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

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

The peroxisome proliferator-activated receptor alpha (PPARalpha) agonist ciprofibrate inhibits apolipoprotein B mRNA editing in low density lipoprotein receptor-deficient mice: effects on plasma lipoproteins and the development of atherosclerotic lesions

Low density lipoprotein receptor (LDLR)-deficient mice fed a chow diet have a mild hypercholesterolemia caused by the abnormal accumulation in the plasma of apolipoprotein B (apoB)-100- and apoB-48-carrying intermediate density lipoproteins (IDL) and low density lipoproteins (LDL). Treatment of LDLR-deficient mice with ciprofibrate caused a marked decrease in plasma apoB-48-carrying IDL and LDL but at the same time caused a large accumulation of triglyceride-depleted apoB-100-carrying IDL and LDL, resulting in a significant increase in plasma cholesterol levels. These plasma lipoprotein changes were associated with an increase in the hepatic secretion of apoB-100-carrying very low density lipoproteins (VLDL) and a decrease in the secretion of apoB-48-carrying VLDL, accompanied by a significant decrease in hepatic apoB mRNA editing. Hepatic apobec-1 complementation factor mRNA and protein abundance were significantly decreased, whereas apobec-1 mRNA and protein abundance remained unchanged. No changes in apoB mRNA editing occurred in the intestine of the treated animals. After 150 days of treatment with ciprofibrate, consistent with the increased plasma accumulation of apoB-100-carrying IDL and LDL, the LDLR-deficient mice displayed severe atherosclerotic lesions in the aorta. These findings demonstrate that ciprofibrate treatment decreases hepatic apoB mRNA editing and alters the pattern of hepatic lipoprotein secretion toward apoB-100-associated VLDL, changes that in turn lead to increased atherosclerosis.     

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.     

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.     

Epigallocatechin gallate increases the formation of cytosolic lipid droplets and decreases the secretion of apoB-100 VLDL

Epigallocatechin gallate (EGCG) increases the formation of cytosolic lipid droplets by a mechanism that is independent of the rate of triglyceride biosynthesis and involves an enhanced fusion between lipid droplets, a process that is crucial for their growth in size. EGCG treatment reduced the secretion of both triglycerides and apolipoprotein B-100 (apoB-100) VLDLs but not of transferrin, albumin, or total proteins, indicating that EGCG diverts triglycerides from VLDL assembly to storage in the cytosol. This is further supported by the observed increase in both intracellular degradation of apoB-100 and ubiquitination of the protein (indicative of increased proteasomal degradation) in EGCG-treated cells. EGCG did not interfere with the microsomal triglyceride transfer protein, and the effect of EGCG on the secretion of VLDLs was found to be independent of the LDL receptor. Thus, our results indicate that EGCG promotes the accumulation of triglycerides in cytosolic lipid droplets, thereby diverting lipids from the assembly of VLDL to storage in the cytosol. Our results also indicate that the accumulation of lipids in the cytosol is not always associated with increased secretion of VLDL.     

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

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

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

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

Evidence of increased secretion of apolipoprotein B-48-containing lipoproteins in subjects with type 2 diabetes

Patients with type 2 diabetes have high levels of triglyceride-rich lipoproteins (TRLs), including apolipoprotein B-48 (apoB-48)-containing TRLs of intestinal origin, but the mechanism leading to overaccumulation of these lipoproteins remains to be fully elucidated. Therefore, the objective of this study was to examine the in vivo kinetics of TRL apoB-48 and VLDL, intermediate density lipoprotein (IDL), and LDL apoB-100 in type 2 diabetic subjects (n = 11) and nondiabetic controls (n = 13) using a primed-constant infusion of l-[5,5,5-D(3)]leucine for 12 h in the fed state. Diabetic subjects had significantly higher fasting glycemia, higher fasting insulinemia, higher plasma triglyceride, and lower HDL-cholesterol levels than controls. Compared with controls, diabetic subjects had increased TRL apoB-48, VLDL apoB-100, and IDL apoB-100 pool sizes as a result of increased production rates (PRs) and reduced fractional catabolic rates of these lipoprotein subfractions. Furthermore, multiple linear regression analyses revealed that the diabetic/control status was an independent predictor of TRL apoB-48 PR and represented nearly 35% of its variance. These results suggest that the overaccumulation of TRLs seen in patients with type 2 diabetes is attributable to increased PRs of both intestinally derived apoB-48-containing lipoproteins and TRL apoB-100 of hepatic origin and to decreased catabolism of these subfractions.     

Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006;-1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006;-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [(3)H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006;-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.     

PPARalpha activation increases triglyceride mass and adipose differentiation-related protein in hepatocytes

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed in various tissues. In mice treated with the peroxisome proliferator-activated receptor alpha (PPARalpha) agonist Wy14,643 (Wy), hepatic mRNA and protein levels of ADRP as well as hepatic triglyceride content increased. Also in primary mouse hepatocytes, Wy increased ADRP expression and intracellular triglyceride mass. The triglyceride mass increased in spite of unchanged triglyceride biosynthesis and increased palmitic acid oxidation. However, Wy incubation decreased the secretion of newly synthesized triglycerides, whereas apolipoprotein B secretion increased. Thus, decreased availability of triglycerides for VLDL assembly could help to explain the cellular accumulation of triglycerides after Wy treatment. We hypothesized that this effect could be mediated by increased ADRP expression. Similar to PPARalpha activation, adenovirus-mediated ADRP overexpression in mouse hepatocytes enhanced cellular triglyceride mass and decreased the secretion of newly synthesized triglycerides. In ADRP-overexpressing cells, Wy incubation resulted in a further decrease in triglyceride secretion. This effect of Wy was not attributable to decreased cellular triglycerides after increased fatty acid oxidation because the triglyceride mass in Wy-treated ADRP-overexpressing cells was unchanged. In summary, PPARalpha activation prevents the availability of triglycerides for VLDL assembly and increases hepatic triglyceride content in part by increasing the expression of ADRP.     

Differential labeling of rat hepatic Golgi and serum very low density lipoprotein apoprotein B variants

The synthesis of apoB-100 and apoB-48 by rat liver was investigated by studying the apoB complement of very low density lipoproteins (VLDL) from hepatic perfusates and Golgi fractions. The relative amounts of apoB-100 and apoB-48 in perfusate and Golgi VLDL as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis were similar to those in serum VLDL. To investigate the relative rates of synthesis of the VLDL B proteins, rats were injected intraportally with tritiated amino acid, and hepatic Golgi and serum VLDL were isolated from 7.5 to 120 min later. In hepatic Golgi VLDL, apoB-100 and apoE were maximally labeled at 15 min after the tritiated amino acid pulse. In contrast, VLDL apoB-48 attained maximum radioactivity at 30 min after isotope injection. In serum VLDL, apoB-100 and apoE were maximally labeled at 30 min post-isotope injection, while activity in apoB-48 peaked at 60 min. The data suggest that the synthesis of the B proteins and incorporation into rat liver nascent VLDL are independently regulated. The differential labeling patterns of the VLDL B proteins may be explained by an intracellular pool of apoB-48 that is larger than that of apoB-100. An alternative explanation of the results is that apoB-100 is a precursor to apoB-48.     

The level and compartmentalization of phosphatidate phosphatase-1 (lipin-1) control the assembly and secretion of hepatic VLDL

Phosphatidate phosphatase-1 (PAP-1) converts phosphatidate to diacylglycerol and plays a key role in the biosynthesis of phospholipids and triacylglycerol (TAG). PAP-1 activity is encoded by members of the lipin family, including lipin-1 (1alpha and 1beta), -2, and -3. We determined the effect of lipin-1 expression on the assembly and secretion of very low density lipoproteins (VLDL) using McA-RH7777 cells. Expression of lipin-1alpha or -1beta increased the synthesis and secretion of [(3)H]glycerol-labeled lipids under either basal- or oleate-supplemented conditions. In the presence of oleate, the increased TAG secretion was mainly associated with VLDL(1) (S(f) > 100) and VLDL(2) (S(f) 20-100). Expression of lipin-1alpha or -1beta increased secretion efficiency and decreased intracellular degradation of [(35)S]apolipoprotein B-100 (apoB100). Knockdown of lipin-1 using specific short interfering RNA decreased secretion of [(3)H]glycerolipids and [(35)S]apoB100 even though total PAP-1 activity was not decreased, owing to the presence of lipin-2 and -3 in the cells. Deletion of the nuclear localization signal sequences within lipin-1alpha not only abolished nuclear localization but also resulted in impaired association with microsomal membranes. Cells expressing the cytosolic lipin-1alpha mutant failed to promote [(35)S]apoB100 synthesis or secretion, and showed compromised stimulation in [(3)H]TAG synthesis and secretion. Thus, alteration in hepatic expression of lipin-1 and its compartmentalization control VLDL assembly/secretion.     

Modulation of apolipoprotein B-100 mRNA editing: effects on hepatic very low density lipoprotein assembly and intracellular apoB distribution in the rat

We have studied the consequences of alterations to hepatic apoB mRNA editing on the biosynthesis and intracellular distribution of newly synthesized apoB variants together with their mass distribution in nascent Golgi very low density lipoproteins (VLDL). Radiolabeled liver membrane fractions were prepared from control or hypothyroid animals and separated by discontinuous sucrose gradient centrifugation. Hepatic apoB-100 synthesis in these groups accounted for 93-100% of total newly synthesized apoB species of Golgi fractions recovered from the sucrose gradients (G1 and G2). The analogous fractions isolated from the livers of hyperthyroid (treated with 3,3',5-triiodo-L-thyronine, T3) animals revealed that newly synthesized apoB-100 accounted for only 46 +/- 10% (G1) and 24 +/- 11% (G2), respectively, of total newly synthesized apoB. ApoB-100 mass in nascent Golgi VLDL from control and hypothyroid G1 fractions represented 70-78% total apoB as determined by Western blot analysis. By contrast, Golgi VLDL from hyperthyroid animals contained predominantly (greater than 78%) apoB-48 as the apoB species. Electron microscopy revealed that the morphology and size distribution of hyperthyroid G1 VLDL were similar to particles isolated from control animals. Thus, despite a profound reduction in the proportion of apoB-100 mRNA species containing an unmodified codon (CAA, B-GLN) at position 2153 in hyperthyroid animals (6 +/- 1% vs 50-61% in control and hypothyroid animals) apoB-100 biosynthesis was detectable in a defined membrane fraction isolated by discontinuous sucrose gradient centrifugation. However, no apoB-100 synthesis was detectable in liver samples prepared by Polytron disruption in Triton-containing buffers. These data suggest that effective hepatic VLDL assembly and secretion in the T3-treated rat continues despite a profound reduction in apoB-100 biosynthesis and implies that apoB-48 contains the requisite domains to direct this process, a situation analogous to that in the intestine.     

Synthesis, modification, and flotation properties of rat hepatocyte apolipoproteins

We have studied apolipoprotein synthesis, intracellular modification and secretion by primary adult rat hepatocyte cultures using continuous pulse or pulse chase labeling with [35S]methionine, immunoprecipitation and two-dimensional isoelectric focusing/polyacrylamide gel electrophoresis. The flotation properties of the newly secreted apolipoproteins were studied by discontinuous density gradient ultracentrifugation and one- and two-dimensional polyacrylamide gel electrophoresis. These studies showed that rat hepatocyte apoE is modified intracellularly to produce minor isoproteins that differ in size and charge. One of these minor isoproteins represents a monosialated apoE form (apoE3s1). Similarly, apoCIII is modified intracellularly to produce a disialated apoCIII form (apoCIIIs2), whereas newly synthesized apoA-I and apoA-IV are not glycosylated and overlap on two-dimensional gels with the proapoA-I and the plasma apoA-IV form, respectively. Both unmodified and modified apolipoproteins are secreted into the medium. Separation of secreted apolipoproteins by density gradient ultracentrifugation has shown that 50% of apoE, 80% of apoA-I, and more than 90% of apoA-IV and apoCIII are secreted in a lipid-poor form, whereas apoB-100 and apoB-48 are 100% associated with lipids. ApoB-100 floats in the VLDL and IDL regions, whereas apoB-48 is found in all lipoprotein fractions. ApoE and small amounts of apoA-I, apoA-IV and apoCIII float in the HDL region. Small amounts of apoE and apoCIII are also found in the VLDL and IDL regions, and apoE in the LDL region. Ultracentrifugation of nascent lipoproteins in the presence of rat serum promoted flotation of apoA-I and apoA-IV in the HDL fraction and resulted in increased flotation and distribution of apoE and apoCs in VLDL, IDL and LDL regions. These observations are consistent with the hypothesis that intracellular assembly of lipoproteins involves apoB-48 and apoB-100 forms, whereas a large portion of apoA-I, apoCIII and apoA-IV can be secreted in a lipid-poor form, which associates extracellularly with preexisting lipoproteins.     

Overexpression of apoC-III produces lesser hypertriglyceridemia in apoB-48-only gene-targeted mice than in apoB-100-only mice

The adaptive value of apolipoprotein B-48 (apoB-48), the truncated form of apoB produced by the intestine, in lipid metabolism remains unclear. We crossed human apoC-III transgenic mice with mice expressing either apoB-48 only (apoB48/48) or apoB-100 only (apoB100/100). Cholesterol levels were higher in apoB48/48 mice than in apoB100/100 mice but triglyceride levels were similar. Lipid levels were increased by the apoC-III transgene. However, triglyceride levels were significantly higher in apoB100/100C-III than in apoB48/48C-III mice (895 +/- 395 mg/dl vs. 690 +/- 252 mg/dl; P <0.01), whereas cholesterol levels were higher in the apoB48/48C-III mice than in apoB100/100C-III (144 +/- 35 mg/dl vs. 94 +/- 30 mg/dl; P <0.00001). Triglyceride clearance from VLDL was impaired to a greater extent in apoB100/100C-III vs. apoB100/100 mice than in apoB48/48C-III vs. apoB48/48 mice. Triglyceride secretion rates were no different in apoC-III transgenic mice than in their nontransgenic littermates. ApoB-48 triglyceride-rich lipoproteins were more resistant to the triglyceride-increasing effects of apoC-III but appeared more sensitive to the remnant clearance inhibition. Our findings support a coordinated role for apoB-48 in facilitating the delivery of dietary triglycerides to the periphery. Consistent with such a mechanism, glucose levels were significantly higher in apoB48/48 mice vs. apoB100/100 mice, perhaps on the basis of metabolic competition.     

Inhibition of phosphatidylcholine synthesis via the phosphatidylethanolamine methylation pathway impairs incorporation of bulk lipids into VLDL in cultured rat hepatocytes

Inhibition of phosphatidylcholine (PC) synthesis via the phosphatidylethanolamine (PE) methylation pathway was shown to decrease the secretion of VLDL from primary rat hepatocytes (Nishimaki-Mogami et al. 1996. BIOCHIM: Biophys. Acta. 1304: 21-31). To understand further the role of PE methylation, we determined the effect of bezafibrate, an inhibitor of PE methylation, on VLDL assembly within the microsomal lumen. Bezafibrate was shown to decrease VLDL (triacylglycerol) secretion only when cellular PE methylation was active in the presence of methionine. Pulse-chase experiments showed that bezafibrate treatment did not impair the movement of [(35)S]apolipoprotein (apo)B-48 from microsomal membranes into the lumen. However, bezafibrate treatment resulted in reduced VLDL-[(35)S]apoB-48 and increased [(35)S]apoB-48-containing particles in the HDL density range (HDL-[(35)S]apoB-48) within the lumen. Inhibition of PE methylation by bezafibrate or 3-deazaadenosine after the completion of HDL-[(35)S]apoB-48 assembly effectively decreased VLDL-[(35)S]apoB-48 secretion with a concomitant increase in HDL-[(35)S]apoB-48 secretion. These findings suggest that inhibition of PC synthesis via the PE methylation pathway impairs the stage of bulk triacylglycerol incorporation during the assembly of VLDL.     

Postprandial changes of apoB-100 and apoB-48 in TG rich lipoproteins in familial combined hyperlipidemia

Impaired chylomicron (chylo) remnant clearance and small VLDL overproduction are major metabolic abnormalities in familial combined hyperlipidemia (FCHL). Quantitative data on postprandial apolipoprotein B-48 (apoB-48) and apolipoprotein B-100 (apoB-100) in TG rich lipoproteins (TRL) in FCHL have not been reported before. Eight untreated FCHL patients and 10 matched controls underwent a 24 h oral fat load. Fasting apoB-48 and apoB-100 were significantly higher in all TRL in FCHL. Maximal concentrations of chylo-[Svedberg's flotation rate (Sf) >400] apoB-48 and apoB-100 were reached later in FCHL (at t = 6 h), in contrast to controls (t = 4 h). Maximal VLDL1-(Sf60-400)-apoB-48 occurred at the same time point (t = 2 h) in both, whereas VLDL1-apoB-100 was maximal at t = 4 h in both, most likely representing delayed VLDL clearance by preferential clearance of chylo and their remnants by competition for the same clearance mechanisms. VLDL2-(Sf20-60)-apoB-48 and VLDL2- apoB-100 decreased in FCHL, in contrast to an increase of apoB-48, and no change of apoB-100 in controls, suggesting impaired conversion of VLDL1-apoB-48 into VLDL2-apoB-48 in FCHL, and partly also of VLDL1-apoB-100. In conclusion, in FCHL clearance of large postprandial Sf >400 apoB-48 and apoB-100 TRL is delayed. ApoB-100 accumulates in the VLDL1 range postprandially in both FCHL and controls, reaching higher levels in FCHL and thereby conferring a higher atherogenic burden in the postprandial situation in FCHL.