ApoA-IV modulates the secretory trafficking of apoB and the size of triglyceride-rich lipoproteins

Although the evidence linking apoA-IV expression and triglyceride (TG)-rich lipoprotein assembly and secretion is compelling, the intracellular mechanisms by which apoA-IV could modulate these processes remain poorly understood. We therefore examined the functional impact of apoA-IV expression on endogenous apoB, TG, and VLDL secretion in stably transfected McA-RH7777 rat hepatoma cells. Expression of apoA-IV modified with the endoplasmic reticulum (ER) retention signal KDEL (apoA-IV-KDEL) dramatically decreased both the rate and efficiency of endogenous apoB secretion, suggesting a presecretory interaction between apoA-IV-KDEL and apoB or apoB-containing lipoproteins. Expression of native apoA-IV using either a constitutive or tetracycline-inducible promoter delayed the initial rate of apoB secretion and reduced the final secretion efficiency by ～40%. However, whereas apoA-IV-KDEL reduced TG secretion by 75%, expression of native apoA-IV caused a 20-35% increase in TG secretion, accompanied by a ～55% increase in VLDL-associated apoB, an increase in the TG:phospholipid ratio of secreted d < 1.006 lipoproteins, and a 10.1 nm increase in peak VLDL(1) particle diameter. Native apoA-IV expression had a negligible impact on expression of the MTP gene. These data suggest that by interacting with apoB in the secretory pathway, apoA-IV alters the trafficking kinetics of apoB-containing TG-rich lipoproteins through cellular lipidation compartments, which in turn, enhances particle expansion and increases TG secretion.     

Short-term overexpression of DGAT1 or DGAT2 increases hepatic triglyceride but not VLDL triglyceride or apoB production

Increased triglyceride synthesis resulting from enhanced flux of fatty acids into liver is frequently associated with VLDL overproduction. This has led to the common belief that hepatic triglyceride synthesis can directly modulate VLDL production. We used adenoviral vectors containing either murine acyl-coenzyme A:diacylglycerol transferase 1 (DGAT1) or DGAT2 cDNA to determine the effect of a short-term increase in hepatic triglyceride synthesis on VLDL triglyceride and apolipoprotein B (apoB) production in female wild-type mice. Hepatic DGAT1 and DGAT2 overexpression resulted in 2.0-fold and 2.4-fold increases in the triglyceride content of liver, respectively. However, the increase in hepatic triglyceride content had no effect on the production rate of VLDL triglyceride or apoB in either case. Liver subfractionation showed that DGAT1 and DGAT2 overexpression significantly increased the content of triglyceride within the cytoplasmic lipid fraction, with no change in the triglyceride content of the microsomal membrane or microsomal VLDL. The increased cytoplasmic triglyceride content was observed in electron micrographs of liver sections from mice overexpressing DGAT1 or DGAT2. Overexpression of DGAT1 or DGAT2 resulted in enhanced [(3)H]glycerol tracer incorporation into triglyceride within cytoplasmic lipids. These results suggest that increasing the cytoplasmic triglyceride pool in hepatocytes does not directly influence VLDL triglyceride or apoB production. In the presence of adequate cytoplasmic lipid stores, factors other than triglyceride synthesis are rate-limiting for VLDL production.     

Secretion of triacylglycerol-poor VLDL particles from McA-RH7777 cells expressing human hepatic lipase

Hepatic lipase (HL) plays a role in the catabolism of apolipoprotein (apo)B-containing lipoproteins through its lipolytic and ligand-binding properties. We describe a potential intracellular role of HL in the assembly and secretion of VLDL. Transient or stable expression of HL in McA-RH7777 cells resulted in decreased (by 40%) incorporation of [(3)H]glycerol into cell-associated and secreted triacylglycerol (TAG) relative to control cells. However, incorporation of [(35)S]methionine/cysteine into cell and medium apoB-100 was not decreased by HL expression. The decreased (3)H-TAG synthesis/secretion in HL expressing cells was not attributable to decreased expression of genes involved in lipogenesis. Fractionation of medium revealed that the decreased [(3)H]TAG from HL expressing cells was mainly attributable to decreased VLDL. Expression of catalytically-inactive HL (HL(SG)) (Ser-145 at the catalytic site was substituted with Gly) in the cells also resulted in decreased secretion of VLDL-[(3)H]TAG. Examination of lumenal contents of microsomes showed a 40% decrease in [(3)H]TAG associated with lumenal lipid droplets in HL or HL(SG) expressing cells as compared with control. The microsomal membrane-associated [(3)H]TAG was decreased by 50% in HL expressing cells but not in HL(SG) expressing cells. Thus, expression of HL, irrespective of its lipolytic function, impairs formation of VLDL precursor [(3)H]TAG in the form of lumenal lipid droplets. These results suggest that HL expression in McA-RH7777 cells result in secretion of [(3)H]TAG-poor VLDL.     

The apoE isoform binding properties of the VLDL receptor reveal marked differences from LRP and the LDL receptor

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptor-related protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors.     

Effect of endurance training upon lipid metabolism in the liver of cachectic tumour-bearing rats

The syndrome of cancer cachexia is accompanied by several alterations in lipid metabolism, and the liver is markedly affected. Previous studies showed that moderate exercise training may prevent liver fat accumulation through diminished delivery of lipids to the liver, increased hepatic oxidation and increased incorporation of triacylglycerol (TAG) into very low density lipoprotein (VLDL). Our aim was to examine the influence of moderate intensity training (8 weeks) upon TAG content, VLDL assembly and secretion, apolipoprotein B (apoB) and microsomal transfer protein (MTP) gene expression in the liver of cachectic tumour-bearing rats. Animals were randomly assigned to a sedentary control (SC), sedentary tumour-bearing (ST) or exercise-trained control (EC) or to an exercise trained tumour-bearing (ET) group. Trained rats ran on a treadmill (60% VO(2max)) for 60 min day(-1), 5 day week(-1), for 8 weeks. TAG content and the rate of VLDL secretion (followed for 3 h), as well as mRNA expression of apoB and MTP, and total cholesterol, VLDL-TAG, VLDL-cholesterol, high density lipoprotein cholesterol (HDL-cholesterol) and tumour weight were evaluated. VLDL-cholesterol showed a decrease in ST (p < 0.05) in relation to SC. Serum TAG, VLDL-TAG and tissue TAG content were all increased in ST (p < 0.01), when compared with SC. ST showed a lower rate of VLDL secretion (p < 0.05) and reduced expression of apoB (p < 0.001) and MTP (p < 0.001), when compared with SC. These parameters were restored to control values (p < 0.05) when the animals were submitted to the exercise training protocol. Tumour weight decreased 10-fold after training (p < 0.001). It is possible to affirm, therefore, that endurance training promoted the re-establishment of lipid metabolism in cachectic tumour-bearing animals, especially in relation to VLDL secretion and assembly.     

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

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

Effect of fenofibrate and atorvastatin on VLDL apoE metabolism in men with the metabolic syndrome

We examined the effects of fenofibrate and atorvastatin on very low density lipoprotein (VLDL) apolipoprotein (apo)E metabolism in the metabolic syndrome (MetS). We studied 11 MetS men in a randomized, double-blind, crossover trial. VLDL-apoE kinetics were examined using stable isotope methods and compartmental modeling. Compared with placebo, fenofibrate (200 mg/day) and atorvastatin (40 mg/day) decreased plasma apoE concentrations (P < 0.05). Fenofibrate decreased VLDL-apoE concentration and production rate (PR) and increased VLDL-apoE fractional catabolic rate (FCR) compared with placebo (P < 0.05). Compared with placebo, atorvastatin decreased VLDL-apoE concentration and increased VLDL-apoE FCR (P < 0.05). Fenofibrate and atorvastatin had comparable effects on VLDL-apoE concentration. The increase in VLDL-apoE FCR with fenofibrate was 22% less than that with atorvastatin (P < 0.01). With fenofibrate, the change in VLDL-apoE concentration was positively correlated with change in VLDL-apoB concentration, and negatively correlated with change in VLDL-apoB FCR. In MetS, fenofibrate and atorvastatin decreased plasma apoE concentrations. Fenofibrate decreased VLDL-apoE concentration by lowering VLDL-apoE production and increasing VLDL-apoE catabolism. By contrast, atorvastatin decreased VLDL-apoE concentration chiefly by increasing VLDL-apoE catabolism. Our study provides new insights into the mechanisms of action of two different lipid-lowering therapies on VLDL-apoE metabolism in MetS.     

Effects of apoA-V on HDL and VLDL metabolism in APOC3 transgenic mice

Apolipoprotein A-V (apoA-V) and apoC-III are exchangeable constituents of VLDL and HDL. ApoA-V counteracts the effect of apoC-III on triglyceride (TG) metabolism with poorly defined mechanisms. To better understand the effects of apoA-V on TG and cholesterol metabolism, we delivered apoA-V cDNA into livers of hypertriglyceridemic APOC3 transgenic mice by adenovirus-mediated gene transfer. In response to hepatic apoA-V production, plasma TG levels were reduced significantly as a result of enhanced VLDL catabolism without alternations in VLDL production. This effect was associated with reduced apoC-III content in VLDL. Increased apoA-V production also resulted in decreased apoC-III and increased apoA-I content in HDL. Furthermore, apoA-V-enriched HDL was associated with enhanced LCAT activity and increased cholesterol efflux. This effect, along with apoE enrichment in HDL, contributed to HDL core expansion and alpha-HDL formation, accounting for significant increases in both the number and size of HDL particles. As a result, apoA-V-treated APOC3 transgenic mice exhibited decreased VLDL-cholesterol and increased HDL-cholesterol levels. ApoA-V-mediated reduction of apoC-III content in VLDL represents an important mechanism by which apoA-V acts to ameliorate hypertriglyceridemia in adult APOC3 transgenic mice. In addition, increased apoA-V levels accounted for cholesterol redistribution from VLDL to larger HDL particles. These data suggest that in addition to its TG-lowering effect, apoA-V plays a significant role in modulating HDL maturation and cholesterol metabolism.     

ACAT2 contributes cholesteryl esters to newly secreted VLDL, whereas LCAT adds cholesteryl ester to LDL in mice

The relative contributions of ACAT2 and LCAT to the cholesteryl ester (CE) content of VLDL and LDL were measured. ACAT2 deficiency led to a significant decrease in the percentage of CE (37.2 +/- 2.1% vs. 3.9 +/- 0.8%) in plasma VLDL, with a concomitant increase in the percentage of triglyceride (33.0 +/- 3.2% vs. 66.7 +/- 2.5%). Interestingly, the absence of ACAT2 had no apparent effect on the percentage CE in LDL, whereas LCAT deficiency significantly decreased the CE percentage (38.6 +/- 4.0% vs. 54.6 +/- 1.9%) and significantly increased the phospholipid percentage (11.2 +/- 0.9% vs. 19.3 +/- 0.1%) of LDL. When both LCAT and ACAT2 were deficient, VLDL composition was similar to VLDL of the ACAT2-deficient mouse, whereas LDL was depleted in core lipids and enriched in surface lipids, appearing discoidal when observed by electron microscopy. We conclude that ACAT2 is important in the synthesis of VLDL CE, whereas LCAT is important in remodeling VLDL to LDL. Liver perfusions were performed, and perfusate apolipoprotein B accumulation rates in ACAT2-deficient mice were not significantly different from those of controls; perfusate VLDL CE decreased from 8.0 +/- 0.8% in controls to 0 +/- 0.7% in ACAT2-deficient mice. In conclusion, our data establish that ACAT2 provides core CE of newly secreted VLDL, whereas LCAT adds CE during LDL particle formation.     

Effects of the cholesteryl ester transfer protein inhibitor torcetrapib on VLDL apolipoprotein E metabolism

Cholesteryl ester transfer protein (CETP) inhibition leads to changes in lipoprotein metabolism. We studied the effect of the CETP inhibitor torcetrapib on VLDL apolipoprotein E (apoE) metabolism. Subjects, pretreated with atorvastatin (n = 9) or untreated (n = 10), received placebo followed by torcetrapib (4 weeks each). After each treatment, subjects underwent a primed-constant infusion of D(3)-leucine to determine the VLDL apoE production rate (PR) and fractional catabolic rate (FCR). Torcetrapib alone reduced the VLDL apoE pool size (PS) (-28%) by increasing the VLDL apoE FCR (77%) and leaving the VLDL apoE PR unchanged. In subjects pretreated with atorvastatin, torcetrapib increased the VLDL apoE FCR (25%) and PR (21%). This left the VLDL apoE PS unchanged but increased the VLDL apoE content, likely enhancing VLDL clearance and reducing LDL production in this group. Used alone, torcetrapib reduces the VLDL apoE PS by increasing the apoE FCR while leaving the VLDL apoE content unchanged. In contrast, torcetrapib added to atorvastatin treatment increases both the VLDL apoE FCR and PR, leaving the VLDL apoE PS unchanged. Adding torcetrapib to atorvastatin treatment increases the VLDL apoE content, likely leading to decreased conversion of VLDL to LDL, reduced LDL production, and lower levels of circulating VLDL and LDL.     

Upregulation of liver VLDL receptor and FAT/CD36 expression in LDLR-/- apoB100/100 mice fed trans-10,cis-12 conjugated linoleic acid

This study explores the mechanisms responsible for the fatty liver setup in mice fed trans-10,cis-12 conjugated linoleic acid (t10c12 CLA), hypothesizing that an induction of low density lipoprotein receptor (LDLR) expression is associated with lipid accumulation. To this end, the effects of t10c12 CLA treatment on lipid parameters, serum lipoproteins, and expression of liver lipid receptors were measured in LDLR(-/-) apoB(100/100) mice as a model of human familial hypercholesterolemia itself depleted of LDLR. Mice were fed t10c12 CLA over 2 or 4 weeks. We first observed that the treatment induced liver steatosis, even in the absence of LDLR. Mice treated for 2 weeks exhibited hypertriglyceridemia with high levels of VLDL and HDL, whereas a 4 week treatment inversely induced a reduction of serum triglycerides (TGs), essentially through a decrease in VLDL levels. In the absence of LDLR, the mRNA levels of other proteins, such as VLDL receptor, lipoprotein lipase, and fatty acid translocase, usually not expressed in the liver, were upregulated, suggesting their involvement in the steatosis setup and lipoprotein clearance. The data also suggest that the TG-lowering effect induced by t10c12 CLA treatment was attributable to both the reduction of circulating free fatty acids in response to the severe lipoatrophy and the high capacity of liver to clear off plasma lipids.     

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.     

The elevation of apoB in hypercholesterolemic patients is primarily attributed to the relative increase of apoB/Lp-PLA2

Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) is a risk factor of cardiovascular disease. Plasma Lp-PLA₂ is mainly associated with apolipoprotein (apo)B-containing lipoproteins, primarily with low density lipoproteins (LDLs). Importantly, only a proportion of circulating lipoproteins contain Lp-PLA₂. We determined the plasma levels of Lp-PLA₂-bound apoB (apoB/Lp-PLA₂) in patients with primary hypercholesterolemia. The effect of simvastatin therapy was also addressed. The plasma apoB/Lp-PLA₂ concentration in 50 normolipidemic controls and 53 patients with primary hypercholesterolemia at baseline and at 3 months posttreatment with simvastatin (40 mg/day) was determined by an enzyme-linked immunosorbent assay. The concentration of the apoB-containing lipoproteins that do not bind Lp-PLA₂ [apoB/Lp-PLA₂(−)] was calculated by subtracting the apoB/Lp-PLA₂ from total apoB. The apoB/Lp-PLA₂ levels were 3.6-fold higher, while apoB/Lp-PLA₂(−) were 1.3-fold higher in patients compared with controls. After 3 months of simvastatin treatment apoB/Lp-PLA₂ and apoB/Lp-PLA₂(−) levels were reduced by 52% and 33%, respectively. The elevation in apoB-containing lipoproteins in hypercholesterolemic patients is mainly attributed to the relative increase in the proatherogenic apoB/Lp-PLA₂, while simvastatin reduces these particles to a higher extent compared with apoB/Lp-PLA₂(−). Considering that Lp-PLA₂ is proatherogenic, the predominance of apoB/Lp-PLA₂ particles in hypercholesterolemic patients may contribute to their higher atherogenicity and incidence of cardiovascular disease.     

Apolipoprotein A-V association with intracellular lipid droplets

Apolipoprotein A-V (apoA-V) plays a key role in the regulation of triglyceride (TG) metabolism. Given the very low concentration of apoA-V in plasma, we hypothesized that apoA-V may influence plasma TG levels by affecting the assembly and/or secretion of apoB-containing lipoproteins. When apoA-V was overexpressed in cultured Hep3B cells, neither the amount of apoB secreted nor the density distribution of apoB-containing lipoproteins was affected. Fluorescence microscopy and cell lysate immunoprecipitation studies revealed that apoA-V is not associated with apoB intracellularly, yet immunoprecipitation of apoA-V from the cell culture medium resulted in coprecipitation of apoB. These data suggest that the apoA-V association with apoB-containing lipoproteins is a postsecretory event. Confocal fluorescence microscopy revealed the presence of apoA-V in distinct cellular structures. Based on Nile Red staining, we identified these structures to be intracellular lipid droplets. These data suggest that apoA-V has a unique association with cellular lipids and, therefore, may be involved in the storage or mobilization of intracellular lipids.     

The hepatic uptake of VLDL in lrp-ldlr-/-vldlr-/- mice is regulated by LPL activity and involves proteoglycans and SR-BI

LPL activity plays an important role in preceding the VLDL remnant clearance via the three major apolipoprotein E (apoE)-recognizing receptors: the LDL receptor (LDLr), LDL receptor-related protein (LRP), and VLDL receptor (VLDLr). The aim of this study was to determine whether LPL activity is also important for VLDL remnant clearance irrespective of these receptors and to determine the mechanisms involved in the hepatic remnant uptake. Administration of an adenovirus expressing LPL (AdLPL) into lrp(-)ldlr(-/-)vldlr(-/-) mice reduced both VLDL-triglyceride (TG) and VLDL-total cholesterol (TC) levels. Conversely, inhibition of LPL by AdAPOC1 increased plasma VLDL-TG and VLDL-TC levels. Metabolic studies with radiolabeled VLDL-like emulsion particles showed that the clearance and hepatic association of their remnants positively correlated with LPL activity. This hepatic association was independent of the bridging function of LPL and HL, since heparin did not reduce the liver association. In vitro studies demonstrated that VLDL-like emulsion particles avidly bound to the cell surface of primary hepatocytes from lrp(-)ldlr(-/-)vldlr(-/-) mice, followed by slow internalization, and involved heparin-releaseable cell surface proteins as well as scavenger receptor class B type I (SR-BI). Collectively, we conclude that hepatic VLDL remnant uptake in the absence of the three classical apoE-recognizing receptors is regulated by LPL activity and involves heparan sulfate proteoglycans and SR-BI.     

Triglyceride-rich lipoprotein metabolism in unique VLDL receptor, LDL receptor, and LRP triple-deficient mice

The very low density lipoprotein receptor (VLDLR), low density lipoprotein receptor (LDLR), and low density lipoprotein receptor-related protein (LRP) are the three main apolipoprotein E-recognizing endocytic receptors involved in the clearance of triglyceride (TG)-rich lipoproteins from plasma. Whereas LDLR deficiency in mice results in the accumulation of plasma LDL-sized lipoproteins, VLDLR or LRP deficiency alone only minimally affects plasma lipoproteins. To investigate the combined effect of the absence of these receptors on TG-rich lipoprotein levels, we have generated unique VLDLR, LDLR, and LRP triple-deficient mice. Compared with wild-type mice, these mice markedly accumulated plasma lipids and lipases. These mice did not show aggravated hyperlipidemia compared with LDLR and LRP double-deficient mice, but plasma TG was increased after high-fat diet feeding. In addition, these mice showed a severely decreased postprandial TG clearance typical of VLDLR-deficient (VLDLR-/-) mice. Collectively, although VLDLR deficiency in LRP- and LDLR-/- mice does not aggravate hyperlipidemia, these triple-deficient mice represent a unique model of markedly delayed TG clearance on a hyperlipidemic background.     

High-fat/high-cholesterol diet promotes a S1P receptor-mediated antiapoptotic activity for VLDL

Withdrawing growth factors or serum from endothelial cells leads to the activation of effector caspases 3 and 7, resulting in apoptotic cell death. HDL protects against caspase induction through sphingosine-1-phosphate (S1P) receptors. This anti-caspase activity of HDL is antagonized by VLDL from apolipoprotein E4 (apoE4) (genotype, APOE4/4; apolipoprotein, apoE) targeted replacement (TR) mice, but not by VLDL from TR APOE3/3 mice, and requires the binding of apoE4-VLDL to an LDL receptor family member. In the absence of HDL, apoE4-VLDL and apoE3-VLDL from TR mice have limited antiapoptotic activity. In contrast, we show here that a high-fat/high-cholesterol/cholate diet (HFD) radically alters this biological activity of VLDL. On HFD, both apoE3-VLDL and apoE4-VLDL (HFD VLDL) inhibit caspase 3/7 activation initiated by serum withdrawal. This activity of HFD VLDL is independent of an LDL receptor family member but requires the activation of S1P(3) receptors, as shown by the ability of pharmacological block of S1P receptors by VPC 23019 and by small interfering RNA-mediated downregulation of S1P(3) receptors to inhibit HFD VLDL anticaspase activity.     

CETP expression reverses the reconstituted HDL-induced increase in VLDL

Human data suggest that reconstituted HDL (rHDL) infusion can induce atherosclerosis regression. Studies in mice indicated that rHDL infusion adversely affects VLDL levels, but this effect is less apparent in humans. This discrepancy may be explained by the fact that humans, in contrast to mice, express cholesteryl ester transfer protein (CETP). The aim of this study was to investigate the role of CETP in the effects of rHDL on VLDL metabolism by using APOE*3-Leiden (E3L) mice, a well-established model for human-like lipoprotein metabolism. At 1 h after injection, rHDL increased plasma VLDL-C and TG in E3L mice, but not in E3L mice cross-bred onto a human CETP background (E3L.CETP mice). This initial raise in VLDL, caused by competition between rHDL and VLDL for LPL-mediated TG hydrolysis, was thus prevented by CETP. At 24 h after injection, rHDL caused a second increase in VLDL-C and TG in E3L mice, whereas rHDL had even decreased VLDL in E3L.CETP mice. This secondary raise in VLDL was due to increased hepatic VLDL-TG production. Collectively, we conclude that CETP protects against the rHDL-induced increase in VLDL. We anticipate that studies evaluating the anti-atherosclerotic efficacy of rHDL in mice that are naturally deficient for CETP should be interpreted with caution, and that treatment of atherogenic dyslipidemia by rHDL should not be combined with agents that aggressively reduce CETP activity.     

Endogenous apoC-I increases hyperlipidemia in apoE-knockout mice by stimulating VLDL production and inhibiting LPL

Previous studies have shown that overexpression of human apolipoprotein C-I (apoC-I) results in moderate hypercholesterolemia and severe hypertriglyceridemia in mice in the presence and absence of apoE. We assessed whether physiological endogenous apoC-I levels are sufficient to modulate plasma lipid levels independently of effects of apoE on lipid metabolism by comparing apolipoprotein E gene-deficient/apolipoprotein C-I gene-deficient (apoe-/-apoc1-/-), apoe-/-apoc1+/-, and apoe-/-apoc1+/+ mice. The presence of the apoC-I gene-dose-dependently increased plasma cholesterol (+45%; P < 0.001) and triglycerides (TGs) (+137%; P < 0.001), both specific for VLDL. Whereas apoC-I did not affect intestinal [3H]TG absorption, it increased the production rate of hepatic VLDL-TG (+35%; P < 0.05) and VLDL-[35S]apoB (+39%; P < 0.01). In addition, apoC-I increased the postprandial TG response to an intragastric olive oil load (+120%; P < 0.05) and decreased the uptake of [3H]TG-derived FFAs from intravenously administered VLDL-like emulsion particles by gonadal and perirenal white adipose tissue (WAT) (-34% and -25%, respectively; P < 0.05). As LPL is the main enzyme involved in the clearance of TG-derived FFAs by WAT, and total postheparin plasma LPL levels were unaffected, these data demonstrate that endogenous apoC-I suffices to attenuate the lipolytic activity of LPL. Thus, we conclude that endogenous plasma apoC-I increases VLDL-total cholesterol and VLDL-TG dose-dependently in apoe-/- mice, resulting from increased VLDL particle production and LPL inhibition.