Hepatic ABCG5/G8 overexpression reduces apoB-lipoproteins and atherosclerosis when cholesterol absorption is inhibited

We previously reported that liver-specific overexpression of ABCG5/G8 in mice is not atheroprotective, suggesting that increased biliary cholesterol secretion must be coupled with decreased intestinal cholesterol absorption to increase net sterol loss from the body and reduce atherosclerosis. To evaluate this hypothesis, we fed low density lipoprotein receptor-knockout (LDLr-KO) control and ABCG5/G8-transgenic (ABCG5/G8-Tg)xLDLr-KO mice, which overexpress ABCG5/G8 only in liver, a Western diet containing ezetimibe to reduce intestinal cholesterol absorption. On this dietary regimen, liver-specific ABCG5/G8 overexpression increased hepatobiliary cholesterol concentration and secretion rates (1.5-fold and 1.9-fold, respectively), resulting in 1.6-fold increased fecal cholesterol excretion, decreased hepatic cholesterol, and increased (4.4-fold) de novo hepatic cholesterol synthesis versus LDLr-KO mice. Plasma lipids decreased (total cholesterol, 32%; cholesteryl ester, 32%; free cholesterol, 30%), mostly as a result of reduced non-high density lipoprotein-cholesterol and apolipoprotein B (apoB; 36% and 25%, respectively). ApoB-containing lipoproteins were smaller and lipid-depleted in ABCG5/G8-TgxLDLr-KO mice. Kinetic studies revealed similar 125I-apoB intermediate density lipoprotein/LDL fractional catabolic rates, but apoB production rates were decreased 37% in ABCG5/G8-TgxLDLr-KO mice. Proximal aortic atherosclerosis decreased by 52% (male) and 59% (female) in ABCG5/G8-TgxLDLr-KO versus LDLr-KO mice fed the Western/ezetimibe diet. Thus, increased biliary secretion, resulting from hepatic ABCG5/G8 overexpression, reduces atherogenic risk in LDLr-KO mice fed a Western diet containing ezetimibe. These findings identify distinct roles for liver and intestinal ABCG5/G8 in modulating sterol metabolism and atherosclerosis.     

Evolutionary conservation of drug action on lipoprotein metabolism-related targets

Genetic analysis has shown that the slower than normal rhythmic defecation behavior of the clk-1 mutants of Caenorhabditis elegans is the result of altered lipoprotein metabolism. We show here that this phenotype can be suppressed by drugs that affect lipoprotein metabolism, including drugs that affect HMG-CoA reductase activity, reverse cholesterol transport, or HDL levels. These pharmacological effects are highly specific, as these drugs affect defecation only in clk-1 mutants and not in the wild-type and do not affect other behaviors of the mutants. Furthermore, drugs that affect processes not directly related to lipid metabolism show no or minimal activity. Based on these findings, we carried out a compound screen that identified 190 novel molecules that are active on clk-1 mutants, 15 of which also specifically decrease the secretion of apolipoprotein B (apoB) from HepG2 hepatoma cells. The other 175 compounds are potentially active on lipid-related processes that cannot be targeted in cell culture. One compound, CHGN005, was tested and found to be active at reducing apoB secretion in intestinal Caco-2 cells as well as in HepG2 cells. This compound was also tested in a mouse model of dyslipidemia and found to decrease plasma cholesterol and triglyceride levels. Thus, target processes for pharmacological intervention on lipoprotein synthesis, transport, and metabolism are conserved between nematodes and vertebrates, which allows the use of C. elegans for drug discovery.     

Adenovirus-mediated overexpression of sphingomyelin synthases 1 and 2 increases the atherogenic potential in mice

Sphingomyelin synthase 1 (SMS1) and SMS2 are two isoforms of SMS, the last enzyme for sphingomyelin (SM) biosynthesis. To evaluate the role of SMS in vivo in terms of plasma lipoprotein metabolism, we generated recombinant adenovirus vectors containing human SMS1 cDNA (AdV-SMS1), SMS2 cDNA (AdV-SMS2), or the reporter LacZ cDNA (AdV-LacZ) as a control. On day 7 after intravenous infusion of 2 x 10(11) particles of both AdV-SMS1 and AdV-SMS2 into mice, liver SMS1 and SMS2 mRNA levels as well as SMS activity were significantly increased (2.5-, 2.7-, 2.1-, and 2.3-fold, respectively; P < 0.001). Lipoprotein analysis indicated that AdV-SMS1 and AdV-SMS2 treatment caused no changes of total SM and cholesterol levels but significantly decreased HDL-SM and HDL-cholesterol (42% and 38%, and 27% and 25%, respectively; P < 0.05). It also significantly increased non-HDL-SM and non-HDL-cholesterol levels (50% and 35%, and 64% and 61%, respectively; P < 0.05) compared with AdV-LacZ controls. SDS-PAGE showed a significant increase in apolipoprotein B (apoB; P < 0.01) but no changes in apoA-I levels. Moreover, we found that non-HDL from both AdV-SMS1- and AdV-SMS2-treated mice was significantly aggregated after treatment with a mammalian sphingomyelinase, whereas lipoproteins from control animals did not aggregate. To investigate the mechanism of HDL changes, we measured liver scavenger receptor class B type I (SR-BI) levels by Western blot. We found that AdV-SMS1 and AdV-SMS2 mouse liver homogenates contained 50% and 55% higher SR-BI levels than in controls, whereas no change was observed in hepatic ABCA1 levels. An HDL turnover study revealed an increase of plasma clearance rates for [3H]cholesteryl oleyl ether-HDL but not for [125I]HDL in both AdV-SMS1 and AdV-SMS2 mice compared with controls. In conclusion, adenovirus-mediated SMS1 and SMS2 overexpression increased lipoprotein atherogenic potential. Such an effect may contribute to the increased plasma SM levels observed in animal models of atherosclerosis and in human patients with coronary artery disease.     

A decreased expression of angiopoietin-like 3 is protective against atherosclerosis in apoE-deficient mice

KK/Snk mice (previously KK/San) possessing a recessive mutation (hypl) of the angiopoietin-like 3 (Angptl3) gene homozygously exhibit a marked reduction of VLDL due to the decreased Angptl3 expression. Recently, we proposed that Angptl3 is a new class of lipid metabolism modulator regulating VLDL triglyceride (TG) levels through the inhibition of lipoprotein lipase (LPL) activity. In this study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of hypl mutation against hyperlipidemia and atherosclerosis in apolipoprotein E knockout (apoEKO) mice. ApoEKO mice with hypl mutation (apoEKO-hypl) exhibited a significant reduction of VLDL TG, VLDL cholesterol, and plasma apoB levels compared with apoEKO mice. Hepatic VLDL TG secretion was comparable between both apoE-deficient mice. Turnover studies revealed that the clearance of both [3H]TG-labeled and 125I-labeled VLDL was significantly enhanced in apoEKO-hypl mice. Postprandial plasma TG levels also decreased in apoEKO-hypl mice. Both LPL and hepatic lipase activities in the postheparin plasma increased significantly in apoEKO-hypl mice, explaining the enhanced lipid metabolism. Furthermore, apoEKO-hypl mice developed 3-fold smaller atherogenic lesions in the aortic sinus compared with apoEKO mice. Taken together, the reduction of Angptl3 expression is protective against hyperlipidemia and atherosclerosis, even in the absence of apoE, owing to the enhanced catabolism and clearance of TG-rich lipoproteins.     

Huh-7 or HepG2 cells: which is the better model for studying human apolipoprotein-B100 assembly and secretion?

Apolipoprotein-B100 (apoB100) is the essential protein for the assembly and secretion of very low density lipoproteins (VLDL) from liver. The hepatoma HepG2 cell line has been the cell line of choice for the study of synthesis and secretion of human apoB-100. Despite the general use of HepG2 cells to study apoB100 metabolism, they secrete relatively dense, lipid-poor particles compared with VLDL secreted in vivo. Recently, Huh-7 cells were adopted as an alternative model to HepG2 cells, with the implicit assumption that Huh-7 cells were superior in some respects of lipoprotein metabolism, including VLDL secretion. In this study we addressed the hypothesis that the spectrum of apoB100 lipoprotein particles secreted by Huh-7 cells more closely resembles the native state in human liver. We find that Huh-7 cells resemble HepG2 cells in the effects of exogenous lipids, microsomal triglyceride transfer protein (MTP)-inhibition, and proteasome inhibitors of apoB100 secretion, recovery, and degradation. In contrast to HepG2 cells, however, MEK-ERK inhibition does not correct the defect in VLDL secretion. Huh-7 cells do not appear to offer any advantages over HepG2 cells as a general model of human apoB100-lipoprotein metabolism.     

Sex differences in atherosclerosis in mice with elevated phospholipid transfer protein activity are related to decreased plasma high density lipoproteins and not to increased production of triglycerides

Plasma phospholipid transfer protein (PLTP) has atherogenic properties in genetically modified mice. PLTP stimulates hepatic triglyceride secretion and reduces plasma levels of high density lipoproteins (HDL). The present study was performed to relate the increased atherosclerosis in PLTP transgenic mice to one of these atherogenic effects. A humanized mouse model was used which had decreased LDL receptor expression and was transgenic for human cholesterylester transfer protein (CETP) in order to obtain a better resemblance to the plasma lipoprotein profile present in humans. It is well known that female mice are more susceptible to atherosclerosis than male mice. Therefore, we compared male and female mice expressing human PLTP. The animals were fed an atherogenic diet and the effects on plasma lipids and lipoproteins, triglyceride secretion and the development of atherosclerosis were measured. The development of atherosclerosis was sex-dependent. This effect was stronger in PLTP transgenic mice, while PLTP activity levels were virtually identical. Also, the rates of hepatic secretion of triglycerides were similar. In contrast, plasma levels of HDL were about 2-fold lower in female mice than in male mice after feeding an atherogenic diet. We conclude that increased atherosclerosis caused by overexpression of PLTP is related to a decrease in HDL, rather than to elevated hepatic secretion of triglycerides.     

Hepatocyte apoB-containing lipoprotein secretion is decreased by the grapefruit flavonoid,naringenin, via inhibition of MTP-mediated microsomal triglyceride accumulation

Naringenin, the principal flavonoid in grapefruit, reduces plasma lipids in vivo and inhibits apoB secretion, cholesterol esterification, and MTP activity in HepG2 human hepatoma cells. Although naringenin inhibits ACAT, we recently demonstrated that CE availability in the microsomal lumen does not regulate apoB secretion in HepG2 cells. We therefore hypothesized that inhibition of TG accumulation in the ER lumen, secondary to MTP inhibition, is the primary mechanism whereby naringenin blocks lipidation and subsequent secretion of apoB. Multicompartmental modeling of pulse-chase studies was used to compare cellular apoB kinetics in the presence of either naringenin or the specific MTP inhibitor, BMS-197636. At concentrations that reduced apoB secretion by 50%, both compounds selectively enhanced degradation via a kinetically defined, rapid, proteasomal pathway to the same extent. Subcellular fractionation experiments revealed that naringenin and BMS-197636 reduced accumulation of newly synthesized TG in the microsomal lumen by 48% and 54%, respectively. Newly synthesized CE accumulation in the lumen was reduced by 80% and 33% with naringenin and BMS-197636, respectively, demonstrating for the first time that MTP is involved in CE accumulation in the microsomal lumen. Reduced TG availability at this initial site of lipoprotein assembly was associated with significant reductions in the secretion of apoB-containing lipoproteins. Both naringenin and BMS-197636 were most effective in reducing secretion of IDL and LDL, but also inhibited secretion of apoB-containing HDL-sized particles. Furthermore, in McA-RH7777-derived cell lines, naringenin reduced secretion of hapoB72 and hapoB100, which require significant assembly with lipid to be secreted, but did not reduce secretion of hapoB17, hapoB23, and hapoB48, which require only minimal lipidation. Taken together, our results indicate that naringenin inhibits the lipidation and subsequent secretion of apoB-containing lipoproteins primarily by limiting the accumulation of TG in the ER lumen, secondary to MTP inhibition.     

Atherosclerosis: another protein misfolding disease?

The secondary structure and conformation of apo-B 100 in low-density lipoproteins (LDL) are imposed by lipid-protein interactions and dynamics, and affected by the introduction or removal of lipids during the course of lipoprotein metabolism. Following an alteration of the water-lipid interface as a result of, for example, oxidation of lipids, the supramolecular structure becomes destabilized and apoB can misfold. These events have been observed in LDL(-), a fraction of oxidatively modified LDL isolated in vivo. This modified lipoprotein possesses several atherogenic properties and represents an in vivo counterpart of in vitro modified LDL that is implicated in atherosclerosis. The misfolding of apoB, its aggregation, resistance to proteolysis, and cytotoxicity are common motifs shared by LDL(-) and amyloidogenic proteins. Based on these analogies, we propose that atherogenesis could be considered as a disease produced by the accumulation of cytotoxic and pro-inflammatory misfolded lipoproteins.     

Apple procyanidins decrease cholesterol esterification and lipoprotein secretion in Caco-2/TC7 enterocytes

Decrease of plasma lipid levels by polyphenols was linked to impairment of hepatic lipoprotein secretion. However, the intestine is the first epithelium that faces dietary compounds, and it contributes to lipid homeostasis by secreting triglyceride-rich lipoproteins during the postprandial state. The purpose of this study was to examine the effect of apple and wine polyphenol extracts on lipoprotein synthesis and secretion in human Caco-2/TC7 enterocytes apically supplied with complex lipid micelles. Our results clearly demonstrate that apple, but not wine, polyphenol extract dose-dependently decreases the esterification of cholesterol and the enterocyte secretion of lipoproteins. Apple polyphenols decrease apolipoprotein B (apoB) secretion by inhibiting apoB synthesis without increasing the degradation of the newly synthesized protein. Under our conditions, cholesterol uptake, apoB mRNA, and microsomal triglyceride protein activity were not modified by apple polyphenols. The main monomers present in our mixture did not interfere with the intestinal lipid metabolism. By contrast, apple procyanidins reproduced the inhibition of both cholesteryl ester synthesis and lipoprotein secretion. Overall, our results are compatible with a mechanism of action of polyphenols resulting in impaired lipid availability that could induce the inhibition of intestinal lipoprotein secretion and contribute to the hypolipidemic effect of these compounds in vivo.     

Mast cells promote atherosclerosis by inducing both an atherogenic lipid profile and vascular inflammation

Accumulating in vitro and in vivo studies have proposed a role for mast cells in the pathogenesis of atherosclerosis. Here, we studied the role of mast cells in lipoprotein metabolism, a key element in the atherosclerotic disease. Male mice deficient in low‐density lipoprotein receptors and mast cells on a Western diet for 26 weeks had significantly less atherosclerotic changes both in aortic sinus (55%, P = 0.0009) and in aorta (31%, P = 0.049), as compared to mast cell‐competent littermates. Mast cell‐deficient female mice had significantly less atherosclerotic changes in aortic sinus (43%, P = 0.011). Furthermore, we found a significant positive correlation between the extent of atherosclerosis and the number of adventitial/perivascular mast cells in aortic sinus of mast cell‐competent mice (r = 0.615, P = 0.015). Serum cholesterol and triglyceride levels were significantly lower in both male (63%, P = 0.0005 and 57%, P = 0.004) and female (73%, P = 0.00009 and 54%, P = 0.007) mast cell‐deficient mice, with a concomitant decrease in atherogenic apoB‐containing particles and serum preβ‐high‐density lipoprotein and phospholipid transfer protein activity in both male (69% and 24%) and female (74% and 54%) mast cell‐deficient mice. Serum soluble intercellular adhesion molecule was decreased in both male (32%, P = 0.004) and female (28%, P = 0.003) mast cell‐deficient mice, whereas serum amyloid A was similar between mast cell‐deficient and competent mice. In conclusion, mast cells participate in the pathogenesis of atherosclerosis in ldlr^?/? mice by inducing both an atherogenic lipid profile and vascular inflammation. J. Cell. Biochem. 109: 615–623, 2010. © 2009 Wiley‐Liss, Inc.     

Regulation of ApoB secretion by the low density lipoprotein receptor requires exit from the endoplasmic reticulum and interaction with ApoE or ApoB

Apolipoprotein B (apoB) is required for the hepatic assembly and secretion of very low density lipoprotein (VLDL). The LDL receptor (LDLR) promotes post-translational degradation of apoB and thereby reduces VLDL particle secretion. We investigated the trafficking pathways and ligand requirements for the LDLR to promote degradation of apoB. We first tested whether the LDLR drives apoB degradation in an endoplasmic reticulum (ER)-associated pathway. Primary mouse hepatocytes harboring an ethyl-nitrosourea-induced, ER-retained mutant LDLR secreted comparable levels of apoB with LDLR-null hepatocytes, despite reduced secretion from cells expressing the wild-type LDLR. Additionally, treatment of cells with brefeldin A inhibited LDLR-dependent degradation. However, this rescue was reversible, and degradation of apoB occurred upon removal of brefeldin A. To characterize the lipoprotein reuptake pathway of degradation, we employed an LDLR mutant defective in constitutive endocytosis and internalization of apoB. This mutant was as effective in reducing apoB secretion as the wild-type LDLR. However, the effect was dependent on apolipoprotein E (apoE) as only the wild-type LDLR, and not the endocytic mutant, reduced apoB secretion in apoE-null cells. Treatment with heparin rescued a pool of apoB in cells expressing the endocytic mutant, indicating that reuptake of VLDL via apoE still occurs with this mutant. Finally, an LDLR mutant defective in binding apoB but not apoE reduced apoB secretion in an apoE-dependent manner. Together, these data suggest that the LDLR directs apoB to degradation in a post-ER compartment. Furthermore, the reuptake mechanism of degradation occurs via internalization of apoB through a constitutive endocytic pathway and apoE through a ligand-dependent pathway.     

Inducible expression of phospholipid transfer protein (PLTP) in transgenic mice: acute effects of PLTP on lipoprotein metabolism

One main determinant in high-density lipoprotein (HDL) metabolism is phospholipid transfer protein (PLTP), a plasma protein that is associated with HDL. In transgenic mice overexpressing human PLTP we found that elevated plasma PLTP levels dose-dependently increased the susceptibility to diet-induced atherosclerosis. This could be mainly due to the fact that most functions of PLTP are potentially atherogenic, such as decreasing plasma HDL levels. To further elucidate the role of PLTP in lipoprotein metabolism and atherosclerosis we generated a novel transgenic mouse model that allows conditional expression of human PLTP. In this mouse model a human PLTP encoding sequence is controlled by a Tet-On system. Upon induction of PLTP expression, our mouse model showed a strongly increased PLTP activity (from 3.0 ± 0.6 to 11.4 ± 2.8 AU, p < 0.001). The increase in PLTP activity resulted in an acute decrease in plasma cholesterol of 33% and a comparable decrease in phospholipids. The decrease in total plasma cholesterol and phospholipids was caused by a 35% decrease in HDL-cholesterol level and a 41% decrease in HDL-phospholipid level. These results demonstrate the feasibility of our mouse model to induce an acute elevation of PLTP activity, which is easily reversible. As a direct consequence of an increase in PLTP activity, HDL-cholesterol and HDL-phospholipid levels strongly decrease. Using this mouse model, it will be possible to study the effects of acute elevation of PLTP activity on lipoprotein metabolism and pre-existing atherosclerosis.     

An expression system for human apolipoprotein B100 in a rat hepatoma cell line

We have designed an in vitro expression system for human apolipoprotein (apo) B. A full-length human apoB minigene was constructed from cDNA and genomic apoB clones and inserted into a vector where its expression was directed by the cytomegalovirus promoter. The apoB minigene was expressed in a rat hepatoma cell line, McA-RH7777. Human apoB100, which is the ligand for the low density lipoprotein receptor, was secreted in low density lipoprotein or very low density lipoprotein particles, depending on the composition of the medium. A protein with the mobility of apoB48, a structurally related protein involved in cholesterol metabolism, was also produced from the human apoB minigene. This in vitro expression system for human apoB will enable investigators to identify which domains of this protein are involved in processes such as lipoprotein assembly and secretion. This system should also allow investigators to identify definitively the domain in apoB that enables the protein to bind to the low density lipoprotein receptor.     

Decreased secretion of ApoB follows inhibition of ApoB-MTP binding by a novel antagonist

Apolipoprotein B (apoB) and microsomal triglyceride transfer protein (MTP) are essential for the efficient assembly of triglyceride-rich lipoproteins. Evidence has been presented for physical interactions between these proteins. To study the importance of apoB-MTP binding in apoB secretion, we have identified a compound, AGI-S17, that inhibited (60-70% at 40 microM) the binding of various apoB peptides to MTP but not to an anti-apoB monoclonal antibody, 1D1, whose epitope overlaps with an MTP binding site in apoB. AGI-S17 had no significant effect on the lipid transfer activity of the purified MTP. In contrast, another antagonist, BMS-200150, did not affect apoB-MTP binding but inhibited MTP's lipid transfer activity. The differential effects of these inhibitors suggest two functionally independent, apoB binding and lipid transfer, domains in MTP. AGI-S17 was then used to study its effect on the lipid transfer and apoB binding activities of MTP in HepG2 cells. AGI-S17 had no effect on cellular lipid transfer activities, but it inhibited coimmunoprecipitation of apoB with MTP. These studies indicate that AGI-S17 inhibits apoB-MTP binding but has no effect on MTP's lipid transfer activity. Experiments were then performed to study the effect of inhibition of apoB-MTP binding on apoB secretion in HepG2 cells. AGI-S17 (40 microM) did not affect cell protein levels but decreased the total mass of apoB secreted by 70-85%. Similarly, AGI-S17 inhibited the secretion of nascent apoB by 60-80%, but did not affect albumin secretion. These studies indicate that AGI-S17 decreases apoB secretion most likely by inhibiting apoB-MTP interactions. Thus, the binding of MTP to apoB may be important for the assembly and secretion of apoB-containing lipoproteins and can be a potential target for the development of lipid-lowering drugs. It is proposed that the apoB binding may represent MTP's chaperone activity that assists in the transfer from the membrane to the lumen of the endoplasmic reticulum and in the net lipidation of nascent apoB, and may be essential for lipoprotein assembly and secretion.     

Anti-inflammatory effects of phospholipid transfer protein (PLTP) deficiency in mice

We previously reported that phospholipid transfer protein-deficient (PLTP KO) mice exhibit a lower rate of atherosclerosis. We proposed two possible mechanisms: a reduction in hepatic apoB secretion (Nat Med 7 (2001) 847) and induction of lipoprotein anti-oxidation activity (J Biol Chem 277 (2002) 31850). We now hypothesized that PLTP KO mice may exhibit an anti-inflammatory state per se. First, we found that PLTP KO mice have significantly lower IL-6 levels than wild type (WT) mice. Secondly, we found that IL-6 treatment increased plasma TNFalpha levels in WT mice, but not in PLTP KO mice. Thirdly, we used flow cytometric analyses to measure the mean fluorescence intensity of I-A(b), a MHC-class II molecule, on peripheral monocytes and found that IL-6 treatment significantly increased the I-A(b)-positive cell levels in WT mice, whereas no changes were observed in the cell levels in PLTP KO mice. The results of our experiments demonstrated an anti-inflammatory effect of PLTP deficiency as a further aspect of its proatherogenic potency.     

Fatty acids bound to human serum albumin and its structural variants modulate apolipoprotein B secretion in HepG2 cells

Epidemiologic studies have shown an inverse relationship between human serum albumin (HSA) levels and coronary heart disease (CHD). However, no mechanisms have been identified to explain this relationship. We hypothesized that this relationship is due to differences in binding affinity of fatty acids to HSA and subsequent atherogenic lipoprotein synthesis and secretion from hepatocytes. To test our hypothesis we undertook the current study. Using HepG2 cells, we demonstrated that oleic acid (OA) bound to HSA in a molar ratio of 4:1 and after incubation for 24 h stimulated apolipoprotein B (apoB) secretion. We also tested whether mutant forms of HSA could alter the binding affinity for fatty acids and change the availability of substrate for lipoprotein secretion. Based on the results obtained in this study using 11 HSA mutant proteins complexed with OA, we were able to classify into three major mutant groups based on their effects on apoB secretion. One group in particular (R410Q/Y411W, R410A/Y411A, and W214L/Y411W) showed a significantly diminished effect on apoB secretion when compared to the wild type HSA/OA complex. Furthermore, the amount of free OA internalized in HepG2 cells in the presence of HSA mutant proteins was in good agreement with the effects seen on apoB secretion by the various HSA mutants. This suggests that some mutant forms of HSA might potentially bind fatty acids with a much higher binding affinity and thus deprive fatty acids available for lipoprotein assembly in hepatocytes. In conclusion, our data illustrate that certain HSA polymorphic forms may be protective against the development of CHD and warrants further investigation.     

Lipoprotein kinetics in the metabolic syndrome: pathophysiological and therapeutic lessons from stable isotope studies

Dyslipoproteinaemia is a cardinal feature of the metabolic syndrome that accelerates atherosclerosis. It is usually characterised by high plasma concentrations of triglyceride-rich and apolipoprotein (apo) B-containing lipoproteins, with depressed concentrations of high-density lipoprotein (HDL). Dysregulation of lipoprotein metabolism in these subjects may be due to a combination of overproduction of very-low-density lipoprotein (VLDL) apoB-100, decreased catabolism of apoB-containing particles, and increased catabolism of HDL apoA-I particles. These abnormalities may be consequent on a global metabolic effect of insulin resistance that increases the flux of fatty acids from adipose tissue to the liver, the accumulation of fat in the liver, the increased hepatic secretion of VLDL-triglycerides and the remodelling of both low-density lipoprotein (LDL) and HDL particles in the circulation; perturbations in lipolytic enzymes and lipid transfer proteins contribute to the dyslipidaemia. Our in vivo understanding of the kinetic defects in lipoprotein metabolism in the metabolic syndrome has been chiefly achieved by ongoing developments in the use of stable isotope tracers and mathematical modelling. Knowledge of the pathophysiology of lipoprotein metabolism in the metabolic syndrome is well complemented by extensive cell biological data. Nutritional modifications and increased physical exercise may favourably alter lipoprotein transport in the metabolic syndrome by collectively decreasing the hepatic secretion of VLDL-apoB and the catabolism of HDL apoA-I, as well as by increasing the clearance of LDL-apoB. Pharmacological treatments, such as statins, fibrates or fish oils, can also correct the dyslipidaemia by several mechanisms of action including decreased secretion and increased catabolism of apoB, as well as increased secretion and decreased catabolism of apoA-I. The complementary mechanisms of action of lifestyle and drug therapies support the use of combination regimens to treat dyslipidaemia in the metabolic syndrome.     

Oleate-mediated stimulation of apolipoprotein B secretion from rat hepatoma cells. A function of the ability of apolipoprotein B to direct lipoprotein assembly and escape presecretory degradation.

Physiological concentrations of oleate stimulate apolipoprotein (apo) B-containing lipoprotein secretion from HepG2 cells without increasing apoB mRNA levels. The purpose of this study was to determine whether oleate acts by increasing translation of apoB mRNA or through posttranslational effects on the apoB protein. To address the mechanism of oleate-stimulated secretion of apoB, a series of carboxyl terminally truncated apoB constructs was made. Each contained the SV40 early promoter, the apoB 5'-untranslated region, and SV40 polyadenylation signals. Any difference in the response to oleate between endogenous apoB and the proteins encoded by the constructs or between the constructs themselves should thus depend on the protein sequence. Stable transformants were established for each of the constructs in the rat hepatoma cell line McArdle-RH7777. The effect of oleate on secretion of the apoB protein products was determined by labeling with [35S]methionine, immunoprecipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Carboxyl-terminal truncation of apoB41 resulted in a loss of the ability of apoB secretion to respond to oleate. Ultracentrifugation of secreted proteins on continuous CsCl gradients from 1.0-1.4 g/ml revealed that this correlated with a decrease in the ability of apoB to be recovered as a buoyant lipoprotein particle. Addition of oleate decreased the densities at which the short forms of apoB secreted as lipoproteins were recovered. Pulse-chase analysis of the secretion of apoB100 and of the truncated proteins revealed that they all underwent rapid posttranslational intracellular degradation. We conclude that oleate has no effect on the translation of apoB mRNA but promotes the secretion of apoB-containing lipoproteins by reducing presecretory degradation of those forms of apoB that can produce buoyant lipoproteins.