Hepatitis C virus induces oxidation and degradation of apolipoprotein B to enhance lipid accumulation and promote viral production

Hepatitis C virus (HCV) infection induces the degradation and decreases the secretion of apolipoprotein B (ApoB). Impaired production and secretion of ApoB-containing lipoprotein is associated with an increase in hepatic steatosis. Therefore, HCV infection-induced degradation of ApoB may contribute to hepatic steatosis and decreased lipoprotein secretion, but the mechanism of HCV infection-induced ApoB degradation has not been completely elucidated. In this study, we found that the ApoB level in HCV-infected cells was regulated by proteasome-associated degradation but not autophagic degradation. ApoB was degraded by the 20S proteasome in a ubiquitin-independent manner. HCV induced the oxidation of ApoB via oxidative stress, and oxidized ApoB was recognized by the PSMA5 and PSMA6 subunits of the 20S proteasome for degradation. Further study showed that ApoB was degraded at endoplasmic reticulum (ER)-associated lipid droplets (LDs) and that the retrotranslocation and degradation of ApoB required Derlin-1 but not gp78 or p97. Moreover, we found that knockdown of ApoB before infection increased the cellular lipid content and enhanced HCV assembly. Overexpression of ApoB-50 inhibited lipid accumulation and repressed viral assembly in HCV-infected cells. Our study reveals a novel mechanism of ApoB degradation and lipid accumulation during HCV infection and might suggest new therapeutic strategies for hepatic steatosis.     

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.     

Genotype rs8099917 near the IL28B gene and amino acid substitution at position 70 in the core region of the hepatitis C virus are determinants of serum apolipoprotein B-100 concentration in chronic hepatitis C

The life cycle of the hepatitis C virus (HCV) is closely related to host lipoprotein metabolism. Serum levels of lipid are associated with the response to pegylated interferon plus ribavirin (PEG-IFN/RBV) therapy, while single nucleotide polymorphisms (SNPs) around the human interleukin 28B (IL28B) gene locus and amino acid substitutions in the core region of the HCV have been reported to affect the efficacy of PEG-IFN/RBV therapy in chronic hepatitis with HCV genotype 1b infection. The aim of this study was to elucidate the relationship between serum lipid and factors that are able to predict the efficacy of PEG-IFN/RB therapy, with specific focus on apolipoprotein B-100 (apoB-100) in 148 subjects with chronic HCV G1b infection. Our results demonstrated that both the aa 70 substitution in the core region of the HCV and the rs8099917 SNP located proximal to the IL28B were independent factors in determining serum apoB-100 and low-density lipoprotein (LDL) cholesterol levels. A significant association was noted between higher levels of apoB-100 (P = 1.1 × 10(-3)) and LDL cholesterol (P = 0.02) and the subjects having Arg70. A significant association was also observed between subjects carrying the rs8099917 TT responder genotype and higher levels of apoB-100 (P = 6.4 × 10(-3)) and LDL cholesterol (P = 4.2 × 10(-3)). Our results suggest that apoB-100 and LDL cholesterol are markers of impaired cellular lipoprotein pathways and/or host endogenous interferon response to HCV in chronic HCV infection. In particular, serum apoB-100 concentration might be an informative marker for judging changes in HCV-associated intracellular lipoprotein metabolism in patients carrying the rs8099917 responder genotype.     

Iron and Proinflammatory Cytokines in Chronic Hepatitis C Virus Infection

Steatohepatitis is a common finding in chronic hepatitis C virus (HCV) infection. As in other forms of steatohepatitis, oxidative damage may play an outstanding role. However, there are conflicting results relative to the role of iron on hepatic lipogenesis. Proinflammatory cytokines up-regulate ferritin expression, probably reflecting a defensive mechanism against increased oxidative stress, capable to open haem ring and release reactive iron. On the contrary, some adipokines, such as adiponectin, are associated with low ferritin levels. The aim of this study is to analyse the relationships of the amount of liver steatosis with serum iron, transferrin and ferritin as well as with proinflammatory cytokines, such as tumour necrosis factor (TNF)-α and interleukin (IL)-6, and adiponectin levels. We included 82 HCV infected patients and assessed the amount of liver fat by histomorphometry and its relationships with serum iron, ferritin and transferrin, adiponectin and TNF-α and IL-6. Liver steatosis was observed in 67 patients out of 82; in the remaining 15 patients, no steatosis at all was found. Patients with steatosis showed significantly higher serum ferritin levels than patients without steatosis (Z?=?2.14; p?=?0.032). When patients were classified in quartiles according to the intensity of steatosis, we observed that both TNF-α (KW?=?10.6; p?=?0.014) and IL-6 (KW?=?15.2; p?=?0.002) were significantly different among the four groups. Patients with more intense steatosis (highest quartile) showed the highest TNF-α and IL-6 values. Patients with severe hepatitis had higher levels of serum iron than patients with mild to moderate hepatitis. Serum iron also showed a correlation with the proportion of fibrosis (ρ?=?0.30; p?=?0.007). Serum iron levels are related with biochemical and histological parameters derived from liver inflammation in HCV-associated liver disease. Serum ferritin is higher among those with intense steatosis and also shows a (non-significant) trend to be associated with the more severe forms of hepatitis.     

Inhibition of fatty acid synthesis decreases very low density lipoprotein secretion in the hamster.

The hamster was developed as a model to study very low density lipoprotein (VLDL) metabolism, since, as is the case in humans, the hamster liver was found to synthesize apoB-100 and not apoB-48. The effect of inhibiting fatty acid synthesis on the hepatic secretion of VLDL triglyceride (TG) and apolipoprotein (apo) B-100 in this model was then investigated. In an in vivo study, hamsters were fed a chow diet containing 0.15% TOFA (5-tetradecyloxy-2-furancarboxylic acid), an inhibitor of acetyl-CoA carboxylase. After 6 days of treatment, plasma triglyceride and cholesterol levels were decreased by 30.2% and 11.6%, respectively. When the secretion of VLDL-TG by the liver was measured in vivo after injection of Triton WR 1339, TOFA treatment was found to decrease VLDL-TG secretion by 40%. In subsequent in vitro studies utilizing cultured primary hamster hepatocytes, incubation with 20 microM TOFA for 4 h resulted in 98% and 76% inhibition in fatty acid and triglyceride synthesis, respectively; VLDL-TG secretion was decreased by 90%. When hepatocytes were pulsed with [3H]leucine, incubation with TOFA resulted in a 50% decrease in the incorporation of radiolabel into secreted VLDL apoB-100. The results of this study indicate that inhibition of intracellular triglyceride synthesis decreases the secretion of VLDL-TG and apoB-100, and does not result in the secretion of a dense, triglyceride-depleted lipoprotein.     

Oxidative DNA damage in circulating leukocytes occurs as an early event in chronic HCV infection

Chronic hepatitis C virus (HCV) infection is associated with an increased production of reactive oxygen species within the liver that are responsible for the oxidation of intracellular macromolecules. To ascertain whether the increased risk of hepatocellular carcinoma in individuals with chronic HCV infection is related to an accumulation of oxidative DNA damage, the 8-hydroxydeoxyguanosine (8-OHdG) content in the DNA of liver tissue and leukocytes of 87 individuals with HCV- or HBV-related liver disease and of 10 healthy controls was measured. Serum levels of thiobarbituric acid reactive substances (TBARS) were also assessed as an index of lipid peroxidation. RESULTS: The 8-OHdG content in the circulating leukocytes correlated with that of liver tissue (r = 0.618, p < .0004). HCV patients had the highest median 8-OHdG levels (p < .0004). 8-OHdG leukocyte levels in HCV patients were higher than in HBV patients (p < .04) and they significantly correlated with the clinical diagnosis (p < .025), the serum ferritin levels (p < .05), and the amount of liver steatosis (p < .001). No correlation was found with age, gender, history of drinking or smoking, ALT or GGT levels, ESR, alpha-1, or gamma-globulin level and Ishak score. TBARS levels were significantly higher in cirrhotics than in noncirrhotics (p < .01). CONCLUSIONS: The 8-OHdG level in circulating leukocytes is a reliable marker of oxidative stress occurring in the liver of individuals with chronic HCV infection. DNA oxidative damage appears to be an early and unique event in the natural history of HCV-related hepatitis. This injury increases the risk of genomic damage and may be one of the important factors involved in the carcinogenic process in cases of HCV-related chronic liver disease.     

Degradation of newly synthesized apolipoprotein B-100 in a pre-Golgi compartment

The synthesis and secretion of apolipoprotein (apo) B-100 have been studied in a human hepatoblastoma cell line, the Hep G2 cells. Pulse-chase analysis showed that apoB-100 was not quantitatively recovered in the culture medium. To reveal the intracellular degradation of apoB-100 prior to secretion, cells were incubated with 1 microgram/ml Brefeldin A (BFA) which impeded protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus and the fate of apoB-100 retained in the cells was traced at 37 degrees C. A significant amount of intracellular apoB-100 (40-60%/h) was degraded during the chase period, whereas apoA-1 remained intact. ApoB-100 degradation was temperature dependent, no degradation was observed below 20 degrees C. This degradation process was not inhibited by chloroquine, leupeptin, pepstatin, and chymostatin, suggesting that lysosomal proteases were not involved and that apoB-100 was degraded in a pre-Golgi compartment which is either part of, or closely related to, the ER. Preincubation of cells with low density lipoproteins (LDL) induced a 22-32% increase in the degradation of apoB-100. This result raised the possibility that secretion of apoB-100 might be regulated through the intracellular degradation of apoB-100. These results suggest the existence of the degradation pathway for apoB-100 in a pre-Golgi compartment and an unique regulatory mechanism for apoB-100 secretion.     

Hepatic secretion of apoB-100 is impaired in hypobetalipoproteinemic mice with an apoB-38.9-specifying allele

Apolipoprotein B (apoB) truncation-specifying mutations cause familial hypobetalipoproteinemia (FHBL). Lipoprotein kinetics studies have shown that production rates of apoB-100 are reduced by 70-80% in heterozygous FHBL humans, instead of the expected 50%. To develop suitable mouse models to study the underlying mechanism, apoB-38.9-only (Apob(38.9/38.9)) mice were crossbred with Apobec-1 knockout (Apobec-1(-/-)) mice or apoB-100-only (Apob(100/100)) mice to produce two lines of apoB-38.9 heterozygous mice that produce only apoB-38.9 and apoB-100, namely Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice. In vivo rates of apoB-100 secretion were measured using [35S]Met/Cys to label proteins and Triton WR-1339 to block apoB-100 VLDL lipolysis/uptake. Rates of secretion were reduced by 80%, rather than the expected 50%, in both Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) mice compared with those of the respective Apobec-1(-/-)/Apob(+/+) and Apob(100/100) control mice. Continuous labeling and pulse-chase experiments in primary hepatocyte cultures revealed that rates of apoB-100 synthesis by Apobec-1(-/-)/Apob(38.9/+) and Apob(38.9/100) hepatocytes were reduced to the expected 50% of those of the respective controls, but the efficiency of secretion of apoB-100 was significantly lower in apoB-38.9 heterozygous hepatocytes. The greater-than-expected decreases in apoB-100 production rates of FHBL heterozygous humans appear to be attributable to a defect in secretion rather than in the synthesis of apoB-100 from the unaffected apoB allele.     

Metabolism of apoB lipoproteins of intestinal and hepatic origin during constant feeding of small amounts of fat

We aimed to identify mechanisms by which apolipoprotein B-48 (apoB-48) could have an atherogenic role by simultaneously studying the metabolism of postprandial apoB-48 and apoB-100 lipoproteins. The kinetics of apoB-48 and apoB-100, each in four density subfractions of VLDL and intermediate density lipoprotein (IDL), were studied by stable isotope labeling in a constantly fed state with half-hourly administration of almond oil in five postmenopausal women. A non-steady-state, multicompartmental model was used. Despite a much lower production rate, VLDL and IDL apoB-48 shared a similar secretion pattern with apoB-100: both were directly secreted into all fractions with similar percentage mass distributions. Fractional catabolic rates (FCRs) of apoB-48 and apoB-100 were similar in VLDL and IDL. We identified a fast turnover compartment of light VLDL that had a residence time of <30 min for apoB-48 and apoB-100. Finally, a high secretion rate of apoB-48 was associated with a slow FCR of VLDL and IDL apoB-100. In conclusion, the intestine secretes a spectrum of apoB lipoproteins, similar to what the liver secretes, albeit with a much lower secretion rate. Once in plasma, intestinal and hepatic triglyceride-rich lipoproteins have similar rates of clearance and participate interactively in similar metabolic pathways, with high apoB-48 production inhibiting the clearance of apoB-100.     

Ubiquitination regulates the assembly of VLDL in HepG2 cells and is the committing step of the apoB-100 ERAD pathway

Apolipoprotein B-100 (apoB-100) is degraded by endoplasmic reticulum-associated degradation (ERAD) when lipid availability limits assembly of VLDLs. The ubiquitin ligase gp78 and the AAA-ATPase p97 have been implicated in the proteasomal degradation of apoB-100. To study the relationship between ERAD and VLDL assembly, we used small interfering RNA (siRNA) to reduce gp78 expression in HepG2 cells. Reduction of gp78 decreased apoB-100 ubiquitination and cytosolic apoB-ubiquitin conjugates. Radiolabeling studies revealed that gp78 knockdown increased secretion of newly synthesized apoB-100 and, unexpectedly, enhanced VLDL assembly, as the shift in apoB-100 density in gp78-reduced cells was accompanied by increased triacylglycerol (TG) secretion. To explore the mechanisms by which gp78 reduction might enhance VLDL assembly, we compared the effects of gp78 knockdown with those of U0126, a mitogen-activated protein kinase/ERK kinase1/2 inhibitor that enhances apoB-100 secretion in HepG2 cells. U0126 treatment increased secretion of both apoB100 and TG and decreased the ubiquitination and cellular accumu-lation of apoB-100. Furthermore, p97 knockdown caused apoB-100 to accumulate in the cell, but if gp78 was concomitantly reduced or assembly was enhanced by U0126 treatment, cellular apoB-100 returned toward baseline. This indicates that ubiquitination commits apoB-100 to p97-mediated retrotranslocation during ERAD. Thus, decreasing ubiquitination of apoB-100 enhances VLDL assembly, whereas improving apoB-100 lipidation decreases its ubiquitination, suggesting that ubiquitination has a regulatory role in VLDL assembly.     

Early embryonic lethality of H ferritin gene deletion in mice

Ferritin molecules play an important role in the control of intracellular iron distribution and in the constitution of long term iron stores. In vitro studies on recombinant ferritin subunits have shown that the ferroxidase activity associated with the H subunit is necessary for iron uptake by the ferritin molecule, whereas the L subunit facilitates iron core formation inside the protein shell. However, plant and bacterial ferritins have only a single type of subunit which probably fulfills both functions. To assess the biological significance of the ferroxidase activity associated with the H subunit, we disrupted the H ferritin gene (Fth) in mice by homologous recombination. Fth(+/-) mice are healthy, fertile, and do not differ significantly from their control littermates. However, Fth(-/-) embryos die between 3.5 and 9.5 days of development, suggesting that there is no functional redundancy between the two ferritin subunits and that, in the absence of H subunits, L ferritin homopolymers are not able to maintain iron in a bioavailable and nontoxic form. The pattern of expression of the wild type Fth gene in 9.5-day embryos is suggestive of an important function of the H ferritin gene in the heart.     

The production of 85 kDa N-terminal fragment of apolipoprotein B in mutant HepG2 cells generated by targeted modification of apob gene occurs by ALLN-inhibitable protease cleavage during translocation

To study the mechanism of low levels of full length and truncated apoB in individuals heterozygous for apoB truncation, a non-sense mutation was introduced in one of the three alleles of apob gene of HepG2 cells by homologous recombination. Despite very low levels of apoB-82 (1-2%) in the media, a prominent N-terminal apoB protein of 85 kDa (apoB-15) was secreted that fractionated at d > 1.065 in density gradient ultracentrifugation. The mechanism of production of this short protein was studied by ³⁵S-methionine pulse-chase experiment. Oleate prevented presecretory degradation of apoB-100 in the cell and resulted in increased secretion of newly synthesized apoB-100 with decreases in the apoB-15, suggesting that rescue of pre-secretary intracellular degradation of apoB restricted the production and secretion of apoB-15. Further investigation on the degradation of transmembrane forms of apoB, in the presence and absence of a cysteine protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), showed appearance of detectable levels of newly synthesized apoB-82 in the cell and the media together with increased apoB-100 secretion, and reduction in the secretion of apoB-15. Compared to ER membrane, the levels of apoB were higher in the luminal content, and presence of both oleate and ALLN had additive effect on apoB secretion. These results suggest that the presence of improper folding of apoB during translocation led to the cleavage of both apoB-100 and apoB-82 by ALLN-sensitive protease and generation of 85 kDa N-terminal fragment of apoB.     

Influence of peroxisome proliferator-activated receptor alpha agonists on the intracellular turnover and secretion of apolipoprotein (Apo) B-100 and ApoB-48

The peroxisome proliferator-activated receptor (PPAR) alpha agonist WY 14,643 increased the secretion of apolipoprotein (apo) B-100, but not that of apoB-48, and decreased triglyceride biosynthesis and secretion from primary rat hepatocytes. These effects resulted in decreased secretion of apoB-100-very low density lipoprotein (VLDL) and an increased secretion of apoB-100 on low density lipoproteins/intermediate density lipoproteins. ApoB-48-VLDL was also replaced by more dense particles. The proteasomal inhibitor lactacystin did not influence the recovery of apoB-100 or apoB-48 in primary rat hepatocytes, indicating that co-translational (proteasomal) degradation is of less importance in these cells. Treatment with WY 14,643 made the recovery of apoB-100 sensitive to lactacystin, most likely reflecting the decreased biosynthesis of triglycerides. The PPAR alpha agonist induced a significant increase in the accumulation of pulse-labeled apoB-100 even after a short pulse (2-5 min). There was also an increase in apoB-100 nascent polypeptides, indicating that the co-translational degradation of apoB-100 was inhibited. However, a minor influence on an early posttranslation degradation cannot be excluded. This decreased co-translational degradation of apoB-100 explained the increased secretion of the protein. The levels of apoB-48 remained unchanged during these pulse-chase experiments, and albumin production was not affected, indicating a specific effect of PPAR alpha agonists on the co-translational degradation of apoB-100. These findings explain the difference in the rate of secretion of the two apoB proteins seen after PPAR alpha activation. PPAR alpha agonists increased the expression and biosynthesis of liver fatty acid-binding protein (LFABP). Increased expression of LFABP by transfection of McA-RH7777 cells increased the secretion of apoB-100, decreased triglyceride biosynthesis and secretion, and increased PPAR alpha mRNA levels. These findings suggest that PPAR alpha and LFABP could interact to amplify the effect of endogenous PPAR alpha agonists on the assembly of VLDL.     

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.     

Manipulation of cholesterol and cholesteryl ester synthesis has multiple effects on the metabolism of apolipoprotein B and the secretion of very-low-density lipoprotein by primary hepatocyte cultures.

Inhibition of esterified and non-esterified cholesterol synthesis by lovastatin in primary rat hepatocytes suppressed the net synthesis and very-low-density lipoprotein (VLDL) secretion of apolipoprotein B (apoB)-48 and apoB-100. Lovastatin did not alter the rates of apoB-48 and apoB-100 post-translational degradation. 25-Hydroxycholesterol, which inhibited non-esterified cholesterol synthesis but increased the synthesis of cholesteryl ester, showed differential effects on the metabolism of apoB-48 and apoB-100. Whereas the secretion of apoB-48 VLDL was suppressed there was no effect on the secretion of apoB-100 VLDL. The post-translational degradation of apoB-48, but not of apoB-100, was enhanced by 25-hydroxycholesterol. The net synthesis rates of apoB-48 and apoB-100 were unaffected by 25-hydroxycholesterol. The inhibitory effect of lovastatin alone on the net synthesis of apoB-48 and apoB-100 was reversed by the simultaneous presence of 25-hydroxycholesterol, suggesting a role for newly synthesised cholesteryl ester. Prevention of the reversal effect by the acyl-CoA: cholesterol acyltransferase (ACAT) inhibitor YM 17E supported this interpretation. In the presence of lovastatin, restoration of the net synthesis of apoB by 25-hydroxycholesterol was not accompanied by an increased VLDL output of apoB-48 and apoB-100. However, under these conditions there was an increased post-translational degradation of apoB-48 and apoB-100. These results suggest that interference with intracellular cholesterol and cholesteryl ester metabolism interrupts VLDL assembly at sites of both apoB net synthesis and post-translational degradation.     

Apolipoprotein B, a paradigm for proteins regulated by intracellular degradation, does not undergo intracellular degradation in CaCo2 cells

Studies in different liver-derived cells in culture indicate that apolipoprotein (apo) B-100 production is regulated largely by intracellular degradation and the ubiquitin-proteasome pathway is a major mechanism for the degradation. The proteasomal degradation of apoB-100 was postulated to be an intrinsic property of the protein that occurs even in the presence of optimal amounts of lipids supplied to the cell. We examined apoB-100 and apoB-48 biogenesis in CaCo2, a human colon carcinoma cell line. To our surprise, apoB-100 and apoB-48 were quantitatively secreted by CaCo2 cells; essentially none of the newly synthesized apoB was degraded before secretion in a 2-h period whether the cells were cultured on filter or on plastic. Furthermore, although ubiquitin immunoreactivity was readily detected in the intracellular apoB isolated from HepG2 cells, little or no ubiquitin was detectable in the intracellular apoB from CaCo2 cells. The amounts of free ubiquitin and total and non-apoB ubiquitinated proteins were comparable in HepG2 and CaCo2 cells, indicating that CaCo2 cells have the necessary machinery for tagging ubiquitin chains onto cellular proteins for proteasomal degradation. Incubation in lipoprotein-deficient serum did not induce apoB degradation, but the addition of a microsomal triglyceride transfer protein inhibitor led to apoB degradation in CaCo2 cells. Finally, similar proportions of apoB polypeptide in isolated microsomes from CaCo2 and HepG2 cells were accessible to exogenously added trypsin, indicating that the mere exposure of apoB nascent chains to the cytosolic compartment is insufficient to cause the proteasomal degradation. Therefore, the intracellular degradation of apoB is not an intrinsic property of the protein, and the phenomenon is neither universal nor inevitable. The unconditional use of apoB as a paradigm for intracellular protein degradation is not warranted.     

The AAA-ATPase p97 facilitates degradation of apolipoprotein B by the ubiquitin-proteasome pathway

The ATPase associated with various cellular activities (AAA-ATPase) p97 (p97) has been implicated in the retrotranslocation of target proteins for delivery to the cytosolic proteasome during endoplasmic reticulum-associated degradation (ERAD). Apolipoprotein B-100 (apoB-100) is an ERAD substrate in liver cells, including the human hepatoma, HepG2. We studied the potential role of p97 in the ERAD of apoB-100 in HepG2 cells using cell permeabilization, coimmunoprecipitation, and gene silencing. Degradation was abolished when HepG2 cytosol was removed by digitonin permeabilization, and treatment of intact cells with the proteasome inhibitor MG132 caused accumulation of ubiquitinated apoB protein in the cytosol. Cross-linking of intact cells with the thiol-cleavable agent dithiobis(succinimidylpropionate) (DSP), as well as nondenaturing immunoprecipitation, demonstrated an interaction between p97 and intracellular apoB. Small interfering ribonucleic acid (siRNA)-mediated reduction of p97 protein increased the intracellular levels of newly synthesized apoB-100, predominantly because of a decrease in the turnover of newly synthesized apoB-100 protein. However, although the posttranslational degradation of newly synthesized apoB-100 was delayed by p97 knockdown, secretion of apoB-100 was not affected. Knockdown of p97 also impaired the release of apoB-100 and polyubiquitinated apoB into the cytosol. In summary, our results suggest that retrotranslocation and proteasomal degradation of apoB-100 can be dissociated in HepG2 cells, and that the AAA-ATPase p97 is involved in the removal of full-length apoB from the biosynthetic pathway to the cytosolic proteasome.     

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.