Familial hypercholesterolemia (FH) is a genetic disorder characterized by extremely high levels of plasma low-density lipoprotein (LDL), due to defective LDL receptor-Apolipoprotein B (APOB) binding. Current therapies such as statins or LDL apheresis for homozygous FH are insufficiently efficacious at lowering LDL cholesterol or are expensive. Treatments that target APOB100, the structural protein of LDL particles, are potential therapies for FH. We have developed a series of APOB-directed splice-switching oligonucleotides (SSOs) that cause the expression of APOB87, a truncated isoform of APOB100. APOB87, like similarly truncated isoforms expressed in patients with a different condition, familial hypobetalipoproteinemia, lowers LDL cholesterol by inhibiting VLDL assembly and increasing LDL clearance. We demonstrate that these “APO-skip” SSOs induce high levels of exon skipping and expression of the APOB87 isoform, but do not substantially inhibit APOB48 expression in cell lines. A single injection of an optimized APO-skip SSO into mice transgenic for human APOB resulted in abundant exon skipping that persists for more than 6 d. Weekly treatments generated a sustained reduction in LDL cholesterol levels of 34-51% in these mice, superior to Pravastatin in a head-to-head comparison. These results validate APO-skip SSOs as a candidate therapy for FH.
apolipoprotein B; exon skipping; familial hypercholesterolemia; oligoribonucleotides; quantitative reverse-transcription polymerase chain reaction; splice-switching oligonucleotides
Apolipoprotein B (apoB) containing lipoproteins, i.e. VLDL, LDL and Lp(a), are consequently lowered by ACTH treatment in humans. This is also seen as reduced plasma apoB by 20-30% and total cholesterol by 30-40%, mostly accounted for by a decrease in LDL-cholesterol. Studies in hepatic cell line (HepG2) cells showed that apoB mRNA expression is reduced in response to ACTH incubation and is followed by a reduced apoB secretion, which may hypothesize that ACTH lowering apoB containing lipoproteins in humans may be mediated by the inhibition of hepatic apoB synthesis. This was recently confirmed in vivo in a human postprandial study, where ACTH reduced transient apoB48 elevation from the small intestine, however, the exogenic lipid turnover seemed unimpaired. In the present study we investigated if lipid synthesis and/or secretion in HepG2 cells were also affected by pharmacological levels of ACTH to accompany the reduced apoB output. HepG2 cells were incubated with radiolabelled precursors ([14C]acetate and [3H]glycerol) either before or during ACTH stimuli. Cellular and secreted lipids were extracted with chloroform:methanol and separated by the thin layer chromatography (TLC), and [14C]labelled cholesterol and cholesteryl ester and [3H]labelled triglycerides and phospholipids were quantitated by the liquid scintillation counting. It demonstrated that ACTH administration did not result in any significant change in neither synthesis nor secretion of the studied lipids, this regardless of presence or absence of oleic acid, which is known to stabilize apoB and enhance apoB production. The present study suggests that ACTH lowers plasma lipids in humans mainly mediated by the inhibition of apoB synthesis and did not via the reduced lipid synthesis.
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.
kinetics; stable isotopes; triglyceride-rich lipoproteins; apolipoprotein B-48; apolipoprotein B-100
We have developed a double antibody radioimmunoassay (RIA) for human apolipoprotein B (ApoB). The assay measures not only the ApoB content of β-lipoproteins (low density lipoproteins [LDL]) but also that contained in the other lipoproteins in plasma.
Purified lymph and plasma chylomicrons and plasma very low density lipoproteins (VLDL) produced displacement curves in the assay system which paralleled those produced by pure LDL. Thus, the ApoB found in chylomicrons, VLDL, and LDL were immunologically identical. ApoB accounted for about 25 and 35%, respectively, of the total protein of chylomicrons and VLDL by RIA. VLDL and LDL preparations from normal and hyperlipoproteinemic subjects also produced parallel displacement curves, suggesting that the ApoB of normal and hyperlipoproteinemic subjects were immunologically identical. High density lipoproteins and abetalipoproteinemic plasma displaced no counts, nor did the sera of several animal species produce any useful displacement curves in this system.
The fasting total plasma ApoB concentration of normal subjects was 83±16 mg/dl (mean±SD). ApoB levels were high in Type II (162±16), and less so in Type IV (112±24) and Type V (105±17).
When plasma ApoB concentration in Type IV patients was graphed against plasma glycerides, two subpopulations, which may represent different genetic or biochemical subgroups, were apparent.
ApoB concentration in individuals on constant diet and drug regimen was stable over weeks to months. Greater than 90% of ApoB of normal and Type II subjects was in the d > 1.006 plasma fraction. By contrast, only 50-80% of ApoB was in the d > 1.006 fraction in Types IV and V. Thus, hypertriglyceridemia was associated primarily with a redistribution of ApoB to the lighter density fractions; by contrast, in hypercholesterolemia absolute ApoB concentration was markedly increased.
Apolipoprotein (apo) B is essential for the assembly and secretion of triglyceride-rich lipoproteins made by the liver. As the sole protein component in LDL, apoB is an important determinant of atherosclerosis susceptibility and a potential pharmaceutical target. Single-chain antibodies (sFvs) are the smallest fragment of an IgG molecule capable of maintaining the antigen binding specificity of the parental antibody. In the present study, we describe the cloning and construction of two intracellular antibodies (intrabodies) to human apoB. We targeted these intrabodies to the endoplasmic reticulum for the purpose of retaining nascent apoB within the ER, thereby preventing its secretion. Expression of the 1D1 intrabody in the apoB-secreting human hepatoma cell line HepG2 resulted in marked reduction of apoB secretion. This study demonstrates the utility of an intrabody to specifically block the secretion of a protein determinant of plasma LDL as a therapeutic strategy for the treatment of hyperlipidemia.
Single-chain antibodies; apoB; lipoproteins; HepG2 cells
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.
non-alcoholic fatty liver disease; glycosyltransferase; GLT8D2; apoB100
Apolipoprotein (apo) AI and apoB are the major apolipoproteins of high-density lipoprotein (HDL) and low-density lipoprotein (LDL), respectively. ApoB assembles the precursor of LDL, very-low-density lipoprotein (VLDL), in the liver. The assembly starts with the formation of a primordial particle, which is converted to VLDL2. The VLDL2 particle is then transferred to the Golgi apparatus and can either be secreted or converted to triglyceride-rich VLDL1. We have reviewed this assembly process, the process involved in the storage of triglycerides in cytosolic lipid droplets, and the relationship between these two processes. We also briefly discuss the formation of HDL. ApoB mediates the interaction between LDL and the arterial wall. Two regions in apoB are involved in this binding. This interaction and its role in the development of atherosclerosis are reviewed. ApoB can be used to measure the number of LDL or VLDL particles present in plasma, as there is one molecule of apoB on each particle. By contrast, the amount of cholesterol and other lipids on each particle varies under different conditions. We address the possibility of using apoAI and apoB levels to estimate the risk of development of cardiovascular diseases and to monitor intervention to treat these diseases.
Apolipoprotein AI; atherosclerosis; cardiovascular disease
The synthesis of apolipoprotein B (apoB) dictates the formation of chylomicrons and very low density lipoproteins (VLDL), two major lipoprotein precursors in the human plasma. Despite its biological significance, the mechanism of the assembly of these apoB-containing lipoproteins remains elusive. An essential obstacle is the lack of systems that allow fine dissection of key components during assembly, including nascent apoB peptide, lipids in defined forms, chaperones, and microsomal triglyceride transfer protein (MTP). In this study, we use a prokaryotic cell-free expression system to reconstitute early events in the assembly of apoB-containing lipoprotein that involve the N-terminal domains of apoB. Our study shows that the N-terminal domains larger than 20.5% of apoB (B20.5) have an intrinsic ability to remodel vesicular phospholipid bilayers into discrete protein-lipid complexes. The presence of appropriate lipid substrates during apoB translation plays a pivotal role for successful lipid recruitment, and similar lipid recruitment fails to occur if the lipids are added posttranslationally. Cotranslational presence of MTP can dramatically promote the folding of B6.4–20.5 and B6.4–22. Furthermore, apoB translated in the presence of MTP retains its phospholipid recruitment capability posttranslationally. Our data suggest that during the synthesis of apoB, the N-terminal domain has a short window for intrinsic phospholipid recruitment, the timeframe of which is predetermined by the environment where apoB synthesis occurs. The presence of MTP prolongs this window of time by acting as a chaperone. The absence of either proper lipid substrate or MTP may result in the improper folding of apoB and consequently its degradation.
apolipoprotein B; microsomal triglyceride transfer protein; cell free; cotranslational; low density lipoprotein
Familial hypobetalipoproteinaemia (FHBL) is a codominant disorder characterised by fatty liver and reduced plasma levels of low‐density lipoprotein (LDL) and its protein constituent apolipoprotein B (apoB). FHBL is linked to the APOB gene in some but not all known cases. In a group of 59 patients with FHBL genotyped for APOB gene mutations, we found three novel splice‐site mutations: c.904+4A→G in intron 8, c.3843−2A→G in intron 24 and c.4217−1G→T in intron 25.
To assess the effects of these mutations on apoB pre‐mRNA splicing.
ApoB mRNA was analysed in the liver of one proband and in cells expressing APOB minigenes harbouring the mutations found in the other probands.
In the liver of the c.3843−2A→G carrier, an apoB mRNA devoid of exon 25 was identified, predicted to encode a truncated peptide of 1260 amino acids. The analysis of minigene transcripts in COS‐1 cells showed that the c.904+4A→G mutation caused the formation of an mRNA devoid of exon 8, predicted to encode a short apoB of 247 amino acids. The minigene harbouring the c.4217−1G→T mutation in intron 25 generated an mRNA in which exon 25 joined to a partially deleted exon 26, resulting from the activation of an acceptor site in exon 26; this mRNA is predicted to encode a truncated protein of 1380 amino acids. All these truncated apoBs were not secreted as constituents of plasma lipoproteins.
These findings demonstrate the pathogenic effect of rare splice‐site mutations of the APOB gene found in FHBL.
Lipid esters stored in cytoplasmic lipid droplets (CLDs) of hepatocytes are used to synthesize very low-density lipoproteins (VLDLs), into which apolipoprotein B (ApoB) is integrated cotranslationally. In the present study, by using Huh7 cells, derived from human hepatoma and competent for VLDL secretion, we found that ApoB is highly concentrated around CLDs to make “ApoB-crescents.” ApoB-crescents were seen in <10% of Huh7 cells under normal conditions, but the ratio increased to nearly 50% after 12 h of proteasomal inhibition by N-acetyl-l-leucinyl-l-leucinyl-l-norleucinal. Electron microscopy showed ApoB to be localized to a cluster of electron-lucent particles 50–100 nm in diameter adhering to CLDs. ApoB, proteasome subunits, and ubiquitinated proteins were detected in the CLD fraction, and this ApoB was ubiquitinated. Interestingly, proteasome inhibition also caused increases in autophagic vacuoles and ApoB in lysosomes. ApoB-crescents began to decrease after 12–24 h of proteasomal inhibition, but the decrease was blocked by an autophagy inhibitor, 3-methyladenine. Inhibition of autophagy alone caused an increase in ApoB-crescents. These observations indicate that both proteasomal and autophagy/lysosomal degradation of ApoB occur around CLDs and that the CLD surface functions as a unique platform for convergence of the two 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.
Familial hypercholesterolemia (FH) is an autosomal dominant condition with a population prevalence of one in 300–500 (heterozygous) that is characterized by high levels of low-density lipoprotein (LDL) cholesterol, tendon xanthomata, and premature atherosclerosis and coronary heart disease (CHD). FH is caused mainly by mutations in the LDLR gene. However, mutations in other genes including APOB and PCSK9, can give rise to a similar phenotype. Homozygous FH with an estimated prevalence of one in a million is associated with severe hypercholesterolemia with accelerated atherosclerotic CHD in childhood and without treatment, death usually occurs before the age of 30 years. Current approaches for the treatment of homozygous FH include statin-based lipid-lowering therapies and LDL apheresis. Mipomersen is a second-generation antisense oligonucleotide (ASO) targeted to human apolipoprotein B (apoB)-100. This review provides an overview of the pathophysiology and current treatment options for familial hypercholesterolemia and describes novel therapeutic strategies focusing on mipomersen, an antisense apoB synthesis inhibitor. Mipomersen is distributed mainly to the liver where it silences apoB mRNA, thereby reducing hepatic apoB-100 and giving rise to reductions in plasma total cholesterol, LDL-cholesterol, and apoB concentrations in a dose-and time-dependent manner. Mipomersen has been shown to decrease apoB, LDL-cholesterol and lipoprotein(a) in patients with heterozygous and homozygous FH on maximally tolerated lipid-lowering therapy. The short-term efficacy and safety of mipomersen has been established, however, injection site reactions are common and concern exists regarding the long-term potential for hepatic steatosis with this ASO. In summary, mipomersen given alone or in combination with standard lipid-lowering medications shows promise as an adjunct therapy in patients with homozygous or refractory heterozygous FH at high risk of atherosclerotic CHD, who are not at target or are intolerant of statins.
antisense oligonucleotide; apolipoprotein B; familial hypercholesterolemia; LDL-cholesterol; metabolism; mipomersen
Liver dominates the production and secretion of apolipoprotein B (apoB) and evidence shows that liver malfunction induced by hepatitis B virus (HBV) infection could lead to apolipoprotein metabolism disorders. The present study was undertaken to assess the effects of HBV on apoB expression.
Clinical examination: serum apoB levels in patients with chronic HBV infection and in healthy individuals were measured by immunoturbidimetry using biochemical analyzer Olympus 5400. Cell study: mRNA and protein expression levels of apoB in HepG2 and HepG2.2.15 cells were measured by RT-PCR and Western blot. Alternatively, HBV infectious clone pHBV1.3 or control plasmid pBlue-ks were tranfected into HepG2 cells, and mRNA and protein expression levels of apoB, as well as the microsomal triglyceride transfer protein (MTP) in tranfected HepG2 cells were also measured by RT-PCR and western blot.
Serum apoB level was much lower in chronic HBV patients as compared to healthy individuals (P < 0.05). Expression of apoB mRNA and protein was lower in HepG2.2.15 cells than in HepG2 cells. Similarly, expression of apoB mRNA and protein was lower in pHBV1.3 transfected HepG2 cells than in pBlue-ks transfected HepG2 cells. Expression of MTP mRNA and protein in pHBV1.3 transfected HepG2 cells was reduced in a dose-dependent fashion.
HBV infection plays an inhibitory effect on apoB expression.
hepatitisB virus; chronic HBV infection;lipid metabolism; apolipoprotein B; microsomal triglyceride transfer protein
Apolipoprotein B (apoB) is an essential component of chylomicrons,
very low density lipoproteins, and low density lipoproteins. ApoB is a
palmitoylated protein. To investigate the role of palmitoylation in
lipoprotein function, a palmitoylation site was mapped to Cys-1085 and
removed by mutagenesis. Secreted lipoprotein particles formed by
nonpalmitoylated apoB were smaller and denser and failed to assemble a
proper hydrophobic core. Indeed, the relative concentrations of
nonpolar lipids were three to four times lower in lipoprotein particles
containing mutant apoB compared with those containing wild-type apoB,
whereas levels of polar lipids isolated from wild-type or mutant apoB
lipoprotein particles appeared identical. Palmitoylation localized apoB
to large vesicular structures corresponding to a subcompartment of the
endoplasmic reticulum, where addition of neutral lipids was postulated
to occur. In contrast, nonpalmitoylated apoB was concentrated in a
dense perinuclear area corresponding to the Golgi compartment. The
involvement of palmitoylation as a structural requirement for proper
assembly of the hydrophobic core of the lipoprotein particle and its
intracellular sorting represent novel roles for this posttranslational
Apolipoprotein B mRNA is edited at cytidine 6666 in the enterocytes lining the small intestine of all mammals; converting a CAA codon to a UAA stop codon. The conversion is ~80% efficient in this tissue and leads to the expression of the truncated protein, ApoB48, essential for secretion of dietary lipid as chylomicrons. Caco-2 cell raft cultures have been used as an in vitro model for the induction of editing activity during human small intestinal cell differentiation. This induction of apoB mRNA editing has been ascribed to the expression of APOBEC-1. In agreement our data demonstrated differentiation-dependent induction of expression of the editing enzyme APOBEC-1 and in addition we show alternative splicing of the essential auxiliary factor ACF. However transfection of these editing factors in undifferentiated proliferating Caco-2 cells was not sufficient to induce robust apoB mRNA editing activity. Only differentiation of Caco-2 cells could induce more physiological like levels of apoB mRNA editing. The data suggested that additional regulatory mechanism(s) were induced by differentiation that controlled the functional activity of editing factors.
ACF; alternative splicing; apolipoprotein B; APOBEC-1; differentiation; Caco-2 cells; mRNA editing
Animal studies investigating the beneficial effects of Puerariae radix on cardiovascular disease have suggested this plant possesses anti-diabetic and lipid lowering properties. However, the exact mechanism by which Puerariae radix affects lipid metabolism is currently unknown. The aim of this study was to investigate the effect of the water extract of Puerariae radix on the secretion of VLDL and chylomicrons from HepG2 liver cells and CaCo2 cells, respectively, in humans.
The amount of apoB100 (a protein marker for VLDL) and apoB48 (a protein marker for chylomicrons) in cells and media were quantified by Western Blotting and enhanced chemiluminescence (ECL). Total, free and esterified cholesterol concentrations were measured by gas liquid chromatography.
Treatment of cells with water extract of Puerariae radix significantly decreased apoB100 production and secretion from HepG2 cells up to 66% in a dose dependent manner. The intracellular total cholesterol and free cholesterol concentration in HepG2 cells also decreased with increasing concentration of the Puerariae radix. In contrast, water extract of Puerariae radix attenuated apoB48 concentrations in cells, but not apoB48 secretion from CaCo2 enterocytes.
Collectively, our findings suggest that the water extract of Puerariae radix attenuates the hepatic lipoprotein production and secretion. Our present cell culture findings may explain why circulating VLDL and LDL levels were attenuated in animals supplemented with Puerariae radix. Since decreasing the production and secretion of atherogenic lipoproteins decreases the risk of development of cardiovascular disease, diets supplemented with radix may provide a safe and effective beneficial cardioprotective effects in humans.
Puerariae radix; ApoB100; ApoB48; HepG2 cells; CaCo2 cells; cholesterol
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.
The association between hypercholesterolemia and elevated serum apolipoprotein B (APOB) has generated interest in APOB as a therapeutic target for patients at risk of developing cardiovascular disease. In the clinic, mipomersen, an antisense oligonucleotide (ASO) APOB inhibitor, was associated with a trend toward increased hepatic triglycerides, and liver steatosis remains a concern. We found that siRNA-mediated knockdown of ApoB led to elevated hepatic triglycerides and liver steatosis in mice engineered to exhibit a human-like lipid profile. Many genes required for fatty acid synthesis were reduced, suggesting that the observed elevation in hepatic triglycerides is maintained by the cell through fatty acid uptake as opposed to fatty acid synthesis. Fatty acid transport protein 5 (Fatp5/Slc27a5) is required for long chain fatty acid (LCFA) uptake and bile acid reconjugation by the liver. Fatp5 knockout mice exhibited lower levels of hepatic triglycerides due to decreased fatty acid uptake, and shRNA-mediated knockdown of Fatp5 protected mice from diet-induced liver steatosis. Here, we evaluated if siRNA-mediated knockdown of Fatp5 was sufficient to alleviate ApoB knockdown-induced steatosis. We determined that, although Fatp5 siRNA treatment was sufficient to increase the proportion of unconjugated bile acids 100-fold, consistent with FATP5's role in bile acid reconjugation, Fatp5 knockdown failed to influence the degree, zonal distribution, or composition of the hepatic triglycerides that accumulated following ApoB siRNA treatment.
Electronic supplementary material
The online version of this article (doi:10.1007/s11745-011-3596-3) contains supplementary material, which is available to authorized users.
APOB; Liver steatosis; siRNA; FATP5; Slc27a5; siRNA combinations
A unique kindred with premature cardiovascular disease, tubo-eruptive xanthomas, and type III hyperlipoproteinemia (HLP) associated with familial apolipoprotein (apo) E deficiency was examined. Homozygotes (n = 4) had marked increases in cholesterol-rich very low density lipoproteins (VLDL) and intermediate density lipoproteins (IDL), which could be effectively lowered with diet and medication (niacin, clofibrate). Homozygotes had only trace amounts of plasma apoE, and accumulations of apoB-48 and apoA-IV in VLDL, IDL, and low density lipoproteins. Radioiodinated VLDL apoB and apoE kinetic studies revealed that the homozygous proband had markedly retarded fractional catabolism of VLDL apoB-100, apoB-48 and plasma apoE, as well as an extremely low apoE synthesis rate as compared to normals. Obligate heterozygotes (n = 10) generally had normal plasma lipids and mean plasma apoE concentrations that were 42% of normal. The data indicate that homozygous familial apoE deficiency is a cause of type III HLP, is associated with markedly decreased apoE production, and that apoE is essential for the normal catabolism of triglyceride-rich lipoprotein constituents.
We used wild-type (WT) mice and mice engineered to express either apoB-100 only (B100 mice) or apoB-48 only (B48 mice) to examine the effects of streptozotocin-induced diabetes (DM) on apoB-100– and apoB-48–containing lipoproteins. Plasma lipids increased with DM in WT mice, and fat tolerance was markedly impaired. Lipoprotein profiles showed increased levels and cholesterol enrichment of VLDL in diabetic B48 mice but not in B100 mice. C apolipoproteins, in particular apoC-I in VLDL, were increased. To investigate the basis of the increase in apoB-48 lipoproteins in streptozotocin-treated animals, we characterized several parameters of lipoprotein metabolism. Triglyceride and apoB production rates were normal, as were plasma lipase activity, VLDL glycosaminoglycan binding, and VLDL lipolysis. However, β-VLDL clearance decreased due to decreased trapping by the liver. Whereas LRP activity was normal, livers from treated mice incorporated significantly less sulfate into heparan sulfate proteoglycans (HSPG) than did controls. Hepatoma (HepG2) cells and endothelial cells cultured in high glucose also showed decreased sulfate and glucosamine incorporation into HSPG. Western blots of livers from diabetic mice showed a decrease in the HSPG core protein, perlecan. Delayed clearance of postprandial apoB-48–containing lipoproteins in DM appears to be due to decreased hepatic perlecan HSPG.
Free fatty acids released from adipose tissue affect the synthesis of apolipoprotein B-containing lipoproteins and glucose metabolism in the liver. Whether there also exists a reciprocal metabolic arm affecting energy metabolism in white adipose tissue is unknown.
Methods and Findings
We investigated the effects of apoB-containing lipoproteins on catecholamine-induced lipolysis in adipocytes from subcutaneous fat cells of obese but otherwise healthy men, fat pads from mice with plasma lipoproteins containing high or intermediate levels of apoB100 or no apoB100, primary cultured adipocytes, and 3T3-L1 cells. In subcutaneous fat cells, the rate of lipolysis was inversely related to plasma apoB levels. In human primary adipocytes, LDL inhibited lipolysis in a concentration-dependent fashion. In contrast, VLDL had no effect. Lipolysis was increased in fat pads from mice lacking plasma apoB100, reduced in apoB100-only mice, and intermediate in wild-type mice. Mice lacking apoB100 also had higher oxygen consumption and lipid oxidation. In 3T3-L1 cells, apoB100-containing lipoproteins inhibited lipolysis in a dose-dependent fashion, but lipoproteins containing apoB48 had no effect. ApoB100-LDL mediated inhibition of lipolysis was abolished in fat pads of mice deficient in the LDL receptor (Ldlr−/−Apob100/100).
Our results show that the binding of apoB100-LDL to adipocytes via the LDL receptor inhibits intracellular noradrenaline-induced lipolysis in adipocytes. Thus, apoB100-LDL is a novel signaling molecule from the liver to peripheral fat deposits that may be an important link between atherogenic dyslipidemias and facets of the metabolic syndrome.
Familial hypercholesterolemia is caused by mutations in the LDL receptor gene (Ldlr). Elevated plasma LDL levels result from slower LDL catabolism and a paradoxical lipoprotein overproduction. We explored the relationship between the presence of the LDL receptor and lipoprotein secretion in hepatocytes from both wild-type and LDL receptor–deficient mice. Ldlr–/– hepatocytes secreted apoB100 at a 3.5-fold higher rate than did wild-type hepatocytes. ApoB mRNA abundance, initial apoB synthetic rate, and abundance of the microsomal triglyceride transfer protein 97-kDa subunit did not differ between wild-type and Ldlr–/– cells. Pulse-chase analysis and multicompartmental modeling revealed that in wild-type hepatocytes, approximately 55% of newly synthesized apoB100 was degraded. However, in Ldlr–/– cells, less than 20% of apoB was degraded. In wild-type hepatocytes, approximately equal amounts of LDL receptor–dependent apoB100 degradation occured via reuptake and presecretory mechanisms. Adenovirus-mediated overexpression of the LDL receptor in Ldlr–/– cells resulted in degradation of approximately 90% of newly synthesized apoB100. These studies show that the LDL receptor alters the proportion of apoB that escapes co- or post-translational presecretory degradation and mediates the reuptake of newly secreted apoB-containing lipoprotein particles.
The apolipoprotein B-100 (apoB-100) gene in leukocytes and the apoB-100 messenger RNA (mRNA) and translated apolipoprotein in the livers from normal and abetalipoproteinemic individuals were evaluated. Four complementary DNA probes for apoB-100 covering the 5', middle, and 3' regions of the apoB-100 mRNA were utilized and Southern blot analysis indicated that the apoB-100 gene is present in abetalipoproteinemia without major insertions or deletions. Polyadenylated hepatic apoB-100 mRNA from two abetalipoproteinemic patients was normal in size, and the concentration of apoB-100 mRNA was increased sixfold compared with control hepatic apoB-100 mRNA levels. ApoB-100 was detected in hepatocytes of abetalipoproteinemic patients by immunohistochemical techniques. These results indicate that the biochemical defect in abetalipoproteinemic patients studied is most consistent with a posttranslational defect in apoB-100 processing or secretion with an up-regulation of the apoB-100 mRNA.
Immune responses to oxidized low-density lipoprotein (oxLDL) are proposed to be important in atherosclerosis. To identify the mechanisms of recognition that govern T cell responses to LDL particles, we generated T cell hybridomas from human ApoB100 transgenic (huB100tg) mice that were immunized with human oxLDL. Surprisingly, none of the hybridomas responded to oxidized LDL, only to native LDL and the purified LDL apolipoprotein ApoB100. However, sera from immunized mice contained IgG antibodies to oxLDL, suggesting that T cell responses to native ApoB100 help B cells making antibodies to oxLDL. ApoB100 responding CD4+ T cell hybridomas were MHC class II–restricted and expressed a single T cell receptor (TCR) variable (V) β chain, TRBV31, with different Vα chains. Immunization of huB100tgxLdlr−/− mice with a TRBV31-derived peptide induced anti-TRBV31 antibodies that blocked T cell recognition of ApoB100. This treatment significantly reduced atherosclerosis by 65%, with a concomitant reduction of macrophage infiltration and MHC class II expression in lesions. In conclusion, CD4+ T cells recognize epitopes on native ApoB100 protein, this response is associated with a limited set of clonotypic TCRs, and blocking TCR-dependent antigen recognition by these T cells protects against atherosclerosis.
Substitutional RNA editing represents an important posttranscriptional enzymatic pathway for increasing genetic plasticity by permitting production of different translation products from a single genomically encoded template. One of the best-characterized examples in mammals is C to U deamination of the nuclear apolipoprotein B (apoB) mRNA. ApoB mRNA undergoes a single, site-specific cytidine deamination event yielding an edited transcript that results in tissue-specific translation of two distinct isoforms, referred to as apoB100 and apoB48. Tissue- and site-specific cytidine deamination of apoB mRNA is mediated by an incompletely characterized holoenzyme containing a minimal core complex consisting of an RNA-specific cytidine deaminase, Apobec-1 and a requisite cofactor, apobec-1 complementation factor (ACF). The underlying biochemical and genetic mechanisms regulating tissue-specific apoB mRNA editing have been accelerated through development and characterization of physiological rodent models as well as knockout and transgenic animal strains.
Lipid metabolism; RNA editing; Apobec-1; Hepatocytes; Hormonal regulation; Diet; Primer extension; Subcellular distribution