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Background: The objective of this study was to investigate whether apoC3 (apolipoprotein C3) inhibition with an antisense oligonucleotide (ASO) modulates intestinal triglyceride secretion., Methods: Sprague-Dawley rats were treated with subcutaneous injections of apoC3 ASO 25 mg/kg twice weekly or inactive ASO for 4 weeks before the assessment of lymph flow, triglyceride and apoB48 (apolipoprotein B48) appearance in the lymph. Rats were surgically implanted with catheters in the mesenteric lymph duct and duodenum. Following an overnight fast, an intraduodenal lipid bolus (1.5-mL intralipid) was administered. Lymph fluid was collected for the following 4 hours to compare effects on lymph flow, lymph triglyceride and apoB48 concentration, and secretion. To assess suppression of apoC3 expression and protein abundance by apoC3 ASO compared with inactive ASO (placebo), intestinal and hepatic tissues were collected from a subset of animals before (fasting) and after an enteral lipid bolus (post-lipid)., Results: ApoC3 ASO significantly reduced apoC3 mRNA expression in the liver compared with inactive ASO (fasting: 42%, P =0.0048; post-lipid: 66%, P <0.001) and in the duodenum (fasting: 29%, P =0.0424; post-lipid: 53%, P =0.0120). As expected, plasma triglyceride also decreased significantly (fasting: 74%, P <0.001; post-lipid: 33%, P =0.0276). Lymph flow and cumulative lymph volume remained unchanged following apoC3 ASO therapy; however, lymph triglyceride, but not apoB48 output, increased by 38% (ANOVA, P <0.001). Last, no changes were observed in stool triglyceride, intestinal fat (quantified via oil red O staining), and expression of mRNAs involved in triglyceride synthesis, lipid droplet formation, and chylomicron transport and secretion., Conclusions: Despite the marked reduction in plasma triglyceride concentration that occurs with apoC3 ASO inhibition, intestinal triglyceride output surprisingly increased rather than decreased. These data demonstrate that the reduction of intestinal triglyceride output does not contribute to the potent plasma triglyceride-lowering observed with this novel therapy for hypertriglyceridemia. Further studies are required to explore the mechanism of this intestinal effect., Competing Interests: Disclosures ApoC3 (antisense oligonucleotide; both the inactive and active forms) was provided free of charge by Ionis Pharmaceuticals. No grant funding was received from Ionis Pharmaceuticals.

Objective: Nobiletin is a dietary flavonoid that improves insulin resistance and atherosclerosis in mice with metabolic dysfunction. Dysregulation of intestinal lipoprotein metabolism contributes to atherogenesis. The objective of the study was to determine if nobiletin targets the intestine to improve metabolic dysregulation in both male and female mice. Approach and Results: Triglyceride-rich lipoprotein (TRL) secretion, intracellular triglyceride kinetics, and intestinal morphology were determined in male and female LDL (low-density lipoprotein) receptor knockout ( Ldlr -/- ), and male wild-type mice fed a standard laboratory diet or high-fat, high-cholesterol (HFHC) diet ± nobiletin using an olive oil gavage, radiotracers, and electron microscopy. Nobiletin attenuated postprandial TRL levels in plasma and enhanced TRL clearance. Nobiletin reduced fasting jejunal triglyceride accumulation through accelerated TRL secretion and lower jejunal fatty acid synthesis with no impact on fatty acid oxidation. Fasting-refeeding experiments revealed that nobiletin led to higher levels of phosphorylated AKT (protein kinase B) and FoxO1 (forkhead box O1) and normal Srebf1c expression indicating increased insulin sensitivity. Intestinal length and weight were diminished by HFHC feeding and restored by nobiletin. Both fasting and postprandial plasma GLP-1 (glucagon-like peptide-1; and likely GLP-2) were elevated in response to nobiletin. Treatment with a GLP-2 receptor antagonist, GLP-2(3-33), reduced villus length in HFHC-fed mice but did not impact TRL secretion in any diet group. In contrast to males, nobiletin did not improve postprandial lipid parameters in female mice., Conclusions: Nobiletin opposed the effects of the HFHC diet by normalizing intestinal de novo lipogenesis through improved insulin sensitivity. Nobiletin prevents postprandial lipemia because the enhanced TRL clearance more than compensates for increased TRL secretion.

Purpose of Review: LDL, triglyceride-rich lipoprotein (TRL) and lipoprotein(a) [Lp(a)] particles are the key atherogenic lipoproteins. Deranged metabolism of these lipoproteins accounts for a spectrum of clinically important dyslipidemias, such as FH, elevated Lp(a) and diabetic dyslipidemia. We review the findings from recent dynamic and tracer studies that have contributed to expanding knowledge in this field., Recent Findings: Deficiency in LDL receptor activity does not only impair the catabolism of LDL-apoB-100 in FH, but also induces hepatic overproduction and decreases catabolism of TRLs. Patients with elevated Lp(a) are characterized by increased hepatic secretion of Lp(a) particles. Elevation of TRLs in diabetes is partly mediated by increased production of apoB-48 and apoC-III, and impaired clearance of apoB-48 in the postprandial state. Tracer kinetic studies show that proprotein convertase subtilisin/kexin type 9 mAbs alone or in combination with statin can increase the catabolism and decrease production of LDL and Lp(a) particles. By contrast, angiopoietin-like protein 3 inhibitors (e.g. evinacumab) reduce VLDL production and increase LDL clearance in FH. Glucagon-like peptide-1 receptor agonists can improve diabetic dyslipidemia by increasing the catabolism of apoB-48 and decreasing the production of apoB-48 and apoC-III., Summary: Dynamic studies of the metabolism of atherogenic lipoproteins provide new insight into the nature of dyslipidemias and point to how new therapies with complementary modes of action may have maximal clinical impact., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Objective- Treatment with liraglutide, a GLP-1 (glucagon-like peptide-1) agonist, has been shown to reduce postprandial lipidemia, an important feature of diabetic dyslipidemia. However, the underlying mechanisms for this effect remain unknown. This prompted us to study the effect of liraglutide on the metabolism of ApoB48 (apolipoprotein B48). Approach and Results- We performed an in vivo kinetic study with stable isotopes (D 8 -valine) in the fed state in 10 patients with type 2 diabetes mellitus before treatment and 6 months after the initiation of treatment with liraglutide (1.2 mg/d). We also evaluated, in mice, the effect of a 1-week liraglutide treatment on postload triglycerides and analysed in vitro on jejunum, the direct effect of liraglutide on the expression of genes involved in the biosynthesis of chylomicron. In diabetic patients, liraglutide treatment induced a dramatic reduction of ApoB48 pool (65±38 versus 162±87 mg; P=0.005) because of a significant decrease in ApoB48 production rate (3.02±1.33 versus 6.14±4.27 mg kg -1 d -1 ; P=0.009) and a significant increase in ApoB48 fractional catabolic rate (5.12±1.35 versus 3.69±0.75 pool d -1 ; P=0.005). One-week treatment with liraglutide significantly reduced postload plasma triglycerides in mice and liraglutide, in vitro, reduced the expression of ApoB48, DGAT1 (diacylglycerol O-acyltransferase 1), and MTP (microsomal transfer protein) genes. Conclusions- We show that treatment with liraglutide induces a significant reduction of the ApoB48 pool because of both a reduction of ApoB48 production and an increase in ApoB48 catabolism. In vitro, liraglutide reduces the expression of genes involved in chylomicron synthesis. These effects might benefit cardiovascular health. Clinical Trial Registration- URL: https://www.clinicaltrials.gov . Unique identifier: NCT02721888.

Objective- Although postprandial hypertriglyceridemia can be a risk factor for coronary artery disease, the extent of its significance remains unknown. This study aimed to investigate the correlation between the postprandial lipid profiles rigorously estimated with the meal tolerance test and the presence of lipid-rich plaque, such as thin-cap fibroatheroma (TCFA), in the nonculprit lesion. Approach and Results- A total of 30 patients with stable coronary artery disease who underwent a multivessel examination using optical coherence tomography during catheter intervention for the culprit lesion were enrolled. Patients were divided into 2 groups: patients with TCFA (fibrous cap thickness ≤65 µm) in the nonculprit lesion and those without TCFA. Serum remnant-like particle-cholesterol and ApoB-48 (apolipoprotein B-48) levels were measured during the meal tolerance test. The value of remnant-like particle-cholesterol was significantly greater in the TCFA group than in the non-TCFA group ( P=0.045). Although the baseline ApoB-48 level was similar, the increase in the ApoB-48 level was significantly higher in the TCFA group than in the non-TCFA group ( P=0.028). In addition, the baseline apolipoprotein C-III levels was significantly greater in the TCFA group ( P=0.003). These indexes were independent predictors of the presence of TCFA (ΔApoB-48: odds ratio, 1.608; 95% confidence interval, 1.040-2.486; P=0.032; apolipoprotein C-III: odds ratio, 2.581; 95% confidence interval, 1.177-5.661; P=0.018). Conclusions- Postprandial hyperchylomicronemia correlates with the presence of TCFA in the nonculprit lesion and may be a residual risk factor for coronary artery disease.

Objective: Intestinal overproduction of atherogenic chylomicron particles postprandially is an important component of diabetic dyslipidemia in insulin-resistant states. In addition to enhancing insulin secretion, peripheral glucagon-like peptide-1 (GLP-1) receptor stimulation has the added benefit of reducing this chylomicron overproduction in patients with type 2 diabetes mellitus. Given the presence of central GLP-1 receptors and GLP-1-producing neurons, we assessed whether central GLP-1 exerts an integral layer of neuronal control during the production of these potentially atherogenic particles., Approach and Results: Postprandial production of triglyceride-rich lipoproteins was assessed in Syrian hamsters administered a single intracerebroventricular injection of the GLP-1 receptor agonist exendin-4. Intracerebroventricular exendin-4 reduced triglyceride-rich lipoprotein-triglyceride and -apolipoprotein B48 accumulation relative to vehicle-treated controls. This was mirrored by intracerebroventricular MK-0626, an inhibitor of endogenous GLP-1 degradation, and prevented by central exendin9-39, a GLP-1 receptor antagonist. The effects of intracerebroventricular exendin-4 were also lost during peripheral adrenergic receptor and central melanocortin-4 receptor inhibition, achieved using intravenous propranolol and phentolamine and intracerebroventricular HS014, respectively. However, central exendin9-39 did not preclude the effects of peripheral exendin-4 treatment on chylomicron output., Conclusions: Central GLP-1 is a novel regulator of chylomicron production via melanocortin-4 receptors. Our findings point to the relative importance of central accessibility of GLP-1-based therapies and compel further studies examining the status of this brain-gut axis in the development of diabetic dyslipidemia and chylomicron overproduction., (© 2015 American Heart Association, Inc.)

Objective: We investigated the role of adipocyte differentiation-related protein (ADRP) in triglyceride turnover and in the secretion of very low-density lipoprotein (VLDL) from McA-RH7777 cells and primary rat hepatocytes., Methods and Results: An increase in the expression of ADRP increased triglyceride accumulation in cytosolic lipid droplets and prevented the incorporation of fatty acids into secretable triglycerides, thereby reducing the secretion of triglycerides as well as of apolipoprotein B-100 (apoB-100) and apoB-48 VLDL. The ability of ADRP to block the secretion of apoB-100 VLDL1 decreased with increasing quantities of fatty acids in the medium, indicating a saturable process and emphasizing the importance of sequestering of fatty acids for the effect of ADRP on VLDL secretion. Knockdown (small interfering RNA) of ADRP decreased the pool of cytosolic lipid droplets but increased only the secretion of apoB-48 VLDL1. Additionally, there was an increased flow of fatty acids into beta-oxidation., Conclusions: ADRP is essential for the accumulation of triglycerides in cytosolic lipid droplets. An increase in ADRP prevents the formation of VLDL by diverting fatty acids from the VLDL assembly pathway into cytosolic triglycerides, whereas a decrease of the protein increases the sorting of fatty acids to beta-oxidation and promotes the secretion of apoB-48 VLDL1.

Objective: Whereas postprandial hyperlipidemia is a well-described feature of insulin-resistant states and type 2 diabetes, no previous studies have examined intestinal lipoprotein production rates (PRs) in relation to hyperinsulinemia or insulin resistance in humans., Methods and Results: Apolipoprotein B-48 (apoB-48)-containing lipoprotein metabolism was examined in the steady-state fed condition with a 15-hour primed constant infusion of [D3]-l-leucine in 14 nondiabetic men with a broad range of body mass index (BMI) and insulin sensitivity. To examine the relationship between indices of insulin resistance and intestinal lipoprotein PR data were analyzed in 2 ways: by correlation and by comparing apoB-48 PRs in those whose fasting plasma insulin concentrations were above or below the median for the 14 subjects studied (60 pmol/L). ApoB-48 PR was significantly higher in hyperinsulinemic, insulin-resistant subjects (1.73+/-0.39 versus 0.88+/-0.13 mg/kg per day; P<0.05) and correlated with fasting plasma insulin concentrations (r=0.558; P=0.038), despite great heterogeneity in apoB-48 kinetic parameters, particularly among the obese subjects. There was no significant difference in clearance of apoB-48 between the 2 groups, nor was there a significant correlation between apoB-48 fractional clearance rate and fasting insulin or homeostasis model assessment-insulin resistance., Conclusions: These are the first human data to conclusively demonstrate that intestinal apoB-48-containing triglyceride-rich lipoprotein PR is increased in hyperinsulinemic, insulin-resistant humans. Intestinal lipoprotein particle overproduction is a newly described feature of insulin resistance in humans.

Objective: To examine the impact of ezetimibe, a selective inhibitor of intestinal cholesterol absorption, on the in vivo kinetics of apolipoproteins (apo) B-48 and B-100 in humans., Methods and Results: Kinetics of triglyceride-rich lipoprotein (TRL) apoB-48 and very-low-density lipoprotein (VLDL), intermediate-density lipoprotein (IDL), and low-density lipoprotein (LDL) apoB-100 labeled with a stable isotope were assessed at baseline and at the end of 8 weeks of treatment with 10 mg/d of ezetimibe in 8 men with moderate primary hypercholesterolemia. Data were fit to a multicompartmental model using SAAMII to calculate fractional catabolic rate (FCR) and production rate (PR). Ezetimibe significantly decreased total and LDL cholesterol concentrations by -14.5% and -22.0% (P=0.004), respectively, with no significant change in plasma triglyceride and high-density lipoprotein (HDL) cholesterol levels. Ezetimibe had no significant effect on TRL apoB-48 kinetics and pool size (PS). However, VLDL and IDL apoB-100 FCRs were significantly increased (+31.2%, P=0.02 and +20.8%, P=0.04, respectively) with a concomitant elevation of VLDL apoB-100 PR (+20.9%, P=0.04). Furthermore, LDL apoB-100 PS was significantly reduced by -23.2% (P=0.004), caused by a significant increase in FCR of this lipoprotein fraction (+24.0%, P=0.04)., Conclusions: These results indicate that reduction of plasma LDL cholesterol concentration after treatment with ezetimibe is associated with an increase in FCR of apoB-100-containing lipoproteins.

Objective: Experiments in cells and animal models show that lipoprotein lipase (LpL) bound to apolipoprotein (apo)B lipoproteins enhances their uptake by receptor mediated pathways. It is unknown whether this pathway is important in humans., Methods and Results: ApoB lipoproteins with LpL were isolated from normal subjects after oral fat loading by immunoaffinity chromatography and were further separated into apoB100 and apoB48 lipoproteins. Postprandially, apoB lipoproteins with LpL had significantly greater increases (4- to 10-fold) and faster rates of clearance (5- to 8-fold) percentage-wise than those without LpL. apoB lipoproteins with LpL had enhanced clearance regardless of whether they also contained apoE. LpL was particularly important for the clearance of apoB48 lipoproteins, of which 25% (range, 11% to 31%) could be removed from circulation together with LpL during the postprandial state. apoB lipoproteins with LpL were larger in size and were enriched in triglyceride, cholesterol, and apoE compared with those without LpL. However, neither size nor apoE content explained the faster clearance rates of LpL-containing lipoproteins., Conclusions: Plasma LpL may act like an apolipoprotein to enhance the clearance of apoB lipoproteins in humans, a mechanism particularly important for intestinal lipoproteins in the postprandial state.

Background: Hypercholesterolemia has been reported to inhibit ischemia-induced angiogenesis. To address its effects on arteriogenesis, we investigated arterial growth in hypercholesterolemic low-density lipoprotein receptor(-/-)/ApoB-48(-/-) (HCE) mice., Methods and Results: The extent and the time course of arteriogenesis after femoral artery ligation was evaluated in HCE and strain-matched control mice. Distal limb perfusion was measured by laser Doppler imaging, whereas MRI was used to visualize arterial flow and micro-computed tomography to assess vascular growth. After femoral artery ligation, serial laser Doppler imaging demonstrated significantly delayed restoration of perfusion in untreated HCE compared with control mice (day 3, 0.09 versus 0.19, P<0.05). Treatment with Ad-PR39 in control mice led to a significant restoration of arterial blood flow and tissue perfusion at day 3, whereas in HCE mice, hindlimb perfusion began increasing only by day 7. Micro-CT analysis confirmed increased growth of smaller arterioles (16 to 63 microm in diameter) in the Ad-PR39-treated control compared with HCE mice. The delay in arteriogenesis in HCE mice correlated with delayed tissue appearance of F4/80+ cells. Analysis of gene expression after Ad-PR39 treatment demonstrated that HCE mice had significantly reduced expression of FGF receptor 1, hypoxia-inducible factor-1alpha, vascular cell adhesion molecule-1, macrophage scavenger receptor-1, and cyclophilin A compared with controls 3 days after arterial ligation that equalized by day 7, mimicking relative changes in arteriogenesis and tissue perfusion., Conclusions: Hypercholesterolemia results in delayed native arteriogenesis because of reduced early monocyte/macrophage influx and delayed and impaired arterial growth response to growth factor therapy.

Background: The role of vascular endothelial growth factors (VEGFs) in large arteries has been proposed to be either vasculoprotective or proatherogenic. Because VEGF family members are used for human therapy, it is important to know whether they could enhance atherogenesis. We tested the effects of the members of the VEGF gene family on atherogenesis in LDL-receptor/apolipoprotein (apo) B48 double-knockout (LDLR/apoB48) mice using systemic adenoviral gene transfer., Methods and Results: Six groups of LDLR/apoB48-deficient mice (n=110) were kept 3 months on a Western-type diet. After 6 weeks of diet, mice were injected via tail vein with recombinant adenoviruses expressing VEGF-A, -B, -C, or -D or LacZ (1 x 10(9) PFU) or rhVEGF-A protein (2 microg/kg) and euthanized 6 weeks later. Also, older mice (n=36) were injected after 4 months on the diet and euthanized 6 weeks later (total time on the diet, 22 weeks) to evaluate the effects of gene transfers on the development of more mature lesions. Aortas were analyzed for the presence of macroscopic lesions, cross-sectional lesion areas, neovascularization, and cellular composition of the lesions. All groups had equivalent plasma cholesterol and triglyceride levels. Gene transfers with recombinant adenoviruses or administration of rhVEGF-A protein had no statistically significant effects on en face atherosclerotic lesions in the aorta, cross-sectional lesion area, neovascularization, or cellular composition of the lesions., Conclusions: This study shows no proatherogenic effects of adenovirus-mediated gene transfers of VEGF-A, -B, -C, or -D in the LDLR/apoB48-deficient hypercholesterolemic mice, in which lipoprotein profile and atherosclerosis closely resemble those in human disease.

Objective: Humans with high expression of apolipoprotein(a) [apo(a)] and high plasma levels of lipoprotein(a) [Lp(a)] are at increased risk for atherosclerosis, but the mechanism is not known. We have previously shown that the KIV(5-8) domain of apo(a) has unique cell-surface binding properties, and naturally occurring fragments of apo(a) encompassing this domain are thought to be atherogenic in humans. To investigate the effect of KIV(5-8) on lipoprotein metabolism and atherosclerosis in vivo, we created several independent lines of liver-targeted KIV(5-8) transgenic mice., Methods and Results: The transgenic mice have plasma apo(a) peptide concentrations that are similar to Lp(a) concentrations in humans at risk for coronary artery disease. Remarkably, the transgenic mice had a 2- to 4-fold increase in cholesterol-rich remnant lipoproteins (RLPs) when fed a cholesterol-rich diet, and a 5- to 20-fold increase in atherosclerosis lesion area in the aortic root. Using an in vivo clearance study, we found only slight differences in the triglyceride and apolipoprotein B secretion rates between the 2 groups of mice, suggesting an RLP clearance defect. Using an isolated perfused mouse liver system, we showed that transgenic livers had a slower rate of RLP removal, which was retarded further when KIV(5-8), full-length apo(a), or Lp(a) were added to the perfusate. An apo(a) peptide that does not interact with cells, K(IV2)3, did not retard RLP removal, and low-density lipoprotein (LDL) had a much smaller effect than Lp(a)., Conclusions: We propose that high levels of apo(a)/Lp(a), perhaps acting via a specific cell-surface binding domain, inhibit hepatic clearance of remnants, leading to high plasma levels of RLPs and markedly enhanced atherosclerosis. We speculate that the KIV(5-8) region of apo(a) competes with one or more receptors for remnant clearance in the liver and that this process may represent one mechanism accounting for increased atherosclerosis in humans with high secretion levels of apo(a).

Objective: Our purpose was to determine the relationship between apolipoprotein (apo) A-I and apoB-48 and apoB-100 metabolism in moderately hypercholesterolemic humans., Methods and Results: The kinetics of apoA-I within high-density lipoprotein (HDL), apoB-48 and apoB-100 within triglyceride-rich lipoproteins, and apoB-100 within intermediate-density lipoprotein and low density-lipoprotein (LDL) were examined with a primed constant infusion of [5,5,5-(2)H(3)] leucine in the fed state (hourly feeding) in 23 subjects after consumption of a 36% total fat diet. Lipoproteins were isolated by ultracentrifugation; apolipoproteins by SDS-PAGE gels; and isotope enrichment assessed by gas chromatograph/mass spectrometry. Kinetic parameters were calculated by multicompartmental modeling of the data with SAAM II. ApoA-I production rate (PR) was correlated with LDL apoB-100 pool size (PS; r=0.49; P=0.017) and LDL cholesterol (r=0.61; P=0.002), whereas apoA-I fractional catabolic rate (FCR) was inversely correlated with apoB-48 FCR (r=-0.40; P=0.05) but not with very low-density lipoprotein apoB-100 FCR., Conclusions: Two links exist between apoA-I and apoB kinetics: 1) when LDL apoB-100 PS is high, there is increased apoA-I PR; and 2) delayed chylomicron remnant clearance (represented by apoB-48 FCR) is associated with enhanced apoA-I FCR, a finding indicating that alterations in intestinal lipoproteins may be more important in determining HDL cholesterol levels than changes in liver lipoproteins. Using stable isotopes in humans, 2 links were observed between apoA-I and apoB kinetics: (1) when LDL apoB-100 PS is high, there is increased apoA-I PR; and (2) delayed chylomicron remnant clearance is associated with enhanced apoA-I FCR, indicating that alterations in intestinal lipoproteins may be more important in determining HDL-C levels than changes in liver lipoprotein particles.

Objective: The arterial retention of apolipoprotein (apo) B100- and apoB48-containing lipoproteins was simultaneously determined in a rabbit model of human hypercholesterolemia using 3D confocal microscopy., Methods and Results: Lipoproteins containing apoB100 (LDL) and apoB48 (chylomicron remnants) were differentially conjugated with fluorophores and simultaneously perfused at equivalent concentrations under physiological conditions in situ through carotid vessels of Watanabe heritable hyperlipidemic rabbits and compared with controls. Retention of lipoproteins was defined as the amount remaining after an extensive washout phase. LDL and chylomicron remnants were both retained, primarily within the subendothelial space. Without a concomitant increase in exposure to lipoproteins, we found a marked increase in the retention of cholesterol within the intima of Watanabe heritable hyperlipidemic rabbits compared with controls, specifically because of increased entrapment of apoB48-containing lipoproteins., Conclusions: Collectively, our data suggest that hypercholesterolemia induced as a consequence of LDL receptor deficiency differentially influences retention of LDL and of chylomicron remnants.

Purpose of Review: The "response to retention" hypothesis of atherosclerosis suggests that the arterial deposition of cholesterol is directly proportional to the concentration of circulating plasma lipoproteins. However, there is increasing evidence to support the concept that specific lipoproteins may be preferentially retained within the arterial wall, possibly as a result of greater affinity for cell surface and extracellular matrices., Recent Findings: Recently, key studies have provided insight into mechanisms involved in the interaction of apolipoprotein B (apoB)-containing lipoproteins with extracellular matrices. In addition, novel methods and innovative experimental design has enabled us to differentiate between the delivery, retention and efflux of apoB(48)- and apoB(100)-containing lipoproteins. Other studies have demonstrated a relationship between extracellular matrix proteoglycan expression and the development of atherosclerosis. Discussion in the present review also extends to the mechanisms that are involved in the relative intimal retention of apoB(48)- and apoB(100)-containing lipoproteins in order to explain the atherogenicity of these macromolecules., Summary: The perspective of this review is to highlight recent advances in the area of arterial lipoprotein retention and the physiological significance these processes may have in the aetiology of cardiovascular disease. Importantly, an understanding of the mechanisms responsible for the retention of apoB(48)/B(100)-containing lipoproteins will enable new strategies to be developed for the future management of cardiovascular disease.

Objectives: Recently, we reported that exaggerated postprandial triglyceridemia in normolipidemic patients with coronary artery disease is associated with enrichment of remnant lipoproteins with apolipoprotein C-I (apoC-I). In this study, the number and composition of chylomicron remnants and very low density lipoproteins (VLDLs) were examined in 30 asymptomatic normolipidemic 50-year-old men with and without early carotid atherosclerotic lesions., Methods and Results: Intima-media thickness of the far wall of the common carotid artery was determined by B-mode ultrasound. Triglyceride-rich lipoproteins were subfractionated by density gradient ultracentrifugation and separated into VLDL and chylomicron remnant fractions by immunoaffinity chromatography. The postprandial triglyceridemia and increase in triglyceride-rich lipoprotein particle number (ie, apolipoprotein B concentrations) were not exaggerated in men with early atherosclerosis. In contrast, their large (Svedberg flotation rate 60 to 400) and small (Svedberg flotation rate 20 to 60) chylomicron remnants and VLDL were greatly enriched with apoC-I, and their small chylomicron remnants and VLDL particles were relatively enriched with cholesterol. Moreover, the number of apoC-I molecules on small chylomicron remnants was strongly associated with the degree of atherosclerosis., Conclusions: Early asymptomatic atherosclerosis in normolipidemic men without exaggerated postprandial triglyceridemia is associated with the enrichment of postprandial chylomicron and VLDL particles with apoC-I. Therefore, it is conceivable that the apoC-I content of lipoprotein remnants may serve as an early marker of coronary artery disease risk.

The microsomal triglyceride transfer protein (MTP) plays a key role in the secretion of apolipoprotein B (apoB)-containing lipoproteins. The rare variant of a functional polymorphism in the promoter region of the MTP gene has been associated with elevated transcriptional activity of the gene in vitro (MTP-493G/T). With use of a "recruit-by-genotype" approach, we investigated one of the potentially complex phenotypes of this polymorphism, the appearance in plasma of apoB-48 after a meal intake. A total of 12 homozygous carriers of the rare MTP-493T variant were identified from a population-based screening of 50-year-old healthy white men. All subjects were of the apoE3/3 genotype. Along with 48 baseline well-matched heterozygotes (n=24) plus homozygotes (n=24) for the common variant, they were given a standardized oral fat meal. Postprandial plasma concentrations of apoB-48 were determined by the combination of density gradient ultracentrifugation and analytical SDS-PAGE. The postprandial plasma concentrations of triglycerides did not differ between the groups, but homozygous carriers of the rare MTP-493T variant showed a >100% greater increase in apoB-48 in the smallest (Svedberg flotation rate constant 20 to 60) triglyceride-rich lipoprotein fraction (P=0.005). These data support the notion that elevated transcriptional activity of MTP leads to an increased generation of the smallest triglyceride-rich lipoprotein from the intestine.

We tested the hypothesis that daily aerobic exercise opposes the fasting hypertriglyceridemia and exaggerated postprandial lipemia observed after substituting dietary fat with carbohydrate. Eight healthy postmenopausal women aged 51 to 66 years consumed the same high-fat mixed meal on 3 occasions: (1) after 3 days on a low-carbohydrate diet (35%, 50%, and 15% energy from carbohydrate, fat, and protein, respectively); (2) after 3 days on an isoenergetic high-carbohydrate diet (corresponding values 70%, 15%, and 15%); and (3) after 3 days on the same high-carbohydrate diet with 60 minutes of brisk walking daily. Plasma triglycerides were higher after the high-carbohydrate diet than after the low-carbohydrate diet: fasting, 1.58+/-0.19 versus 0.96+/-0.12 mmol/L, respectively; 6-hour postprandial area under concentration versus time curve, 13.74+/-1.57 versus 10.12+/-1.15 (mmol/L)xhour, respectively (both P<0.01). In the fasted and postprandial states, concentrations of apolipoproteins B-48 and B-100 in the triglyceride-rich lipoprotein fraction were significantly higher after the high-carbohydrate diet, as was the concentration of remnant-like lipoprotein particle cholesterol (a measure of lipoprotein remnants). These carbohydrate-induced increases in the number of circulating triglyceride-rich particles and their remnants were abolished when subjects had exercised daily during the high-carbohydrate diet.

Atherosclerosis is an inflammatory disease, and the involvement of immune mechanisms in disease progression is increasingly recognized. Immunization with oxidized low density lipoprotein (LDL) decreases atherosclerosis in several animal models. To explore humoral and cellular immune reactions involved in this protection, we immunized apolipoprotein E knockout mice with either homologous plaque homogenates or homologous malondialdehyde (MDA)-LDL. Immunization with both these antigen preparations reduced lesion development. The plaques contained immunogen(s) sharing epitopes on MDA-LDL, MDA-very low density lipoprotein, and oxidized cardiolipin. This shows that a T-cell-dependent antibody response was associated with protection against atherosclerosis. The protection was associated with specific T-cell-dependent elevation of IgG antibodies against MDA-LDL and oxidized phospholipids, and the increased titers of IgG antibodies were correlated with decreased lesion formation and lower serum cholesterol levels.

The pathogenesis for atherosclerosis is still unclear, and several hypotheses have been articulated to explain the initiating events in atherogenesis. Although these hypotheses are by no means mutually exclusive, there is a growing body of recent evidence that has led to the concept that subendothelial retention of apolipoprotein B100-containing lipoproteins is the initiating event in atherogenesis. Subsequently, a series of biological responses to this retained material leads to specific molecular and cellular processes that promote lesion formation. The present review assesses some of the studies that support this concept.

Subjects with the apolipoprotein (apo) E4 allele have been shown to have higher low density lipoprotein (LDL) cholesterol and apoB levels than do subjects with the other alleles. To elucidate the metabolic mechanisms responsible for this finding, we examined the kinetics of apoB-48 within triglyceride-rich lipoproteins (TRLs) and of apoB-100 within very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), and LDL by using a primed constant infusion of [5,5,5-(2)H(3)]leucine in the fed state (hourly feeding) during consumption of an average American diet in 18 normolipidemic subjects, 12 of whom had the apoE3/E3 genotype and 6, the apoE3/E4 genotype. Lipoproteins were isolated by ultracentrifugation and apolipoproteins, by sodium dodecyl sulfate gels; isotope enrichment was assessed by gas chromatography-mass spectrometry. Kinetic parameters were calculated by multicompartmental modeling of the data with SAAM II software. Compared with the apoE3/E3 subjects, the apoE3/E4 subjects had significantly higher levels of total apoB, 100. 1+/-17.8 versus 135.4+/-34.0 mg/dL (P=0.009), and significantly higher levels of LDL apoB-100, 88.1+/-19.2 versus 127.5+/-32.7 mg/dL (P=0.005), respectively. The pool size of TRL apoB-48 was 17.4% lower for apoE3/E4 subjects compared with apoE3/E3 subjects due to a 33.3% lower production rate (P=0.28). There was no significant difference in the TRL apoB-48 fractional catabolic rate (5.1+/-2.2 versus 5.0+/-2.1 pools per day). The pool size for VLDL apoB-100 was 36% lower for apoE3/E4 subjects compared with apoE3/E3 subjects due entirely to a 30% lower production rate (P=0.04). The LDL apoB-100 pool size was 57.8% higher (P=0.003) for apoE3/E4 subjects compared with apoE3/E3 subjects due to a 35.5% lower fractional catabolic rate of LDL apoB-100 (P=0.003), with no significant difference in production rate. In addition, 77% of VLDL apoB-100 was converted to LDL apoB-100 in apoE3/E4 subjects compared with 58% in apoE3/E3 subjects (P=0.05). In conclusion, the presence of 1 E4 allele was associated with higher LDL apoB-100 levels owing to lower fractional catabolism of LDL apoB-100 and a 33% increase in the conversion of VLDL apoB-100 to LDL apoB-100.

Background: Exaggerated postprandial triglyceridemia is common in normolipidemic patients with coronary artery disease (CAD). Alterations in the composition of triglyceride-rich lipoproteins (TRLs) are likely to underlie this metabolic disturbance. However, the composition of very-low-density lipoproteins (VLDLs), which are the most abundant postprandial TRLs, has never been defined in CAD patients., Methods and Results: We examined postprandial changes in the number and composition of VLDLs in middle-aged, normolipidemic CAD patients and control subjects. TRLs from 14 patients and 14 control subjects aged 45 to 55 years were subfractionated by density gradient ultracentrifugation into Svedberg flotation rate (Sf) fractions >400, 60 to 400, and 20 to 60. The VLDLs were separated from chylomicron remnants by immunoaffinity chromatography. In CAD patients, the postprandial concentrations of triglycerides and large (Sf 60 to 400) VLDL particles were elevated. In addition, their postprandial large VLDLs were enriched in apolipoprotein (apo) C-I and their postprandial small (Sf 20 to 60) VLDL remnants were enriched with apo C-I and cholesterol., Conclusions: Perturbed handling of postprandial triglycerides in normolipidemic CAD patients involves the accumulation of apo C-I-rich large VLDL particles and the generation of small, apo C-I- and cholesterol-rich VLDL remnants.

The kinetics of apolipoprotein (apo) B-100 and apoB-48 within triglyceride-rich lipoproteins (TRLs) and of apoB-100 within IDL and LDL were examined with a primed-constant infusion of (5,5,5-(2)H(3)) leucine in the fed state (hourly feeding) in 19 subjects after consumption of an average American diet (36% fat). Lipoproteins were isolated by ultracentrifugation and apolipoproteins by SDS gels, and isotope enrichment was assessed by gas chromatography/mass spectrometry. Kinetic parameters were calculated by multicompartmental modeling of the data with SAAM II. The pool sizes (PS) of TRL apoB-48, VLDL apoB-100, and LDL apoB-100 were 17+/-10, 273+/-167, and 3325+/-1146 mg, respectively. There was a trend toward a faster fractional catabolic rate (FCR) for VLDL apoB-100 than for TRL apoB-48 (6.73+/-3.48 versus 5.02+/-2.07 pools/d, respectively, P=0.06). The mean FCRs for IDL and LDL apoB-100 were 10.07+/-7.28 and 0.27+/-0.08 pools/d, respectively. The mean production rate (PR) of TRL apoB-48 was 6.5% of VLDL apoB-100 (1. 3+/-0.90 versus 20.06+/-6.53 mg. kg(-1). d(-1), P<0.0001). TRL apoB-48 PS was correlated with apoB-48 PR (r=0.780, P<0.0001) but not FCR (r=-0.1810, P=0.458). VLDL apoB-100 PS was correlated with both PR (r=0.713, P=0.0006) and FCR (r=-0.692, P=0.001) of VLDL apoB-100 and by apoB-48 PR (r=0.728, P=0.0004). LDL apoB-100 PS was correlated with FCR (r=-0.549, P=0.015). These data indicate that (1) the FCRs of TRL apoB-48 and VLDL apoB-100 are similar in the fed state, (2) TRL apoB-48 PS is correlated with TRL apoB-48 PR, (3) VLDL apoB-100 PS is correlated with both PR and FCR of VLDL apoB-100 and PR of TRL apoB-48, and (4) LDL apoB-100 PS is correlated with LDL FCR.

Background: Alimentary lipemia has been associated with coronary heart disease and common carotid artery intima-media thickness (IMT). This study was designed to investigate the relations of subclasses of postprandial triglyceride-rich lipoproteins (TRLs) with IMT., Methods and Results: Ninety-six healthy 50-year-old men with an apolipoprotein (apo) E3/E3 genotype underwent an oral fat tolerance test and B-mode carotid ultrasound examination. The apo B-48 and apo B-100 contents of each fraction of TRLs were determined as a measure of chylomicron remnant and VLDL particle concentrations. In the fasting state, LDL cholesterol (P<0.05) and basal proinsulin (P<0. 05) were significantly related to IMT, whereas HDL cholesterol, plasma triglycerides, and insulin were not. In the postprandial state, plasma triglycerides at 1 to 4 hours (P<0.01 at 2 hours), total triglyceride area under the curve (AUC) (P<0.05), incremental triglyceride AUC (P<0.01), and the large VLDL (Sf 60 to 400 apo B-100) concentration at 3 hours (P<0.05) were significantly related to IMT. Multivariate analyses showed that plasma triglycerides at 2 hours, LDL cholesterol, and basal proinsulin were consistently and independently related to IMT when cumulative tobacco consumption, alcohol intake, waist-to-hip circumference ratio, and systolic blood pressure were included as confounders., Conclusions: These results provide further evidence for postprandial triglyceridemia as an independent risk factor for early atherosclerosis and also suggest that the postprandial triglyceridemia is a better predictor of IMT than particle concentrations of individual TRLs.

Hepatocytes derived either from rats fed a diet enriched in n-3 fatty acids or from rats fed a low-fat diet and cultured with an n-3 fatty acid (eicosapentaenoic acid, EPA) in vitro were used to distinguish between the dietary effects and the direct effects of n-3 fatty acids on hepatocellular apolipoprotein (apo) B metabolism and secretion. ApoB-48 and apoB-100 synthesis, degradation, and secretion as large (d<1.006) and small (d>1.006) particles were determined after a pulse label with [35S]methionine. These effects were compared with changes in triacylglycerol (TAG) synthesis and secretion and with changes in de novo fatty acid synthesis (using 3H2O incorporation) under identical conditions. When n-3 fatty acid was given via the dietary route, apoB-48 very low density lipoprotein (VLDL) secretion was inhibited, but there was no effect on the secretion of apoB-100 VLDL. There was no effect on the secretion of either apoB-48 or apoB-100 as small, dense particles (d>1.006). Cellular TAG synthesis was significantly inhibited under these conditions, and fatty acid synthesis de novo was inhibited by 80%. By contrast, after direct addition of EPA to hepatocytes from normal rats, the secretion of both apoB-48 and apoB-100 VLDL was suppressed. The secretion of apoB-48, but not of apoB-100, as dense particles was also inhibited. However, there was little or no effect on TAG synthesis nor on fatty acid synthesis de novo. In addition, whereas dietary administration of n-3 fatty acid gave rise to decreased net synthesis and degradation of apoB-48, direct administration in vitro resulted in increased degradation with no effect on net synthesis. We conclude that the effects of n-3 fatty acids on hepatic lipid and apoB metabolism differ according to whether they are administered in vivo, via the dietary route, or in vitro, via direct addition to hepatocyte cultures.

Studies in animals and humans have demonstrated uptake of plasma chylomicrons (triglyceride-rich lipoprotein [TGRLP] of Sf>400) by accessible macrophages in vivo. One potential mechanism is via a unique receptor pathway we previously identified in human blood and THP-1 monocytes and macrophages for the lipoprotein lipase (LpL)- and apolipoprotein (apo) E-independent, high-affinity, specific binding of plasma chylomicrons and hypertriglyceridemic VLDL (HTG-VLDL) to cell-surface membrane-binding proteins (MBP 200, 235; apparent Mr 200, 235 kD on SDS-PAGE) that leads to lipid accumulation in vitro. Competitive binding studies reported here demonstrate that anti-apoB antibodies specifically block the high-affinity binding of TGRLP to this receptor on THP-1 cells and on ligand blots. LpL, which binds to an N-terminal domain of apoB, also inhibits TGRLP binding both to this site on THP-1s and to MBP 200, 235 by binding to apoB. Chylomicrons of Sf>1100 that contain apoB-48, but not apoB-100, bind specifically to MBP 200, 235, and this binding is blocked by anti-apoB IgG. In contrast, lactoferrin and heparin do not inhibit TGRLP binding. We conclude that the receptor-binding domain is within apoB-48 (or an equivalent in apoB-100) near the LpL-binding domain, but not a heparin-binding domain. Uptake of TGRLP by this mechanism could provide essential nutrients or, in HTG, cause excess lipid accumulation and foam cell formation.

Intestinal cells secrete apoB48-containing very low density lipoproteins (VLDLs) and chylomicrons for the transport of biliary and dietary lipids. The molecular mechanisms regulating the assembly of intestinal lipoproteins are not known due to a lack of reliable and specific cell culture models. Caco-2 (a human colon carcinoma) cells have been used to study intestinal lipid metabolism. These cells have been shown to secrete both apoB100- and apoB48-containing triglyceride (TG)-rich lipoproteins only after differentiation into enterocyte-like cells. To study lipoprotein assembly in nondifferentiated Caco-2 cells, we stably expressed human recombinant apoB48 cDNA under the control of a constitutive cytomegalovirus promoter. Pulse-chase analysis revealed that the majority (> 50%) of apoB48 synthesized was degraded intracellularly in the presence or absence of oleic acid. Transfected nondifferentiated cells secreted lipoproteins with flotation densities similar to those of plasma HDL or LDL when cultured in serum-free or serum-containing media, respectively. Incubation of cells with media containing serum and oleic acid resulted in the secretion of VLDL-like particles. Secretion of VLDL was inhibited (> 80%) by triacsin C due to > 60% inhibition of oleate-induced TG synthesis. However, inhibition of cholesteryl ester synthesis by 70% with an acyl coenzyme A:cholesterol acyltransferase inhibitor did not affect VLDL secretion. Efficient assembly of lipoproteins usually requires the microsomal TG transfer protein (MTP). The presence of MTP in transfected Caco-2 cells was investigated by measuring TG transfer activity in microsomal fractions. Microsomal fractions had 0.2% TG transfer activity per hour per microgram of protein, which corresponds to 30% to 60% of the MTP activity present in liver-derived cells. To determine whether MTP activity was required for lipoprotein assembly, transfected cells were incubated in the presence of the MTP inhibitor CP-10,447. This compound completely abolished the secretion of apoB. These data show that the transfected cell lines secrete lipoproteins of different densities under different culture conditions and that the assembly of larger VLDL particles requires active TG synthesis and MTP activity. Thus, in nondifferentiated Caco-2 cells, the amount of apoB secreted and not the MTP activity is the limiting factor for lipoprotein assembly.

Smokers have an increased risk for coronary artery disease (CAD), which can only partly be explained by fasting lipoprotein changes. Recent studies have indicated that smokers express metabolic abnormalities characteristic of insulin resistance syndrome. A preliminary study reported an increased postprandial triglyceride (TG) response in smokers compared with nonsmokers. To investigate the effect of smoking on postprandial lipemia, a fat-rich mixed meal (837 kcal, 63 g of fat) was served to 12 healthy smokers and 12 controls with similar fasting lipoprotein profiles, body composition, and lifestyles. Blood was drawn before and 3, 4, 6, and 8 hours postprandially, and triglyceride-rich lipoprotein (TRL) fractions (chylomicrons, VLDL1, VLDL2, and IDL) were separated with density gradient ultracentrifugation. Pre- and postprandial TG, retinyl esters (RE), apolipoprotein B-48 (apoB-48) and B-100 (apoB-100) were measured in each fraction. Smokers showed a significantly increased postprandial TG response in chylomicrons, VLDL1, and VLDL2. The areas under the incremental curve (AUIC) of apoB-48 in chylomicrons (2.83 +/- 0.84 versus 0.56 +/- 0.17; P < .05) and VLDL1 (10.17 +/- 1.96 versus 2.95 +/- 2.44; P = < .01) were markedly higher in smokers than in controls. Changes of RE responses of all TRL fractions were consistent with those of apoB-48. Postprandial apoB-100 concentrations and lipolytic enzymes were similar between the two groups. In conclusion, smokers have the syndrome of impaired TG tolerance because of defective clearance of chylomicrons and their remnants. Prolonged residence time of atherogenic remnant particles may constitute a significant risk factor for CAD in smokers.

Apolipoprotein B (apo B) plays a dominant role in cholesterol homeostasis. Several polymorphic sites within or adjacent to the gene locus for apo B have been detected. The X+ allele (XbaI restriction site present) of the XbaI restriction fragment polymorphism on the apo B gene has been found in some studies to be associated with higher serum cholesterol and/or triglyceride levels and with greater dietary response. The present study was designed to evaluate whether the apo B XbaI polymorphism was associated with the interindividual variability observed during postprandial lipemia. Fifty-one healthy young male volunteers [20 X-/X- (X-), and 31 X+/X- or X+/X+ (X+)], homozygotes for the apo E3 allele, were subjected to a vitamin A-fat load test. Subjects with the X- genotype had significantly greater retinyl palmitate (RP) and apo B-48 postprandial responses on both the large and the small TRL lipoprotein fractions compared with X+ subjects. In summary, subjects with the X-/X- genotype at the apo B locus have a greater postprandial response than X+ subjects. These differences observed in postprandial lipoprotein metabolism could explain some of the reported associations of this polymorphism to coronary heart disease risk.

Primary rat hepatocyte cultures were enriched in cellular triacylglycerol (TAG) by exposure to extracellular oleate for 3 days. Control cells were cultured for the same time without oleate. The large increase in TAG secretion into the medium of TAG-enriched cells during the final 24 hours (225 +/- 30 versus 40 +/- 10 micrograms/mg cell protein [control cells], P < .01) was not accompanied by a similar change in apolipoprotein B (apoB) secretion (4.22 +/- 0.94 versus 3.72 +/- 0.75 micrograms/mg per 24 hours, respectively). Instead, TAG-enriched cells recruited a larger proportion of apoB for the synthesis of very low density lipoprotein (VLDL), the secretion of which was substantially higher under these circumstances (1.46 +/- 0.39 versus 0.34 +/- 0.06 microgram apoB per milligram cell protein per 24 hours, P < .05). The increase in VLDL assembly was accompanied by a selective 2.5-fold increase (P < .05) in the specific recruitment of apoB-48. There was no significant increase in the amount of apoB-100, which appeared in the VLDL fraction when cells were enriched with TAG. Under these circumstances there was an increase in net cellular synthesis of apoB-48 (5524 +/- 667 versus 2505 +/- 598 disintegrations per minute per milligram protein per hour, P < .05). The net cellular synthesis of apoB-100 was unchanged compared with that observed in control cell cultures (1548 +/- 237 versus 2000 +/- 897 dpm/ mg per hour, respectively). A large proportion of the total secreted apoB was associated with small particles of density higher than VLDL, even when VLDL output was maximally stimulated, suggesting that apoB was oversecreted and in excess of the cells' requirement to transport TAG.

Blood lipid alterations after a fatty meal may be atherogenic, but there is little information regarding their associations with disease independent of fasting lipids. Asymptomatic atherosclerosis cases (n = 229) and 373 control subjects free of atherosclerosis, as defined by carotid intima-media thickness on ultrasound images, were given a fatty meal with vitamin A, followed by 3.5- and 8-hour measurements of triglycerides (TGs), TG-rich lipoprotein TGs, apoproteinB48, and retinyl palmitate. Among white men and women but not among blacks, case status was associated with greater postprandial responses of TGs and TG-rich lipoprotein TGs, but only in nonobese persons (body mass index < 30 kg/m2). The associations were strong and significant after controlling for coronary risk factors (odds ratio, approximately 2.0) and fasting TGs (odds ratio, 1.5). Associations with other postprandial lipid measurements did not persist after controlling for fasting lipids. Elevated postprandial TGs appear to be an independent risk factor for carotid intimal thickening in nonobese whites. The lack of such a relation in obese subjects and the lipid profile they manifest suggest that postprandial TGs must be accompanied by accumulation of TG-rich lipoprotein remnants to be atherogenic.

The effects of diets rich in saturated fatty acids (SFA) (total polyunsaturated fatty acids [PUFA] [g]/total SFA [g][P/S ratio], 0.2) or omega-6 PUFA (P/S ratio, 1.3) on the postprandial response of triglyceride-rich lipoproteins (TRL) was determined in normolipidemic young men. After 15 and 29 days of diet intervention, the postabsorptive concentrations of apolipoprotein (apo) B-48 and apoB-100 were higher in the SFA group than in the PUFA group, but the absolute increase in apoB-48 was similar 3 hours after a challenge meal containing one third of daily energy and returned to postabsorptive values at 6 hours; this response was closely coupled to that of TRL triglycerides. In both groups, the percent increase in TRL apoB-48 and triglycerides was greater after the PUFA meal than after the SFA meal. The concentration of TRL apoB-100 also increased at 3 hours in both diet groups but returned to postabsorptive values at 6 hours only in those fed the PUFA diet; in the SFA group, apoB-100 remained high at 6 hours and fell below postabsorptive values only 9 hours after the meal. This apoB-100 response was affected primarily by the fatty acid composition of the diet and not by that of the challenge meal. The postprandial response of apoB-100 was closely coupled to that of cholesterol and apoE. These observations suggest that in healthy young men, neither the fatty acid composition of the diet nor that of the challenge meal affects the clearance of chylomicron remnants after a fat-containing meal. By contrast, the postprandial accumulation of hepatogenous TRL is prolonged in individuals fed a diet rich in SFA.

Oversecretion of apoB and decreased removal of apoB-containing lipoproteins by the liver results in hyperapobetalipoproteinemia, which is a risk factor for atherosclerosis. We investigated how dexamethasone, a synthetic glucocorticoid, affects the synthesis, degradation, and secretion of apoB-100 and apoB-48. Primary rat hepatocytes were incubated with dexamethasone for 16 hours. Incorporation of [35S]methionine into apoB-48 and apoB-100 was increased by 36% and 50%, respectively, with 10 nmol/L dexamethasone, despite a 28% decrease of incorporation into total cell proteins. However, Northern blot analysis revealed that dexamethasone (1 to 1000 nmol/L) did not significantly alter the steady-state concentrations of apoB mRNA, suggesting that the net increase in apoB synthesis may involve increased translational efficiency. The intracellular retention and the rate and efficiency of apoB secretion were determined by pulse-chase experiments in which the hepatocytes were labeled with [35S]methionine for 10 minutes or 1 hour, and the disappearance of labeled apoB from the cells and its accumulation in the medium were monitored. Degradation of labeled apoB-100 after a 3-hour chase in both protocols was decreased from about 50% to 30%, whereas degradation of apoB-48 was decreased from 30% to 10% to 20% by treatment with 10 or 100 nmol/L dexamethasone. Additionally, the half-life of decay (time required for 50% of labeled cell apoB-100 to disappear from the peak of radioactivity following a 10-minute pulse) was increased by treatment with 10 nmol/L dexamethasone from 77 to 112 minutes, and the value for apoB-48 increased from 145 to 250 minutes. Treatment with 100 nmol/L dexamethasone also stimulated secretion of 35S-labeled apoB-100 and apoB-48 by twofold and 1.5-fold, respectively. The increased secretion of apoB-100 and apoB-48 after dexamethasone treatment was confirmed by immunoblot analysis for apoB mass, and the effect was relatively specific since albumin secretion was not significantly changed. We conclude that glucocorticoids promote the secretion of hepatic apoB-containing lipoproteins by increasing the net synthesis of apoB-100 and apoB-48 and by decreasing the intracellular degradation of newly synthesized apoB. An increased action of glucocorticoids coupled with a decreased ability of insulin to suppress these effects in insulin resistance can lead to hyperapobetalipoproteinemia and an increased risk of atherosclerosis.

We studied the effect of overexpression of apolipoprotein (apo) B-48 on the synthesis and secretion of endogenous apoB-100 in rat hepatoma McA-RH7777 cell lines stably transfected with human apoB-48 cDNA under the control of the cytomegalovirus promoter. Three cell lines that secrete 40 to 60 ng human apoB.mg cell protein-1.h-1 were used. The recombinant human apoB-48 exhibited physicochemical characteristics (buoyant density, 1.06 to 1.21 g/mL; beta-electrophoretic mobility and diameters, 16 to 20 nm) indistinguishable from those of endogenous rat apoB-48. Overexpression of the recombinant human apoB-48 resulted in a 50% decrease in the secretion of endogenous apoB-100 but did not affect the secretion of apoE or apoA-I. Several possible mechanisms for the decreased secretion of apoB-100 were evaluated. First, recruitment of lipids into lipoproteins was shown to be unaffected since no major changes in the physicochemical properties of apoB-100-containing lipoproteins were observed. Second, the intracellular degradation of apoB-100 was not altered as the intracellular retention half-time and secretion efficiency remained unaffected by apoB-48 overexpression. Third, the posttranslational regulatory mechanisms for apoB-100 remained normal, as demonstrated by a twofold increase in apoB-100 secretion after supplementation with oleic acid. Unexpectedly, a 35% to 50% decrease in the steady-state synthesis of endogenous apoB-100 was observed in apoB-48-transfected cells compared with control cells. These data suggested that decreased secretion of apoB-100 was secondary to decreased synthesis. The decreased apoB-100 synthesis was not due to decreased steady-state levels of rat apoB-100 mRNA. These results suggest that overexpression of recombinant human apoB-48 may interfere with posttranscriptional events, possibly at the translation-translocation level, and decrease translational yield of apoB-100. These posttranscriptional events prior to the complete synthesis of the apoB-100 polypeptide can be important in the control of apoB-100 secretion.

The metabolism of chylomicrons, very-low-density lipoprotein (VLDL), and their remnants in the postprandial state was studied in normolipidemic healthy men by measuring apoB-48 and apoB-100 and retinyl palmitate (RP) in fractions of triglyceride-rich lipoproteins after a mixed meal type of oral fat load supplemented with vitamin A. ApoB-48 was present at low concentrations in the fasting plasma samples in most subjects and increased in response to the test meal in Svedberg's flotation rate (Sf) > 20 lipoprotein fractions. Concomitantly, the level of Sf 60 to 400 apoB-100 (large VLDL) had doubled at 3 hours and returned to baseline at 9 hours. The number of apoB-48-containing lipoprotein particles did not exceed 20% of the total number of apoB-containing lipoproteins contained in Sf 12 to 400 fractions at any time point after fat intake. The peak plasma level of RP was delayed compared with the peak plasma concentration of apoB-48, suggesting that retinyl ester labeling of chylomicrons is questionable as a means of quantifying postprandial triglyceride-rich lipoproteins of intestinal origin. Approximately 2000 and 4000 RP molecules were carried in each chylomicron particle in the 3- and 6-hour samples, respectively, in contrast to the remnant fractions in which 100 to 600 RP molecules were found for each lipoprotein particle. The limited RP exchange between lipoprotein particles indicates that the smaller intestinal lipoproteins do not originate primarily from larger Sf > 400 chylomicron particles but instead are secreted directly into the Sf 20 to 400 fraction and subsequently converted to smaller chylomicron remnants.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved methods for quantification of organ-specific contributions to postprandial hyperlipidemia indicate substantial increases in the concentration and altered properties of hepatogenous VLDL, in addition to accumulation of chylomicron remnants. Recent clinical studies support the hypothesis that small chylomicron remnants characterized by apolipoprotein (apo)B48 and VLDL remnants characterized by apolipoprotein B100 are atherogenic lipoproteins.

Transgenic mice that express human apolipoprotein (apo)B have been developed by microinjecting fertilized mouse oocytes with an 80 kb genomic DNA fragment that encompasses the entire human APOB gene. In the transgenic mice expressing the largest amounts of human apoB, the concentration of human apoB100 in the plasma is nearly as high as the levels observed in normolipidemic humans (50 mg/dl). Virtually all of the human apoB100 in the transgenic plasma is located in the LDL fraction, resulting in substantially increased levels of LDL cholesterol. These human apoB-transgenic mice should be useful animal models for understanding various aspects of lipoprotein metabolism and for further delineating the role of LDL in atherogenesis.

We describe three patients with Anderson's disease who are members of one family; the father and mother are close relatives and three of seven children show symptoms of the disease. All patients suffered from diarrhea, failure to thrive, and recurrent infections during infancy. Although these symptoms disappeared later in life, biochemical disorders (such as low plasma levels of apolipoproteins A1 and B and cholesterol, resulting in avitaminosis E, plus failure to secrete chylomicrons after a fat meal) persisted. Electron microscopy of enterocytes of one of the patients showed accumulation of lipid vacuoles with no significant aberration of the Golgi apparatus itself. It is possible, therefore, that the disease reflects a defect in chylomicron assembly. We found that low levels of apolipoprotein (apo) B48 were present in the patients' plasma. This suggests that the processing of the B100 message resulting in apo B48 functions normally. The possibility that a mutation in the apo B gene results in an abnormal apo B48 protein is very unlikely since a variable number tandem repeat (VNTR) polymorphism probe mapped to chromosome 2 failed to show correspondence of the parent alleles with the disease. These observations confirm the suggestion that Anderson's disease is not linked to the apo B locus.

For the past 5 years, investigators from many different laboratories have contributed to a greatly increased understanding of two very important lipid-carrying proteins in plasma--apo B-100 and apo B-48. Apo B-100, an extremely large protein composed of 4,536 amino acids, is synthesized by the liver and is crucial for the assembly of triglyceride-rich VLDL particles. Apo B-100 is virtually the only protein of LDL, a cholesteryl ester-enriched class of lipoproteins that are metabolic products of VLDL. The apo B-100 of LDL serves as a ligand for the LDL receptor-mediated uptake of LDL particles by the liver and extrahepatic tissues. The LDL receptor-binding region of apo B-100 is located in the carboxyterminal portion of the molecule, whereas its lipid-binding regions appear to be broadly dispersed throughout its length. Apo B-48 contains the amino-terminal 2,152 amino acids of apo B-100 and is produced by the intestine as a result of editing of a single nucleotide of the apo B mRNA, which changes the codon specifying apo B-100 amino acid 2,153 to a premature stop codon. Apo B-48 has an obligatory structural role in the formation of chylomicrons; therefore, its synthesis is essential for absorption of dietary fats and fat-soluble vitamins. Both apo B-48 and apo B-100 are encoded on chromosome 2 by a single gene that contains 29 exons and 28 introns. An elevated level of apo B-100 in the plasma is a potent risk factor for developing premature atherosclerotic disease. In the past 3 years, many different apo B gene mutations that affect the concentrations of both apo B and cholesterol in the plasma have been characterized. A missense mutation in the codon for apo B-100 amino aid 3,500 is associated with hypercholesterolemia. This mutation results in poor binding of apo B-100 to the LDL receptor, thereby causing the cholesteryl ester-enriched LDL particles to accumulate in the plasma. This disorder is called familial defective apo B-100, and it is probably a cause of premature atherosclerotic disease. Familial hypobetalipoproteinemia is a condition associated with abnormally low levels of apo B and cholesterol; affected individuals may actually have a reduced risk of atherosclerotic disease.(ABSTRACT TRUNCATED AT 400 WORDS)