Expression of apolipoprotein C-III in McA-RH7777 cells enhances VLDL assembly and secretion under lipid-rich conditions

Apolipoprotein (apo) C-III plays a regulatory role in VLDL lipolysis and clearance. In this study, we determined a potential intracellular role of apoC-III in hepatic VLDL assembly and secretion. Stable expression of recombinant apoC-III in McA-RH7777 cells resulted in increased secretion efficiency of VLDL-associated triacylglycerol (TAG) and apoB-100 in a gene-dosage-dependent manner. The stimulatory effect of apoC-III on TAG secretion was manifested only when cells were cultured under lipid-rich (i.e., media supplemented with exogenous oleate) but not lipid-poor conditions. The stimulated TAG secretion was accompanied by increased secretion of apoB-100 and apoB-48 as VLDL₁. Expression of apoC-III also increased mRNA and activity of microsomal triglyceride transfer protein (MTP). Pulse-chase experiments showed that apoC-III expression promoted VLDL₁ secretion even under conditions where the MTP activity was inhibited immediately after the formation of lipid-poor apoB-100 particles, suggesting an involvement of apoC-III in the second-step VLDL assembly process. Consistent with this notion, the newly synthesized apoC-III was predominantly associated with TAG within the microsomal lumen that resembled lipid precursors of VLDL. Introducing an Ala23-to-Thr mutation into apoC-III, a naturally occurring mutation originally identified in two Mayan Indian subjects with hypotriglyceridemia, abolished the ability of apoC-III to stimulate VLDL secretion from transfected cells. Thus, expression of apoC-III in McA-RH7777 cells enhances hepatic TAG-rich VLDL assembly and secretion under lipid-rich conditions.     

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.     

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

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

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.     

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.     

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.     

Lipid metabolism response to a single, prolonged bout of endurance exercise in healthy young men

To discover the alterations in lipid metabolism linked to postexercise hypotriglyceridemia, we measured lipid kinetics, lipoprotein subclass distribution and lipid transfer enzymes in seven healthy, lean, young men the day after 2 h of cycling and rest. Compared with rest, exercise increased fatty acid rate of appearance and whole body fatty acid oxidation by approximately 65 and 40%, respectively (P < 0.05); exercise had no effect on VLDL-triglyceride (TG) secretion rate, increased VLDL-TG plasma clearance rate by 40 +/- 8%, and reduced VLDL-TG mean residence time by approximately 40 min and VLDL-apolipoprotein B-100 (apoB-100) secretion rate by 24 +/- 8% (all P < 0.05). Exercise also reduced the number of VLDL but almost doubled the number of IDL particles in plasma (P < 0.05). Muscle lipoprotein lipase content was not different after exercise and rest, but plasma lipoprotein lipase concentration increased by approximately 20% after exercise (P < 0.05). Plasma hepatic lipase and lecithin:cholesterol acyltransferase concentrations were not affected by exercise, whereas cholesterol ester transfer protein concentration was approximately 10% lower after exercise than after rest (P = 0.052). We conclude that 1) greater fatty acid availability after exercise does not stimulate VLDL-TG secretion, probably because of the increase in fatty acid oxidation and possibly also fatty acid use for restoration of tissue TG stores; 2) reduced secretion of VLDL-apoB-100 lowers plasma VLDL particle concentration; and 3) increased VLDL-TG plasma clearance maintains low plasma TG concentration but is not accompanied by similar increases in subsequent steps of the delipidation cascade. Acutely, therefore, the cardioprotective lowering of plasma TG and VLDL concentrations by exercise is counteracted by a proatherogenic increase in IDL concentration.     

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.     

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.     

Hypolipidemic effect of beta, beta'-methyl-substituted hexadecanedioic acid (MEDICA 16) in normal and nephrotic rats

Treatment of normal or puromycin aminonucleoside-nephrotic rats, kept on a balanced Purina chow diet, with beta, beta'-tetramethyl-substituted hexadecanedioic acid (MEDICA 16) (Bar-Tana, J., G. Rose-Kahn, and M. Srebnik. 1985. J. Biol. Chem. 260: 8404-8410) resulted in an acute reversible inhibition of liver lipogenesis and cholesterogenesis with a concomitant hypolipidemic effect which was sustained as long as the drug was administered. The hypolipidemic effect in normal and nephrotic rats consisted of 70-80% and 40-60% reduction in plasma VLDL-triacylglycerols and cholesterol, respectively, with a respective increase in the HDL-cholesterol/(VLDL + LDL)-cholesterol ratio. The observed hypolipidemic effect was accompanied by a 10-fold decrease in VLDL-apoC-III content with a concomitant enrichment of the VLDL fraction by VLDL remnants having an increased apoB-100/apoB-48 ratio. The pharmacological reduction of VLDL by MEDICA 16 may offer a treatment mode of choice for selected hyperlipidemic states.     

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.     

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.     

Intense exercise stimulates albumin synthesis in the upright posture.

We tested the hypothesis that an elevation in albumin synthetic rate contributes to increased plasma albumin content during exercise-induced hypervolemia. Albumin synthetic rate was measured in seven healthy subjects at 1-5 and 21-22 h after 72 min of intense (85% peak oxygen consumption rate) intermittent exercise and after 5 h recovery in either upright (Up) or supine (Sup) postures. Deuterated phenylalanine (d(5)-Phe) was administrated by a primed-constant infusion method, and fractional synthetic rate (FSR) and absolute synthetic rate (ASR) of albumin were calculated from the enrichment of d(5)-Phe in plasma albumin, determined by gas chromatography-mass spectrometry. FSR of albumin in Up increased significantly (P < 0.05) from 4.9 +/- 0.9%/day at control to 7.3 +/- 0.9%/day at 22 h of recovery. ASR of albumin increased from 87.9 +/- 17.0 to 141.1 +/- 16.6 mg albumin. kg body wt(-1). day(-1). In contrast, FSR and ASR of albumin were unchanged in Sup (3.9 +/- 0.4 to 4.0 +/- 1.4%/day and 74.2 +/- 8.9 to 85.3 +/- 23.9 mg albumin. kg body wt(-1). day(-1) at control and 22 h of recovery, respectively). Increased albumin synthesis after upright intense exercise contributes to the expansion of greater albumin content and its maintenance. We conclude that stimuli related to posture are critical in modulating the drive for albumin synthesis after intense exercise.     

Abnormal in vivo metabolism of apoB-containing lipoproteins in human apoE deficiency

The present study was undertaken to elucidate the metabolic basis for the increased remnants and lipoprotein(a) [Lp(a)] and decreased LDL apolipoprotein B (apoB) levels in human apoE deficiency. A primed constant infusion of (13)C(6)-phenylalanine was administered to a homozygous apoE-deficient subject. apoB-100 and apoB-48 were isolated, and tracer enrichments were determined by gas chromatography-mass spectrometry, then kinetic parameters were calculated by multicompartmental modeling. In the apoE-deficient subject, fractional catabolic rates (FCRs) of apoB-100 in VLDL and intermediate density lipoprotein and apoB-48 in VLDL were 3x, 12x, and 12x slower than those of controls. On the other hand, the LDL apoB-100 FCR was increased by 2.6x. The production rate of VLDL apoB-100 was decreased by 45%. In the Lp(a) kinetic study, two types of Lp(a) were isolated from plasma with apoE deficiency: buoyant and normal Lp(a). (125)I-buoyant Lp(a) was catabolized at a slower rate in the patient. However, (125)I-buoyant Lp(a) was catabolized at twice as fast as (131)I-normal Lp(a) in the control subjects. In summary, apoE deficiency results in: 1) a markedly impaired catabolism of VLDL/chylomicron and their remnants due to lack of direct removal and impaired lipolysis; 2) an increased rate of catabolism of LDL apoB-100, likely due to upregulation of LDL receptor activity; 3) reduced VLDL apoB production; and 4) a delayed catabolism of a portion of Lp(a).     

Effect of weight loss on VLDL-triglyceride and apoB-100 kinetics in women with abdominal obesity

The effects of obesity and weight loss on lipoprotein kinetics were evaluated in six lean women [body mass index (BMI): 21 +/- 1 kg/m(2)] and seven women with abdominal obesity (BMI: 36 +/- 1 kg/m(2)). Stable isotope tracer techniques, in conjunction with compartmental modeling, were used to determine VLDL-triglyceride (TG) and apolipoprotein B-100 (apoB-100) secretion rates in lean women and in obese women before and after 10% weight loss. VLDL-TG and VLDL-apoB-100 secretion rates were similar in lean and obese women. Weight loss decreased the rate of VLDL-TG secretion by approximately 40% (from 0.41 +/- 0.05 to 0.23 +/- 0.03 micromol x kg fat-free mass(-1) x min(-1); P < 0.05). The relative decline in VLDL-TG produced from nonsystemic fatty acids, derived from intraperitoneal and intrahepatic TG, was greater (61 +/- 7%) than the decline in VLDL-TG produced from systemic fatty acids, predominantly derived from subcutaneous TG (25 +/- 8%; P < 0.05). Weight loss did not affect VLDL-apoB-100 secretion rate. We conclude that weight loss decreases the rate of VLDL-TG secretion in women with abdominal obesity, primarily by decreasing the availability of nonsystemic fatty acids. There is a dissociation in the effect of weight loss on VLDL-TG and apoB-100 metabolic pathways that may affect VLDL particle size.     

ApoB-48 and apoB-100 differentially influence the expression of type-III hyperlipoproteinemia in APOE*2 mice

Apolipoprotein E (apoE) is essential for the clearance of plasma chylomicron and VLDL remnants. The human APOE locus is polymorphic and 5-10% of APOE*2 homozygotes exhibit type-III hyperlipoproteinemia (THL), while the remaining homozygotes have less than normal plasma cholesterol. In contrast, mice expressing APOE*2 in place of the mouse Apoe (Apoe(2/2) mice) are markedly hyperlipoproteinemic, suggesting a species difference in lipid metabolism (e.g., editing of apolipoprotein B) enhances THL development. Since apoB-100 has an LDLR binding site absent in apoB-48, we hypothesized that the Apoe(2/2) THL phenotype would improve if all Apoe(2/2) VLDL contained apoB-100. To test this, we crossed Apoe(2/2) mice with mice lacking the editing enzyme for apoB (Apobec(-/-)). Consistent with an increase in remnant clearance, Apoe(2/2). Apobec(-/-) mice have a significant reduction in IDL/LDL cholesterol (IDL/LDL-C) compared with Apoe(2/2) mice. However, Apoe(2/2).Apobec(-/-) mice have twice as much VLDL triglyceride as Apoe(2/2) mice. In vitro tests show the apoB-100-containing VLDL are poorer substrates for lipoprotein lipase than apoB-48-containing VLDL. Thus, despite a lowering in IDL/LDL-C, substituting apoB-48 lipoproteins with apoB-100 lipoproteins did not improve the THL phenotype in the Apoe(2/2).Apobec(-/-) mice, because apoB-48 and apoB-100 differentially influence the catabolism of lipoproteins.     

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.     

Role of the kidney in the metabolism of apolipoprotein A-IV: influence of the type of proteinuria

Increased plasma concentrations of apolipoprotein A-IV (apoA-IV) in chronic renal disease suggest a metabolic role of the kidney for this antiatherogenic protein. Therefore, we investigated patients with various forms of proteinuria and found increased serum concentrations of apoA-IV in 124 nephrotic patients compared with 274 controls (mean 21.9 +/- 9.6 vs. 14.4 +/- 4.0 mg/dl; P < 0.001). Decreasing creatinine clearance showed a strong association with increasing apoA-IV levels. However, serum albumin levels significantly modulated apoA-IV levels in patients with low creatinine clearance, resulting in lower levels of apoA-IV in patients with low compared with high albumin levels (21.4 +/- 8.6 vs. 29.2 +/- 8.4 mg/dl; P = 0.0007). Furthermore, we investigated urinary apoA-IV levels in an additional 66 patients with a wide variety of proteinuria and 30 controls. Especially patients with a tubular type of proteinuria had significantly higher amounts of apoA-IV in urine than those with a pure glomerular type of proteinuria and controls (median 45, 14, and 0.6 ng/mg creatinine, respectively). We confirmed these results in affected members of a family with Dent's disease, who are characterized by an inherited protein reabsorption defect of the proximal tubular system. In summary, our data demonstrate that the increase of apoA-IV caused by renal impairment is significantly modulated by low levels of serum albumin as a measure for the severity of the nephrotic syndrome. From this investigation of apoA-IV in urine as well as earlier immunohistochemical studies, we conclude that apoA-IV is filtered through the normal glomerulus and is subsequently reabsorbed mainly by proximal tubular cells.     

Increased concentration of plasma cholesteryl ester transfer protein in nephrotic syndrome: role in dyslipidemia.

Hyperlipidemia is a prominent feature of the nephrotic syndrome. Lipoprotein abnormalities include increased very low and low density lipoprotein (VLDL and LDL) cholesterol and variable reductions in high density lipoprotein (HDL) cholesterol. We hypothesized that plasma cholesteryl ester transfer protein (CETP), which influences the distribution of cholesteryl esters among the lipoproteins, might contribute to lipoprotein abnormalities in nephrotic syndrome. Plasma CETP, apolipoprotein and lipoprotein concentrations were measured in 14 consecutive untreated and 7 treated nephrotic patients, 5 patients with primary hypertriglyceridemia, and 18 normolipidemic controls. Patients with nephrotic syndrome displayed increased plasma concentrations of apoB, VLDL, and LDL cholesterol. The VLDL was enriched with cholesteryl ester (CE), shown by a CE/triglyceride (TG) ratio approximately twice that in normolipidemic or hypertriglyceridemic controls (P < 0.001). Plasma CETP concentration was increased in patients with untreated nephrotic syndrome compared to controls (3.6 vs. 2.3 mg/l, P < 0.001), and was positively correlated with the CE concentration in VLDL (r = 0.69, P = 0.004) and with plasma apoB concentration (r = 0.68, P = 0.007). Treatment with corticosteroids resulted in normalization of plasma CETP and of the CE/TG ratio in VLDL. An inverse correlation between plasma CETP and HDL cholesterol was observed in hypertriglyceridemic nephrotic syndrome patients (r = -0.67, P = 0.03). The dyslipidemia of nephrotic syndrome includes increased levels of apoB-lipoproteins and VLDL that are unusually enriched in CE and likely to be atherogenic. Increased plasma CETP probably plays a significant role in the enrichment of VLDL with CE, and may also contribute to increased concentrations of apoB-lipoproteins and decreased HDL cholesterol in some patients.     

Regional specificities of monoclonal anti-human apolipoprotein B antibodies

The usefulness of monoclonal antibodies as probes of protein structure is directly related to knowledge of the structures and locations of the epitopes with which they interact. In this report we provide a detailed map of 13 epitopes on apoB-100 defined by our anti-apoB monoclonal antibodies based on current information on the amino acid sequence of apoB-100. To localize antibody specificities to smaller regions along the linear sequence of the apoB-100 molecule we used a) thrombin- and kallikrein-generated fragments of apoB-100; b) beta-galactosidase- apoB fusion proteins; c) heparin; and d) antibody versus antibody competition experiments. Most of the monoclonal antibodies elicited by immunization with LDL were directed towards epitopes within the first 1279 amino terminal (T4/K2 fragments) or last 1292 carboxyl terminal amino acid residues (T2/K4 fragments) of apoB-100. One epitope localized to the mid-portion of apoB-100 was elicited by immunization with VLDL (D7.2). Saturating amounts of heparin bound to LDL did not inhibit the binding of any of the monoclonal antibodies to their respective epitopes on apoB-100, indicating that none of the antibody determinants is situated close to any of the reported heparin binding sites on LDL apoB. We examined the expression of apoB epitopes on VLDL subfractions and LDL isolated from a normolipidemic donor. The apparent affinities with which the antibodies interacted with their respective epitopes on the VLDL subfractions and LDL uniformly increased as follows: LDL greater than VLDL3 greater than VLDL2 greater than VLDL1, suggesting that each of the major regions of apoB-100 is progressively more exposed as normal VLDL particles become smaller in size and epitopes are most exposed in LDL. Previous experiments utilizing hypertriglyceridemic VLDL subfractions yielded similar results, but the rank order of VLDL subfractions and LDL was not the same for all antibodies tested. Thus, differences in apoB epitope expression on VLDL particles of differing sizes is a general phenomenon, but the expression of apoB epitopes in hypertriglyceridemic VLDL appears to be more heterogeneous than is the case for VLDL from normolipidemic donors.(ABSTRACT TRUNCATED AT 400 WORDS)