Reduced intestinal fat absorptive capacity but enhanced susceptibility to diet-induced fatty liver in mice heterozygous for ApoB38.9 truncation

Fatty liver is prevalent in apolipoprotein B (apoB)-defective familial hypobetalipoproteinemia (FHBL). Similar to humans, mouse models of FHBL produced by gene targeting (apob(+/38.9)) manifest low plasma cholesterol and increased hepatic triglycerides (TG) even on a chow diet due to impaired hepatic VLDL-TG secretive capacity. Because apoB truncations shorter than apoB48 are expressed in the intestine, we examined whether FHBL mice may have limited capacity for intestinal dietary TG absorption. In addition, we investigated whether FHBL mice are more susceptible to diet-induced hepatic TG accumulation. Fat absorption capacity was impaired in apoB38.9 mice in a gene dose-dependent manner. Relative fractional fat absorption coefficients for apob(+/+), apob(+/38.9), and apob(38.9/38.9) were 1.00, 0.96, and 0.71, respectively. To raise hepatic TG, we fed high-fat (HF) and low-fat (LF) pellets. Hepatic TG level was observed in rank order: HF > LF > chow. On both LF and HF, liver TG level was higher in the apob(+/38.9) than in apob(+/+). Hepatic TG secretion remained impaired in the apob(+/38.9) on the HF diet. Thus the FHBL mice are more susceptible to diet-induced fatty liver despite relatively reduced intestinal TG absorption capacity on a HF diet.     

Phospholipid transfer protein deficiency impairs apolipoprotein-B secretion from hepatocytes by stimulating a proteolytic pathway through a relative deficiency of vitamin E and an increase in intracellular oxidants

Genetic deficiency of the plasma phospholipid transfer protein (PLTP) in mice unexpectedly causes a substantial impairment in liver secretion of apolipoprotein-B (apoB), the major protein of atherogenic lipoproteins. To explore the mechanism, we examined the three known pathways for hepatic apoB secretory control, namely endoplasmic reticulum (ER)/proteasome-associated degradation (ERAD), post-ER pre-secretory proteolysis (PERPP), and receptor-mediated degradation, also known as re-uptake. First, we found that ERAD and cell surface re-uptake were not active in PLTP-null hepatocytes. Moreover, ER-to-Golgi blockade by brefeldin A, which enhances ERAD, equalized total apoB recovery from PLTP-null and wild-type cells, indicating that the relevant process occurs post-ER. Second, because PERPP can be stimulated by intracellular reactive oxygen species (ROS), we examined hepatic redox status. Although we found previously that PLTP-null mice exhibit elevated plasma concentrations of vitamin E, a lipid anti-oxidant, we now discovered that their livers contain significantly less vitamin E and significantly more lipid peroxides than do livers of wild-type mice. Third, to establish a causal connection, the addition of vitamin E or treatment with an inhibitor of intracellular iron-dependent peroxidation, desferrioxamine, abolished the elevation in cellular ROS as well as the defect in apoB secretion from PLTP-null hepatocytes. Overall, we conclude that PLTP deficiency decreases liver vitamin E content, increases hepatic oxidant tone, and substantially enhances ROS-dependent destruction of newly synthesized apoB via a post-ER process. These findings are likely to be broadly relevant to hepatic apoB secretory control in vivo.     

Regulation of ApoB secretion by the low density lipoprotein receptor requires exit from the endoplasmic reticulum and interaction with ApoE or ApoB

Apolipoprotein B (apoB) is required for the hepatic assembly and secretion of very low density lipoprotein (VLDL). The LDL receptor (LDLR) promotes post-translational degradation of apoB and thereby reduces VLDL particle secretion. We investigated the trafficking pathways and ligand requirements for the LDLR to promote degradation of apoB. We first tested whether the LDLR drives apoB degradation in an endoplasmic reticulum (ER)-associated pathway. Primary mouse hepatocytes harboring an ethyl-nitrosourea-induced, ER-retained mutant LDLR secreted comparable levels of apoB with LDLR-null hepatocytes, despite reduced secretion from cells expressing the wild-type LDLR. Additionally, treatment of cells with brefeldin A inhibited LDLR-dependent degradation. However, this rescue was reversible, and degradation of apoB occurred upon removal of brefeldin A. To characterize the lipoprotein reuptake pathway of degradation, we employed an LDLR mutant defective in constitutive endocytosis and internalization of apoB. This mutant was as effective in reducing apoB secretion as the wild-type LDLR. However, the effect was dependent on apolipoprotein E (apoE) as only the wild-type LDLR, and not the endocytic mutant, reduced apoB secretion in apoE-null cells. Treatment with heparin rescued a pool of apoB in cells expressing the endocytic mutant, indicating that reuptake of VLDL via apoE still occurs with this mutant. Finally, an LDLR mutant defective in binding apoB but not apoE reduced apoB secretion in an apoE-dependent manner. Together, these data suggest that the LDLR directs apoB to degradation in a post-ER compartment. Furthermore, the reuptake mechanism of degradation occurs via internalization of apoB through a constitutive endocytic pathway and apoE through a ligand-dependent pathway.     

Insulin-Stimulated Degradation of Apolipoprotein B100: Roles of Class II Phosphatidylinositol-3-Kinase and Autophagy

Both in humans and animal models, an acute increase in plasma insulin levels, typically following meals, leads to transient depression of hepatic secretion of very low density lipoproteins (VLDL). One contributing mechanism for the decrease in VLDL secretion is enhanced degradation of apolipoprotein B100 (apoB100), which is required for VLDL formation. Unlike the degradation of nascent apoB100, which occurs in the endoplasmic reticulum (ER), insulin-stimulated apoB100 degradation occurs post-ER and is inhibited by pan-phosphatidylinositol (PI)3-kinase inhibitors. It is unclear, however, which of the three classes of PI3-kinases is required for insulin-stimulated apoB100 degradation, as well as the proteolytic machinery underlying this response. Class III PI3-kinase is not activated by insulin, but the other two classes are. By using a class I-specific inhibitor and siRNA to the major class II isoform in liver, we now show that it is class II PI3-kinase that is required for insulin-stimulated apoB100 degradation in primary mouse hepatocytes. Because the insulin-stimulated process resembles other examples of apoB100 post-ER proteolysis mediated by autophagy, we hypothesized that the effects of insulin in autophagy-deficient mouse primary hepatocytes would be attenuated. Indeed, apoB100 degradation in response to insulin was significantly impaired in two types of autophagy-deficient hepatocytes. Together, our data demonstrate that insulin-stimulated apoB100 degradation in the liver requires both class II PI3-kinase activity and autophagy.     

Missense mutations in APOB within the betaalpha1 domain of human APOB-100 result in impaired secretion of ApoB and ApoB-containing lipoproteins in familial hypobetalipoproteinemia

Familial hypobetalipoproteinemia (FHBL) is associated with mutations in the APOB gene. We reported the first missense APOB mutation, R463W, in an FHBL kindred (Burnett, J. R., Shan, J., Miskie, B. A., Whitfield, A. J., Yuan, J., Tran, K., Mc-Knight, C. J., Hegele, R. A., and Yao, Z. (2003) J. Biol. Chem. 278, 13442-13452). Here we identified a second nonsynonymous APOB mutation, L343V, in another FHBL kindred. Heterozygotes for L343V (n = 10) had a mean plasma apoB at 0.31 g/liter as compared with 0.80 g/liter in unaffected family members (n = 22). The L343V mutation impaired secretion of apoB-100 and very low density lipoproteins. The secretion efficiency was 20% for B100wt and 10% for B100LV and B100RW. Decreased secretion of mutant apoB-100 was associated with increased endoplasmic reticulum retention and increased binding to microsomal triglyceride transfer protein and BiP. Reduced secretion efficiency was also observed with B48LV and B17LV. Biochemical and biophysical analyses of apoB domain constructs showed that L343V and R463W altered folding of the alpha-helical domain within the N terminus of apoB. Thus, proper folding of the alpha-helical domain of apoB-100 is essential for efficient secretion.     

A novel nontruncating APOB gene mutation,R463W,causes familial hypobetalipoproteinemia

Familial hypobetalipoproteinemia (FHBL), an autosomal co-dominant disorder, is associated with reduced plasma concentrations (<5th percentile for age and sex) of apolipoprotein (apo) B and beta-migrating lipoproteins. To date, only mutations in APOB encoding prematurely truncated apoB have been found in FHBL. We discovered a novel APOB gene mutation, namely R463W, in an extended Christian Lebanese FHBL kindred. Heterozygotes for R463W had the typical FHBL phenotype, whereas homozygotes had barely detectable apoB-100. The effect of the R463W mutation on apoB secretion was examined using transfected McA-RH7777 cells that expressed one of two recombinant human apoBs, namely B48 and B17. In both cases, the mutant proteins (B48RW and B17RW) were retained within the endoplasmic reticulum and were secreted poorly compared with their wild-type counterparts. Pulse-chase analysis showed that secretion efficiencies of B48RW and B17RW were, respectively, 45 and 40% lower than those of the wild-types. Substitution of Arg(463) with Ala in apoB-17 (B17RA) decreased secretion efficiency by approximately 50%, but substitution with Lys (B17RK) had no effect on secretion, indicating that the positive charge was important. Molecular modeling of apoB predicted that Arg(463) was in close proximity to Glu(756) and Asp(456). Substitution of Glu(756) with Gln (B17EQ) had no effect on secretion, but substitution of Asp(456) with Asn (B17DN) decreased secretion to the same extent as B17RW. In co-transfection experiments, the mutant B17RW showed increased binding to microsomal triglyceride transfer protein as compared with wild-type B17. Thus, the naturally occurring R463W mutant reveals a key local domain governing assembly and secretion of apoB-containing lipoproteins.     

Fatty liver in familial hypobetalipoproteinemia: triglyceride assembly into VLDL particles is affected by the extent of hepatic steatosis

Familial hypobetalipoproteinemia (FHBL) subjects may develop fatty liver. Liver fat was assessed in 21 FHBL with six different apolipoprotein B (apoB) truncations (apoB-4 to apoB-89) and 14 controls by magnetic resonance spectroscopy (MRS). Liver fat percentages were 16.7 +/- 11.5 and 3.3 +/- 2.9 (mean +/- SD) (P = 0.001). Liver fat percentage was positively correlated with body mass index, waist circumference, and areas under the insulin curves of 2 h glucose tolerance tests, suggesting that obesity may affect the severity of liver fat accumulation in both groups. Despite 5-fold differences in liver fat percentage, mean values for obesity and insulin indexes were similar. Thus, for similar degrees of obesity, FHBL subjects have more hepatic fat. VLDL-triglyceride (TG)-fatty acids arise from plasma and nonplasma sources (liver and splanchnic tissues). To assess the relative contributions of each, [2H2]palmitate was infused over 12 h in 13 FHBL subjects and 11 controls. Isotopic enrichment of plasma free palmitate and VLDL-TG-palmitate was determined by mass spectrometry. Non-plasma sources contributed 51 +/- 15% in FHBL and 37 +/- 13% in controls (P = 0.02). Correlations of liver fat percentage and percent VLDL-TG-palmitate from liver were r = 0.89 (P = 0.0001) for FHBL subjects and r = 0.69 (P = 0.01) for controls. Thus, apoB truncation-producing mutations result in fatty liver and in altered assembly of VLDL-TG.     

Apolipoprotein B100 exit from the endoplasmic reticulum (ER) is COPII-dependent,and its lipidation to very low density lipoprotein occurs post-ER

Hepatic apolipoprotein B100 (apoB100) associates with lipids to form dense lipoprotein particles in the endoplasmic reticulum (ER) and is further lipidated to very low density lipoproteins (VLDL). Because the VLDL diameter can exceed 200 nm, classical ER-derived vesicles may be unable to accommodate VLDLs. Using hepatic membranes and cytosol to reconstitute ER budding, apoB100-containing vesicles sedimented distinct from those harboring more typical cargo but contained Sec23. Moreover, ER exit of apoB was inhibited by dominant-negative Sar1. Budding required Sar1 regardless of whether oleic acid (OA) was added to stimulate apoB lipidation; therefore, either large apoB100-lipoproteins reside in secretory vesicles, or full lipidation occurs post-ER. Using membranes from cells incubated in the presence or absence of OA, we determined that apoB100-lipoproteins in ER vesicles had not become lipidated to VLDLs. VLDL particles resided in the Golgi, but not the ER, after fractionation of OA-treated cells. We conclude that apoB100-lipoproteins exit the ER in COPII vesicles, but under conditions favorable for VLDL formation final lipid loading occurs post-ER.     

LZP is required for hepatic triacylglycerol transportation through maintaining apolipoprotein B stability

The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.     

Fatty liver in familial hypobetalipoproteinemia: roles of the APOB defects, intra-abdominal adipose tissue, and insulin sensitivity

Fatty liver is frequent in the apolipoprotein B (apoB)-defective genetic form of familial hypobetalipoproteinemia (FHBL), but interindividual variability in liver fat is large. To explain this, we assessed the roles of metabolic factors in 32 affected family members with apoB-defective FHBL and 33 related and unrelated normolipidemic controls matched for age, sex, and indices of adiposity. Two hour, 75 g oral glucose tests, with measurements of plasma glucose and insulin levels, body mass index, and waist-hip ratios were obtained. Abdominal subcutaneous, intraperitoneal (IPAT), and retroperitoneal adipose tissue masses were quantified by MR imaging, and hepatic fat was quantified by MR spectroscopy. Mean +/- SD liver fat percentage values of FHBL and controls were 14.8 +/- 12.0 and 5.2 +/- 5.9, respectively (P = 0.001). Means for these measures of obesity and insulin action were similar in the two groups. Important determinants of liver fat percentage were FHBL-affected status, IPAT, and area under the curve (AUC) insulin in both groups, but the strongest predictors were IPAT in FHBL (partial R(2) = 0.55, P < 0.0002) and AUC insulin in controls (partial R(2) = 0.59, P = 0.0001). Regression of liver fat percentage on IPAT fat was significantly greater for FHBL than for controls (P < 0.001). In summary, because apoB-defective FHBL imparts heightened susceptibility to liver triglyceride accumulation, increasing IPAT and insulin resistance exert greater liver fat-increasing effects in FHBL.     

Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins

Glucosamine-induced endoplasmic reticulum (ER) stress was recently shown to specifically reduce apolipoprotein B-100 (apoB-100) secretion by enhancing the proteasomal degradation of apoB-100. Here, we examined the mechanisms linking glucosamine-induced ER stress and apoB-lipoprotein biogenesis. Trypsin sensitivity studies suggested glucosamine-induced changes in apoB-100 conformation. Endoglycosidase H studies of newly synthesized apoB-100 revealed glucosamine induced N-linked glycosylation defects resulting in reduced apoB-100 secretion. We also examined glucosamine-induced changes in VLDL assembly and secretion. A dose-dependent (1-10 mM glucosamine) reduction was observed in VLDL-apoB-100 secretion in primary hepatocytes (24.2-67.3%) and rat McA-RH7777 cells (23.2-89.5%). Glucosamine also inhibited the assembly of larger VLDL-, LDL-, and intermediate density lipoprotein-apoB-100 but did not affect smaller HDL-sized apoB-100 particles. Glucosamine treatment during the chase period (posttranslational) led to a 24% reduction in apoB-100 secretion (P < 0.01; n = 4) and promoted post-ER apoB degradation. However, the contribution of post-ER apoB-100 degradation appeared to be quantitatively minor. Interestingly, the glucosamine-induced posttranslational reduction in apoB-100 secretion could be partially prevented by treatment with desferrioxamine or vitamin E. Together, these data suggest that cotranslational glucosamine treatment may cause defects in apoB-100 N-linked glycosylation and folding, resulting in enhanced proteasomal degradation. Posttranslationally, glucosamine may interfere with the assembly process of apoB lipoproteins, leading to post-ER degradation via nonproteasomal pathways.     

Endoplasmic reticulum stress reduces the export from the ER and alters the architecture of post-ER compartments

In eukaryotic cells several physiologic and pathologic conditions generate the accumulation of unfolded proteins in the endoplasmic reticulum (ER), leading to ER stress. To restore normal function, some ER transmembrane proteins sense the ER stress and activate coordinated signalling pathways collectively called the Unfolded Protein Response (UPR). Little is known on how the UPR relates to post-ER compartments and to the export from the ER of newly synthesized proteins. Here, we report that the ER stress response induced by either thapsigargin or nitric oxide modifies the dynamics of the intracellular distribution of ERGIC-53 and GM130, two markers of the ER Golgi Intermediate Compartment and of the cis-Golgi, respectively. In addition, induction of ER stress alters the morphology of the ERGIC and the Golgi complex and interferes with the reformation of both compartments. Moreover, ER stress rapidly reduces the transport to the Golgi complex of the temperature sensitive mutant of the Vesicular Stomatitis Virus G Glycoprotein (VSV-G) fused with the Green Fluorescent Protein (ts045G), without apparently decreasing the amount of the protein competent for export. Interestingly, a parallel rapid reduction of the number of Sec31 labelled fluorescent puncta on the ER membranes does occur, thus suggesting that the ER stress alters the ER export and the dynamic of post-ER compartments by rapidly targeting the formation of COPII-coated transport intermediates.     

CuZn-SOD deficiency causes ApoB degradation and induces hepatic lipid accumulation by impaired lipoprotein secretion in mice

Elevated hepatic reactive oxygen species play an important role in pathogenesis of liver diseases, such as alcohol-induced liver injury, hepatitis C virus infection, and nonalcoholic steatohepatitis. In the present study, we investigated and compared the hepatic lipid metabolisms of liver-specific Sod2 (superoxide dismutase 2) knock-out (Sod2 KO), Sod1 knock-out (Sod1 KO), and Sod1/liver-specific Sod2 double knock-out mice (double KO). We observed significant increases in lipid peroxidation and triglyceride (TG) in the liver of Sod1 KO and double KO mice but not in the liver of Sod2 KO mice. We also found that high fat diet enhanced fatty changes of the liver in Sod1 KO and double KO mice but not in Sod2 KO mice. These data indicated that CuZn-SOD deficiency caused lipid accumulation in the liver. To investigate the molecular mechanism of hepatic lipid accumulation in CuZn-SOD-deficient mice, we measured TG secretion rate from liver using Triton WR1339. We found significant decrease of TG secretion in CuZn-SOD-deficient mice. Furthermore, we observed marked degradation of apolipoprotein B (apoB) in the liver and plasma of CuZn-SOD-deficient mice, indicating that degradation of apoB impairs secretion of lipoprotein from the liver. Our data suggest that oxidative stress enhances hepatic lipid accumulation by impaired lipoprotein secretion due to the degradation of apoB in liver.     

A targeted apoB38.9 mutation in mice is associated with reduced hepatic cholesterol synthesis and enhanced lipid peroxidation

Familial hypobetalipoproteinemia (FHBL) due to truncation-specifying mutations of apolipoprotein B (apoB), which impair hepatic lipid export in very low-density lipoprotein (VLDL) particles, is associated with fatty liver. In an FHBL-like mouse with the apoB38.9 mutation, fatty liver develops despite reduced hepatic fatty acid synthesis. However, hepatic cholesterol contents in apoB38.9 mice are normal. We found that cholesterogenic enzymes (3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol-C5-desaturase, and 7-dehydrocholesterol reductase) were consistently downregulated in two separate expression-profiling experiments using a total of 19 mice (n = 7 each for apob(+/+) and apob(+/38.9), and n = 5 for apob(38.9/38.9)) and Affymetrix Mu74Av2 GeneChip microarrays. Results were confirmed by real-time PCR. Cholesterol synthesis rates in cultured hepatocytes were reduced by 35% and 25% in apob(38.9/38.9) and apob(+/38.9), respectively, vs. apob(+/+). Hepatic triglycerides and lipid peroxides, the latter measured by thiobarbituric acid-reactive substances (TBARS) assay, were significantly elevated in apob(+/38.9) (117%) and apob(38.9/38.9) (132%) vs. apob(+/+) (100%), as were mRNA expression of the microsomal lipid peroxidizing enzymes Cyp4A10 and Cyp4A14. Hepatic lipid peroxide levels were positively correlated with triglyceride contents (r = 0.601, P = 0.0065). Thus the fatty liver due to a VLDL secretion defect is associated with insufficient adaptation to triglyceride accumulation and with increased lipid peroxidation. In contrast, apoB38.9 mice effectively maintain cholesterol homeostasis in the liver, at least in part, by reducing hepatic cholesterol synthesis.     

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.     

Genotypic associations of the hepatic secretion of VLDL apolipoprotein B-100 in obesity

We examined the effect of genetic polymorphisms of proteins regulating intrahepatic processing of apolipoprotein B-100 (apoB) and the supply of neutral lipids to the liver on the hepatic secretion of very low density lipoprotein (VLDL) apoB in obesity. Hepatic secretion of very low density apolipoprotein B-100 (VLDL apoB) was measured using an infusion of [1-(13)C]leucine in 29 obese men. Isotopic enrichment and turnover of VLDL apoB was determined using gas chromatography-mass spectrometry and multi-compartmental modelling, respectively. Visceral fat was measured by magnetic resonance imaging. Genotypes for the apoB signal peptide (SP27/SP24 alleles), microsomal triglyceride transfer protein promoter (MTP, -493 G/T alleles), apoE (E2, E3, E4 alleles), hepatic lipase promoter (-514 C/T alleles), and cholesteryl ester transfer protein (CETP, Taq1B B1/B2 alleles) were determined using polymerase chain reaction. Statistically significant associations were found between hepatic secretion of apoB and allelic combinations of i) apoB SP with apoE (P = 0.02), hepatic lipase (P = 0.02), and CETP (P = 0. 006) genes, ii) MTP promoter with CETP genes (P = 0.03); the association with apoBSP/MTP promoter allelic combinations just failed to reach significance (P = 0.06), however. The CETP/apoBSP allelic combination was the most significant predictor of apoB secretion, and this was independent of visceral fat, plasma lathosterol and insulin levels, and dietary fat. SP24 carriers who were homozygous for CETP B1 had 60% lower apoB secretion than B2 heterozygotes who were non-carriers of SP24 (10.5 +/- 1.74 mg/kg fat free mass/day, n = 7 vs. 26.1 +/- 3.16, n = 22). The data suggest that variation in both the apoB and CETP genes may be a major genetic determinant of the hepatic secretion of apoB in men with visceral obesity.     

The triple threat to nascent apolipoprotein B. Evidence for multiple, distinct degradative pathways

We previously showed that Omega-3 fatty acids reduce secretion of apolipoprotein B (apoB) from cultured hepatocytes by stimulating post-translational degradation. In this report, we now characterize this process, particularly in regard to the two known processes that degrade newly synthesized apoB, endoplasmic reticulum (ER)-associated degradation and re-uptake from the cell surface. First, we found that Omega-3-induced degradation preferentially reduces the secretion of large, assembled apoB-lipoprotein particles, and apoB polypeptide length is not a determinant. Second, based on several experimental approaches, ER-associated degradation is not involved. Third, re-uptake, the only process known to destroy fully assembled nascent lipoproteins, was clearly active in primary hepatocytes, but Omega-3-induced degradation of apoB continued even when re-uptake was blocked. Cell fractionation showed that Omega-3 fatty acids induced a striking loss of apoB100 from the Golgi, while sparing apoB100 in the ER, indicating a post-ER process. To determine the signaling involved, we used wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, which blocked most, if not all, of the Omega-3 fatty acid effect. Therefore, nascent apoB is subject to ER-associated degradation, re-uptake, and a third distinct degradative pathway that appears to target lipoproteins after considerable assembly and involves a post-ER compartment and PI3K signaling. Physiologic, pathophysiologic, and pharmacologic regulation of net apoB secretion may involve alterations in any of these three degradative steps.     

Different fatty acids inhibit apoB100 secretion by different pathways: unique roles for ER stress, ceramide, and autophagy

Although short-term incubation of hepatocytes with oleic acid (OA) stimulates secretion of apolipoprotein B100 (apoB100), exposure to higher doses of OA for longer periods inhibits secretion in association with induction of endoplasmic reticulum (ER) stress. Palmitic acid (PA) induces ER stress, but its effects on apoB100 secretion are unclear. Docosahexaenoic acid (DHA) inhibits apoB100 secretion, but its effects on ER stress have not been studied. We compared the effects of each of these fatty acids on ER stress and apoB100 secretion in McArdle RH7777 (McA) cells: OA and PA induced ER stress and inhibited apoB100 secretion at higher doses; PA was more potent because it also increased the synthesis of ceramide. DHA did not induce ER stress but was the most potent inhibitor of apoB100 secretion, acting via stimulation of autophagy. These unique effects of each fatty acid were confirmed when they were infused into C57BL6J mice. Our results suggest that when both increased hepatic secretion of VLDL apoB100 and hepatic steatosis coexist, reducing ER stress might alleviate hepatic steatosis but at the expense of increased VLDL secretion. In contrast, increasing autophagy might reduce VLDL secretion without causing steatosis.     

Expression of carboxyl-terminally truncated forms of human apolipoprotein B in rat hepatoma cells. Evidence that the length of apolipoprotein B has a major effect on the buoyant density of the secreted lipoproteins

We examined the relationship between the size of human apolipoprotein (apo) B and the formation and secretion of apoB-containing lipoprotein particles. Stable transformants of the rat hepatoma cell line McA-RH7777 harboring a variety of human apoB cDNA constructs were established, and these produced carboxyl-terminally truncated apoB proteins (apoB18, -B23, -B28, -B31, -B48, and -B53). Immunoblotting of apoB proteins secreted into the culture medium and fractionated by equilibrium density ultracentrifugation revealed that each of the truncated apoB species was secreted from the cells. The peak densities of the apoB-containing particles decreased as the length of the apoB proteins increased. Apolipoproteins B18 and B23 appeared at the bottom of the salt gradient (d = 1.23 g/ml), whereas particles containing apoB28, -B31, -B37, -B48, and -B53 exhibited progressive decreases in density. The density distribution of secreted apolipoproteins was not affected by the expression or secretion of these recombinant apoB species. As determined by nondenaturing gel electrophoresis, apoB28, -B31, -B37, -B48, and -B53 formed their own discrete particles, and there was a direct correlation between the size of the particles and the length of the apoB species. The efficiency and rate of secretion of these truncated forms of apoB were studied by measuring the decrease of immunoprecipitated 35S-labeled apoB proteins in the cells and their accumulation in the medium. Proteins corresponding to apoB28 or larger were rapidly and efficiently secreted, whereas apoB18 and apoB23 were secreted much more slowly. These data imply that the size of these truncated apoB forms governs the lipid content of the apoB-containing lipoproteins formed as well as the kinetics of secretion.     

Familial hypobetalipoproteinemia: a review

We review the genetics and pathophysiology of familial hypobetalipoproteinemia (FHBL), a mildly symptomatic genetically heterogeneous autosomal trait. The minority of human FHBL is caused by truncation-specifying mutations of the APOB gene on chromosome 2. In seven families, linkage to chromosome 2 is absent, linkage is instead to chromosome 3 (3p21). In others, linkage is absent to both APOB and to 3p21. Apolipoprotein B-100 (apoB-100) levels are approximately 25% of normal, instead of the 50% expected based on the presence of one normal allele due to reduced rates of production. The presence of the truncating mutation seems to have a "dominant recessive" effect on apoB-100 secretion. Concentrations of apoB truncations in plasma differ by truncation but average at approximately 10% of normal levels. Lipoproteins bearing truncated forms of apoB are cleared more rapidly than apoB-100 particles. In contrast with apoB-100 particles cleared primarily in liver via the LDL receptor, most apoB truncation particles are cleared in renal proximal tubular cells via megalin. Since apoB defects cause a dysfunctional VLDL-triglyceride transport system, livers accumulate fat. Hepatic synthesis of fatty acids is reduced in compensation. Informational lacunae remain about genes affecting fat accumulation in liver, and the modulation of liver fat in the presence apoB truncation defects.