miR-130b is a potent stimulator of hepatic very-low-density lipoprotein assembly and secretion via marked induction of microsomal triglyceride transfer protein.

miR-130b is a microRNA whose expression is particularly elevated within adipose tissue and in the circulation in diabetic states. Hepatic miR-130b expression has been linked to hepatocellular carcinoma and changes in lipid metabolism. Here, we investigated the role of miR-130b in hepatic lipid homeostasis and lipoprotein export. We observed that overexpression of miR-130b-3p or -5p in HepG2 cells markedly enhanced the secretion of very-low-density lipoprotein (VLDL) particles, enhanced the secretion of [3H]glycerol metabolically labeled triglyceride (TG), and significantly increased the number or the average size of lipid droplets (LDs), respectively. Overexpression of miR-130b also altered the expression of key genes involved in lipid metabolism and in particular markedly increased both mRNA and protein expression levels of microsomal triglyceride transfer protein (MTP). Conversely, the miR-130b inhibitor decreased mRNA levels of MTP and fatty acid synthase (FAS) in HepG2 cells. However, dual-luciferase reporter assays indicated that MTP is not a direct target of miR-130b-3p. miR-130b overexpression did not alter de novo synthesized TG or the stability and secretion of apolipoprotein B 100. Interestingly, knockdown of phosphatase and tensin homolog (PTEN) blocked the upregulation of MTP mRNA induced by miR-130b. Finally, miR-130b-induced stimulation of VLDL secretion was also observed in a second hepatocyte cell culture model, immortalized human hepatocytes, confirming the effects observed in HepG2 cells. Overall, these data suggest a potential role for miR-130b in promoting hepatic VLDL assembly and secretion mediated by marked stimulation of MTP expression and TG mobilization. Thus miR-130b overexpression corrects the defect in VLDL production in HepG2 cells.

Lomitapide and Mipomersen-Inhibiting Microsomal Triglyceride Transfer Protein (MTP) and apoB100 Synthesis.

PURPOSE OF REVIEW: The goal of this review is to evaluate the role of inhibiting the synthesis of lipoproteins when there is no or little residual LDL-receptor function as in patients with homozygous familial hypercholesterolaemia. Lomitapide is administered orally once a day while mipomersen is given by subcutaneous injection once a week. Lomitapide inhibits microsomal triglyceride transfer protein while mipomersen is an antisense oligonucleotide directed against apoB100. RECENT FINDINGS: The pivotal registration trials for lomitapide and mipomersen were published in 2013 and 2010, respectively. More recently published data from extension trials and cohort studies provides additional information on long-term safety and efficacy. The mean LDL cholesterol reduction was 50% with lomitapide in its single-arm open-label registration trial. Mipomersen reduced LDL cholesterol by approximately 25% in its double-blind, placebo-controlled registration study. Both lomitapide and mipomersen therapy are associated with variable increases in hepatic fat content. The long-term safety of increased hepatic fat content in patients receiving these therapies is uncertain and requires further study. Both drugs may cause elevated transaminase in some patients, but no cases of severe liver injury have been reported. Lomitapide may also cause gastrointestinal discomfort and diarrhoea, especially if patients consume high-fat meals and patients are advised to follow a low-fat diet supplemented with essential fatty acids and fat-soluble vitamins. Mipomersen may cause injection-site and influenza-like reactions. The effect of lomitapide and mipomersen on cardiovascular outcomes has not been studied, but circumstantial evidence suggests that the LDL cholesterol lowering achieved with these two agents may reduce cardiovascular event rates.

The degradation of apolipoprotein B100: multiple opportunities to regulate VLDL triglyceride production by different proteolytic pathways.

Very low density lipoproteins (VLDL) are a major secretory product of the liver. They serve to transport endogenously synthesized lipids, mainly triglycerides (but also some cholesterol and cholesteryl esters) to peripheral tissues. VLDL is also the precursor of LDL. ApoB100 is absolutely required for VLDL assembly and secretion. The amount of VLDL triglycerides secreted by the liver depends on the amount loaded onto each lipoprotein particle, as well as the number of particles. Each VLDL has one apoB100 molecule, making apoB100 availability a key determinant of the number of VLDL particles, and hence, triglycerides, that can be secreted by hepatic cells. Surprisingly, the pool of apoB100 in the liver is typically regulated not by its level of synthesis, which is relatively constant, but by its level of degradation. It is now recognized that there are multiple opportunities for the hepatic cell to intercept apoB100 molecules and to direct them to distinct degradative processes. This mini-review will summarize progress in understanding these processes, with an emphasis on autophagy, the most recently described pathway of apoB100 degradation, and the one with possibly the most physiologic relevance to common metabolic perturbations affecting VLDL production. This article is part of a Special Issue entitled Triglyceride Metabolism and Disease.

Glycosyltransferase GLT8D2 positively regulates ApoB100 protein expression in hepatocytes.

Non-alcoholic fatty liver disease (NAFLD) is characterized by triglyceride (TG) accumulation in hepatocytes. Very low density lipoprotein (VLDL) is a major secretory product of the liver that transports endogenously synthesized TG. Disrupted VLDL secretion may contribute to the accumulation of TG in hepatocytes. ApoB100 (apolipoprotein B100) is a glycoprotein and an essential protein component of VLDL. Its glycosylation may affect VLDL assembly and secretion. However, which glycosyltransferase catalyzes apoB100 glycosylation is unknown. In this study, we cloned the GLT8D2 (glycosyltransferase 8 domain containing 2) gene from HepG2 cells and generated a series of plasmids for in vitro studies of its molecular functions. We discovered that GLT8D2 was localized in the ER, interacted with apoB100, and positively regulated the levels of apoB100 protein in HepG2 cells. Based on these results, we propose that GLT8D2 is a glycosyltransferase of apoB100 that regulates apoB100 levels in hepatocytes.

Measuring H(2)(18)O tracer incorporation on a QQQ-MS platform provides a rapid, transferable screening tool for relative protein synthesis.

Intracellular proteins are in a state of flux, continually being degraded into amino acids and resynthesized into new proteins. The rate of this biochemical recycling process varies across proteins and is emerging as an important consideration in drug discovery and development. Here, we developed a triple-stage quadrupole mass spectrometry assay based on product ion measurements at unit resolution and H(2)(18)O stable tracer incorporation to measure relative protein synthesis rates. As proof of concept, we selected to measure the relative in vivo synthesis rate of ApoB100, an apolipoprotein where elevated levels are associated with an increased risk of coronary heart disease, in plasma-isolated very low density lipoprotein (VLDL) and low density lipoprotein (LDL) in a mouse in vivo model. In addition, serial time points were acquired to measure the relative in vivo synthesis rate of mouse LDL ApoB100 in response to vehicle, microsomal triacylglycerol transfer protein (MTP) inhibitor, and site-1 protease inhibitor, two potential therapeutic targets to reduce plasma ApoB100 levels at 2 and 6 h post-tracer-injection. The combination of H(2)(18)O tracer with the triple quadrupole mass spectrometry platform creates an assay that is relatively quick and inexpensive to transfer across different biological model systems, serving as an ideal rapid screening tool for relative protein synthesis in response to treatment.

Measurement of fractional synthetic rates of multiple protein analytes by triple quadrupole mass spectrometry.

BACKGROUND: Current approaches to measure protein turnover that use stable isotope-labeled tracers via GC-MS are limited to a small number of relatively abundant proteins. We developed a multiplexed liquid chromatography-selected reaction monitoring mass spectrometry (LC-SRM) assay to measure protein turnover and compared the fractional synthetic rates (FSRs) for 2 proteins, VLDL apolipoprotein B100 (VLDL apoB100) and HDL apoA-I, measured by both methods. We applied this technique to other proteins for which kinetics are not readily measured with GC-MS. METHODS: Subjects were given a primed-constant infusion of [5,5,5-D(3)]-leucine (D(3)-leucine) for 15 h with blood samples collected at selected time points. Apolipoproteins isolated by SDS-PAGE from lipoprotein fractions were analyzed by GC-MS or an LC-SRM assay designed to measure the M+3/M+0 ratio at >1% D(3)-leucine incorporation. We calculated the FSR for each apolipoprotein by curve fitting the tracer incorporation data from each subject. RESULTS: The LC-SRM method was linear over the range of tracer enrichment values tested and highly correlated with GC-MS (R(2) > 0.9). The FSRs determined from both methods were similar for HDL apoA-I and VLDL apoB100. We were able to apply the LC-SRM approach to determine the tracer enrichment of multiple proteins from a single sample as well as proteins isolated from plasma after immunoprecipitation. CONCLUSIONS: The LC-SRM method provides a new technique for measuring the enrichment of proteins labeled with stable isotopes. LC-SRM is amenable to a multiplexed format to provide a relatively rapid and inexpensive means to measure turnover of multiple proteins simultaneously.

Nutritional repletion of children with severe acute malnutrition does not affect VLDL apolipoprotein B-100 synthesis rate.

VLDL apo B-100 is essential for the secretion of liver fat. It is thought that synthesis of this lipoprotein is impaired in childhood severe acute malnutrition (SAM), especially in the edematous syndromes, and that this contributes to the common occurrence of hepatic steatosis in this condition. However, to our knowledge, it has not been confirmed that VLDL apo B-100 synthesis is slower in edematous compared with nonedematous SAM and that it is impaired in the malnourished compared with the well-nourished state. Therefore, VLDL apo B-100 kinetics were measured in 2 groups of children with SAM (15 edematous and 7 nonedematous), aged 4-20 mo, at 3 stages during treatment. Measurements were done at 4 ± 1 d postadmission, mid- catch-up growth in weight, and at recovery (normal weight-for-length). VLDL apo B-100 synthesis was determined using stable isotope methodology to measure the rate of incorporation of (2)H(3)-leucine into its apoprotein moiety. The fractional and absolute synthesis of VLDL apo B-100 did not differ between the groups or from the initial malnourished stage to the recovery stage. Concentrations of VLDL apo B-100 were greater in the edematous than in the nonedematous group (P < 0.04) and did not differ from the initial stage to recovery. The data indicate that VLDL apo B-100 synthesis is not reduced when children develop either edematous or nonedematous SAM.

Effects of doxorubicin on myocardial expression of apolipoprotein-B.

OBJECTIVE: Doxorubicin (DOX) is an effective antitumour agent against a variety of human malignancies but is associated with deleterious side effects, including myocardial damage and heart failure. Myocardial apoB-containing lipoprotein (apoB) is upregulated post myocardial infarction and has been shown to be cardioprotective in this setting by unloading excessive lipid. The aim of this study was to investigate whether apoB expression is increased also in DOX-induced heart failure and whether apoB overexpression protects the heart in DOX-induced myocardial injury. DESIGN: Cardiac function and energy metabolism was studied in mice and rats 24 hours after intraperitoneally administered DOX. RESULTS: We found that the content of apoB was decreased in rat myocardium 24 hours after DOX injection. In contrast, apoB content was increased in the infarcted myocardium of rats 24 hours post ischemia-reperfusion. Moreover, transgenic mice overexpressing apoB had better cardiac function and lower intracellular lipid accumulation compared to wild type mice 24 hours post DOX. CONCLUSIONS: Our findings indicate that depression of the myocardial apoB system may contribute to DOX-induced cardiac injury and that overexpression of apoB is protective, not only in ischemically damaged myocardium, but also in DOX-induced heart failure.

Differentially Regulated Apolipoproteins and Lipid Profiles as Novel Biomarkers for Polypoidal Choroidal Vasculopathy and Neovascular Age-Related Macular Degeneration.

Lipid dyshomeostasis has been implicated in the pathogenesis of various retinal and choroidal vascular diseases. This study aims to investigate whether apolipoprotein (apo) mediated differential regulation of lipid metabolism contributes to the phenotypes of polypoidal choroidal vasculopathy (PCV) and neovascular age-related macular degeneration (nAMD). This study involved 148 subjects including 53 patients with PCV, 44 patients with nAMD, and 51 age-, sex-matched subjects with normal fundus controls. Routine blood biochemistry profile was evaluated. Apolipoproteins was estimated by Luminex technology. After controlling for age, gender, body mass index, duration of hypertension and type 2 diabetes mellitus, apoB/non-high density lipoprotein cholesterol (HDL-C) (p=0.015) was an independent risk factor for nAMD, apoB was an independent risk factor for PCV(p=0.011), compared with control. Low-density lipoprotein cholesterol (LDL-C) was significantly higher in patients with PCV when compared with nAMD (p=0.037). Furthermore, apoB/non-HDL, LDL-C, triglycerides and were significantly correlated with the pathogenesis of subgroups of PCV and nAMD. We concluded that lipid profiles and apos are differential regulated in PCV, nAMD and their subtypes, indicating different pathogenicity contributed to the different phenotypes of PCV and nAMD. Non-pachy PCV shares pathological similarities with nAMD, which is highly correlated with age-related atherosclerosis.

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.

Mipomersen, an apolipoprotein B synthesis inhibitor, for lowering of LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Homozygous familial hypercholesterolaemia is a rare genetic disorder in which both LDL-receptor alleles are defective, resulting in very high concentrations of LDL cholesterol in plasma and premature coronary artery disease. This study investigated whether an antisense inhibitor of apolipoprotein B synthesis, mipomersen, is effective and safe as an adjunctive agent to lower LDL cholesterol concentrations in patients with this disease. METHODS: This randomised, double-blind, placebo-controlled, phase 3 study was undertaken in nine lipid clinics in seven countries. Patients aged 12 years and older with clinical diagnosis or genetic confirmation of homozygous familial hypercholesterolaemia, who were already receiving the maximum tolerated dose of a lipid-lowering drug, were randomly assigned to mipomersen 200 mg subcutaneously every week or placebo for 26 weeks. Randomisation was computer generated and stratified by weight (<50 kg vs >/=50 kg) in a centralised blocked randomisation, implemented with a computerised interactive voice response system. All clinical, medical, and pharmacy personnel, and patients were masked to treatment allocation. The primary endpoint was percentage change in LDL cholesterol concentration from baseline. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00607373. FINDINGS: 34 patients were assigned to mipomersen and 17 to placebo; data for all patients were analysed. 45 patients completed the 26-week treatment period (28 mipomersen, 17 placebo). Mean concentrations of LDL cholesterol at baseline were 11.4 mmol/L (SD 3.6) in the mipomersen group and 10.4 mmol/L (3.7) in the placebo group. The mean percentage change in LDL cholesterol concentration was significantly greater with mipomersen (-24.7%, 95% CI -31.6 to -17.7) than with placebo (-3.3%, -12.1 to 5.5; p=0.0003). The most common adverse events were injection-site reactions (26 [76%] patients in mipomersen group vs four [24%] in placebo group). Four (12%) patients in the mipomersen group but none in the placebo group had increases in concentrations of alanine aminotransferase of three times or more the upper limit of normal. INTERPRETATION: Inhibition of apolipoprotein B synthesis by mipomersen represents a novel, effective therapy to reduce LDL cholesterol concentrations in patients with homozygous familial hypercholesterolaemia who are already receiving lipid-lowering drugs, including high-dose statins. FUNDING: ISIS Pharmaceuticals and Genzyme Corporation.

Apolipoprotein B100 biogenesis: a complex array of intracellular mechanisms regulating folding, stability, and lipoprotein assembly.

Apolipoprotein B100 (apoB) is a large amphipathic lipid-binding protein that is synthesized by hepatocytes and used to assemble and stabilize very low density lipoproteins (VLDL). It may have been derived through evolution from other lipid-associating proteins such as microsomal triglyceride transfer protein or vitellogenin. The correct folding of apoB requires assistance from chaperone proteins in co-translational lipidation, disulfide bond formation, and glycosylation. Any impairment in these processes results in co-translational targeting of the misfolded apoB molecule for proteasomal degradation. In fact, most of the regulation of apoB production is mediated by intracellular degradation. ApoB that misfolds post-translationally, perhaps as a result of oxidative stress, may be eliminated through autophagy. This review focuses on the proposed pentapartite domain structure of apoB, the role that each domain plays in the binding of lipid species and regulation of apoB synthesis, and the process of VLDL assembly. The factors involved in the recognition, ubiquitination, and proteasomal delivery of defective apoB molecules are also discussed.

Glucosamine-induced endoplasmic reticulum stress attenuates apolipoprotein B100 synthesis via PERK signaling.

Glucosamine impairs hepatic apolipoprotein B100 (apoB100) production by inducing endoplasmic reticulum (ER) stress and enhancing cotranslational and posttranslational apoB100 degradation (Qiu, W., R. K. Avramoglu, A. C. Rutledge, J. Tsai, and K. Adeli. Mechanisms of glucosamine-induced suppression of the hepatic assembly and secretion of apolipoprotein B-100-containing lipoproteins. J. Lipid Res. 2006. 47: 1749-1761). Here, we report that glucosamine also regulates apoB100 protein synthesis via ER-stress-induced PERK activation. Short-term (4 h) glucosamine treatment of HepG2 cells reduced both cellular (by 62%) and secreted apoB100 (by 43%) without altering apoB100 mRNA. Treatment with proteasomal inhibitors only partially prevented the suppressive effects of glucosamine, suggesting that mechanisms other than proteasomal degradation may also be involved. Glucosamine-induced ER stress was associated with a significantly reduced apoB100 synthesis with no significant change in posttranslational decay rates, suggesting that glucosamine exerted its effect early during apoB biosynthesis. The role of PERK and its substrate, alpha-subunit of eukaryotic initiation factor 2 (eIF2alpha), in the suppressive effects of glucosamine on apoB synthesis was then investigated. Coexpression of apoB15 (normally resistant to intracellular degradation) with wild-type double stranded (ds) RNA activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK) in COS-7 cells resulted in a dramatic reduction in the levels of newly synthesized apoB15. Interestingly, cotransfection with apoB15 and a kinase inactive PERK mutant (K618A) increased apoB15 expression. In addition, short-term glucosamine treatment stimulated an increase in phosphorylation of PERK and eIF2alpha. Taken together, these data suggest that in addition to the induction of ER-associated degradation and other degradative pathways, ER stress is associated with suppression of apoB synthesis via a PERK-dependent mechanism.

HPLC analysis of lipoproteins in culture medium of hepatoma cells: an in vitro system for screening antihyperlipidemic drugs.

We isolated a HepG2-derived sub-clone (HepG2-Lipo), which possessed an increased lipoprotein synthesizing ability. HepG2-Lipo cells could secrete triglycerides (TG) and cholesterol at rates 9.4- and 6-fold higher, respectively, when compared to HepG2 cells. Real-time RT-PCR analysis revealed that the expression levels of sterol regulatory element-binding protein-1c and -2 were 2.9- and 1.5-fold higher than in HepG2 cells. Furthermore, two apolipoprotein (apo) genes (apoA-1 and apoB-100) in HepG2-Lipo cells were expressed at 2.8- and 1.9-fold higher levels when compared to those in parental cells. We examined the effects of three antihyperlipidemic agents on the lipoprotein profiles of HepG2-Lipo cells. Simvastatin at 5 microM selectively suppressed cholesterol secretion from HepG2-Lipo cells, and 500 microM fenofibrate inhibited both TG and cholesterol secretion from the cells.

Dissociation between the insulin-sensitizing effect of rosiglitazone and its effect on hepatic and intestinal lipoprotein production.

CONTEXT: Despite its potent, well-documented insulin-sensitizing effects, rosiglitazone (RSG) does not effectively ameliorate the hypertriglyceridemia of insulin-resistant or diabetic individuals and has even been shown to slightly but significantly increase triglyceride-rich lipoproteins (TRL) in some studies. The mechanism of this effect is currently not known. OBJECTIVE: We investigated the effect of RSG treatment on TRL metabolism. DESIGN: This was a 12-wk, single-sequence, cross-over study of rosiglitazone vs. placebo for 6 wk. PARTICIPANTS: Participants included 17 nondiabetic men with a broad range of insulin sensitivity. INTERVENTION: INTERVENTION included rosiglitazone 8 mg/d vs. placebo for 6 wk. MAIN OUTCOME MEASURE: TRL metabolism (concentration, production and catabolic rates) was assessed in a constant fed state with a 12-h primed constant infusion of [D3]l-leucine and multicompartmental modeling. RESULTS: RSG treatment resulted in significant insulin sensitization with no change in body weight. Fasting plasma triglyceride (TG) concentration, however, was higher with RSG vs. placebo (P = 0.0006), as were fasting and fed TRL-TG, TRL-apoB-48, and TRL-apoB-100 (fed TRL-apoB-48: 0.93 +/- 0.08 vs. 0.76 +/- 0.07 mg/dl, P =0.017, and fed TRL-apoB-100: 15.57 +/- 0.90 vs. 13.71 +/- 1.27 mg/dl, P = 0.029). This small but significant increase in plasma TRL concentration was explained by a tendency for RSG to increase TRL production and reduce particle clearance, as indicated by the significantly increased production to clearance ratios for both apoB-48-containing (0.43 +/- 0.03 vs. 0.34 +/- 0.03, P = 0.048) and apoB-100-containing (7.0 +/- 0.4 vs. 6.2 +/- 0.6, P = 0.029) TRL. CONCLUSION: These data indicate dissociation between the insulin-sensitizing effects of RSG and absence of anticipated reductions in production rates of apoB-100- and apoB-48-containing-TRL particles, which may explain the absence of TG lowering seen in humans treated with this agent.

Comparison of effects of anagliptin and alogliptin on serum lipid profile in type 2 diabetes mellitus patients.

INTRODUCTION: Anagliptin (ANA) improves dyslipidemia in addition to blood glucose levels. However, there are no comparative studies on the effects of ANA and other dipeptidyl peptidase-4 inhibitors on serum lipid profile. We compared the effects of ANA on serum lipid profile with those of alogliptin (ALO) in type 2 diabetes mellitus outpatients. MATERIALS AND METHODS: The study participants were 87 type 2 diabetes mellitus patients who had been treated with dipeptidyl peptidase-4 inhibitors for ≥8 weeks and had a low-density lipoprotein cholesterol (LDL-C) level of ≥120 mg/dL. Participants were switched to either 200 mg/day ANA or 25 mg/day ALO for 24 weeks. RESULTS: There was no significant difference in percentage change in LDL-C level at 24 weeks between the ANA and ALO groups. Treatment with ANA for 12 weeks significantly decreased LDL-C levels, one of the secondary end-points. Treatment with ANA for 24 weeks significantly improved apolipoprotein B-100 levels, and the percentage change in LDL-C levels at 24 weeks correlated significantly with the percentage change in apolipoprotein B-100 levels in the ANA group. CONCLUSIONS: The LDL-C-lowering effects of ANA and ALO at 24 weeks were almost similar in patients with type 2 diabetes mellitus. However, the results showed a tendency for a decrease in LDL-C level at 24 weeks in the ANA group, and that such improvement was mediated, at least in part, through the suppression of apolipoprotein B-100 synthesis.

[Expression profiles of lipid metabolism-related genes in liver of apoE(-/-)/LDLR(-/-) mice].

OBJECTIVE: To explore the relationship between the expression characteristics of lipid metabolism-related genes in the liver and early atherosclerotic lesions in apolipoprotein E and low density lipoprotein receptor gene double knockout (apoE(-/-)/LDLR(-/-)) mice. METHODS: RT-PCR was used to detect the differential expression of lipid metabolism-related genes in the liver of apoE(-/-)/LDLR(-/-) and wild type (WT) mice. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) level as well as aortic morphology were also analyzed. RESULTS: Among the 11 lipid metabolism-related genes, apolipoprotein B100 (apoB100) mRNA levels were significantly higher in apoE(-/-)/LDLR(-/-)mice compared with WT mice. At 14 days, 1, 2 and 3 months of age, the level of mRNA expression were 1.55, 1.47, 1.50 and 2.42 folds of those of the age matched WT mice respectively. The fatty acid transporter (FAT/CD36) mRNA expression levels were higher in 14-day and 3-month old mice at 1.30 and 1.35 folds of those of the age matched WT mice, respectively. Apolipoprotein A IV (apoA IV) and Apolipoprotein AV (apoAV) mRNA levels were significantly down-regulated (0.89 fold decrease in 14-day, and 0.90 folds decrease in 3-month, respectively). The mRNA expression levels of apolipoprotein AI (apo AI), apolipoprotein F (apo F), peroxidase proliferator-activated receptor alpha (PPAR-alpha), liver X receptor alpha (LXRalpha), angiopoietin-like protein 3 (ANGPTL3), acyl-coenzymeA oxidase 1 (ACOX1) and carnitine palmitoyl transferase 1 (CPT1) had no significant changes. Serum TC, TG and LDL-C were higher than those of age matched WT mice at 7, 2 and 30 folds, respectively. Furthermore, apoE(-/-)/LDLR(-/-) mice demonstrated typical early atherosclerotic lesions at sinus and root regions of aorta in an age dependent manner. CONCLUSION: Alterations of the expression of lipid metabolism-related genes in liver play important roles in the development of AS in the apoE(-/-)/LDLR(-/-) mice at early ages.

Distinct metabolism of apolipoproteins (a) and B-100 within plasma lipoprotein(a).

OBJECTIVES: Lipoprotein(a) [Lp(a)] is mainly similar in composition to LDL, but differs in having apolipoprotein (apo) (a) covalently linked to apoB-100. Our purpose was to examine the individual metabolism of apo(a) and apoB-100 within plasma Lp(a). MATERIALS AND METHODS: The kinetics of apo(a) and apoB-100 in plasma Lp(a) were assessed in four men with dyslipidemia [Lp(a) concentration: 8.9-124.7nmol/L]. All subjects received a primed constant infusion of [5,5,5-(2)H3] L-leucine while in the constantly fed state. Lp(a) was immunoprecipitated directly from whole plasma; apo(a) and apoB-100 were separated by gel electrophoresis; and isotopic enrichment was determined by gas chromatography/mass spectrometry. RESULTS: Multicompartmental modeling analysis indicated that the median fractional catabolic rates of apo(a) and apoB-100 within Lp(a) were significantly different at 0.104 and 0.263 pools/day, respectively (P=0.04). The median Lp(a) apo(a) production rate at 0.248nmol/kg·day(-1) was significantly lower than that of Lp(a) apoB-100 at 0.514nmol/kg·day(-1) (P=0.03). CONCLUSION: Our data indicate that apo(a) has a plasma residence time (11days) that is more than twice as long as that of apoB-100 (4days) within Lp(a), supporting the concept that apo(a) and apoB-100 within plasma Lp(a) are not catabolized from the bloodstream as a unit in humans in the fed state.

Identification of protein disulfide isomerase 1 as a key isomerase for disulfide bond formation in apolipoprotein B100.

Apolipoprotein (apo) B is an obligatory component of very low density lipoprotein (VLDL), and its cotranslational and posttranslational modifications are important in VLDL synthesis, secretion, and hepatic lipid homeostasis. ApoB100 contains 25 cysteine residues and eight disulfide bonds. Although these disulfide bonds were suggested to be important in maintaining apoB100 function, neither the specific oxidoreductase involved nor the direct role of these disulfide bonds in apoB100-lipidation is known. Here we used RNA knockdown to evaluate both MTP-dependent and -independent roles of PDI1 in apoB100 synthesis and lipidation in McA-RH7777 cells. Pdi1 knockdown did not elicit any discernible detrimental effect under normal, unstressed conditions. However, it decreased apoB100 synthesis with attenuated MTP activity, delayed apoB100 oxidative folding, and reduced apoB100 lipidation, leading to defective VLDL secretion. The oxidative folding-impaired apoB100 was secreted mainly associated with LDL instead of VLDL particles from PDI1-deficient cells, a phenotype that was fully rescued by overexpression of wild-type but not a catalytically inactive PDI1 that fully restored MTP activity. Further, we demonstrate that PDI1 directly interacts with apoB100 via its redox-active CXXC motifs and assists in the oxidative folding of apoB100. Taken together, these findings reveal an unsuspected, yet key role for PDI1 in oxidative folding of apoB100 and VLDL assembly.