice have impaired MUFA synthesis. When maintained on a very low-fat (VLF) diet, SCD1(-/-) mice developed severe hypercholesterolemia, characterized by an increase in apolipoprotein B (apoB)-containing lipoproteins and the appearance of lipoprotein X. The rate of LDL clearance was decreased in VLF SCD1(-/-) mice relative to VLF SCD1(+/+) mice, indicating that reduced apoB-containing lipoprotein clearance contributed to the hypercholesterolemia. Additionally, HDL-cholesterol was dramatically reduced in these mice. The presence of increased plasma bile acids, bilirubin, and aminotransferases in the VLF SCD1(-/-) mice is indicative of cholestasis. Supplementation of the VLF diet with MUFA- and PUFA-rich canola oil, but not saturated fat-rich hydrogenated coconut oil, prevented these plasma phenotypes. However, dietary oleate was not as effective as canola oil in reducing LDL-cholesterol, signifying a role for dietary PUFA deficiency in the development of this phenotype. These results indicate that the lack of SCD1 results in an increased requirement for dietary unsaturated fat to compensate for impaired MUFA synthesis and to prevent hypercholesterolemia and hepatic dysfunction. Therefore, endogenous MUFA synthesis is essential during dietary unsaturated fat insufficiency and influences the dietary requirement of PUFA.