Cellular Cholesterol Metabolism Research Articles

Recycling of apolipoprotein E is not associated with cholesterol efflux in neuronal cells

After receptor-mediated endocytosis of apolipoprotein E (apoE)-containing lipoproteins in hepatocytes, the isoform apoE3 is efficiently recycled in a process which is associated with cholesterol efflux. Recycling and cholesterol efflux are greatly reduced when apoE4 is the only isoform present. ApoE is the main apolipoprotein in cerebrospinal fluid, and it plays a pivotal role in maintaining cholesterol homeostasis in the brain. The isoform apoE4 is associated with an increased risk of Alzheimer's disease and it has been postulated that high intracellular cholesterol levels promote the amyloidogenic processing of amyloid precursor protein. Therefore we investigated the cellular processing of different apoE isoforms as well as the associated cholesterol efflux in the murine neuronal cell line HT-22. Uptake of apoE3-containing lipoproteins resulted in the expected recycling while, as seen in non-neuronal cells, recycling of apoE4 was significantly reduced. However, despite these differences in apoE recycling, there was no difference in rates of cholesterol efflux. Therefore we conclude that in this neuronal cell model the reduced recycling of apoE4 does not affect cellular cholesterol metabolism.

Overexpression of A-kinase anchoring protein 12A activates sterol regulatory element binding protein-2 and enhances cholesterol efflux in hepatic cells

A-kinase anchoring protein 12 (AKAP12) is known to function as a scaffold protein and as a putative tumor suppressor. However, little is known about the biological role of AKAP12 in hepatic cells. In this study, we performed micro-array analysis to identify the downstream pathway of AKAP12A, and found that AKAP12A overexpression up-regulates the expressions of several cholesterol-associated genes including HMG-CoA reductase and LDL receptor, which have been reported to be controlled by sterol regulatory element binding protein-2 (SREBP-2). It was found that AKAP12A activates SREBP-2 in hepatic cells, as demonstrated by the presence of its cleavage product, whereas the activation of sterol regulatory element binding protein-1 was not remarkably changed. Moreover, AKAP12A-induced SREBP-2 activation was found to depend on SREBP cleavage-activating protein (SCAP), as inhibition of SCAP by RNAi or sterols blocked SREBP-2 activation in response to AKAP12A overexpression. Interestingly, the hydrophobic amine U18666A caused dramatic movement of AKAP12A from the plasma membrane to cytosol and lysosomal membranes. Moreover, cholesterol depletion from the plasma membrane (using methyl-β-cyclodextrin) caused a shift of AKAP12A from the plasma membrane to the cytoplasm. Cholesterol binding assay revealed that the N-terminal region of AKAP12A binds directly to cholesterol in vitro. Furthermore, AKAP12A overexpression enhanced [ 3H]-cholesterol efflux to extracellular acceptors, suggesting that AKAP12A may activate SREBP-2 by increasing cholesterol efflux. In conclusion, the present study suggests that AKAP12A is a novel regulator of cellular cholesterol metabolism.

Anti-oxidant and anti-atherogenic properties of liposomal glutathione: Studies in vitro, and in the atherosclerotic apolipoprotein E-deficient mice

Liposomal glutathione, but not the control liposomes (with no glutathione), dose-dependently inhibited copper ion-induced low density lipoprotein (LDL) and HDL oxidation. As peroxidase activity was found to be present in both LDL and HDL, it has contributed to the anti-oxidative effects of liposomal glutathione. In-vitro, no significant effect of liposomal glutathione on J774 A.1 macrophage cell-line oxidative stress and on cellular cholesterol metabolism was observed. In contrast, in the atherosclerotic apolipoprotein E-deficient (E 0) mice, consumption of liposomal glutathione (12.5 or 50 mg/kg/day, for 2 months), but not control liposomes, resulted in a significant reduction in the serum susceptibility to AAPH-induced oxidation by 33%. Liposomal glutathione (50 mg/kg/day) consumption also resulted in an increment (by 12%) in the mice peritoneal macrophages (MPM) glutathione content, paralleled by a significant reduction in total cellular lipid peroxides content (by 40%), compared to placebo-treated mice MPM. MPM paraoxonase 2 activity was significantly increased by 27% and by 121%, after liposomal glutathione consumption (12.5 or 50 mg/kg/day, respectively). Analyses of cellular cholesterol fluxes revealed that, liposomal glutathione (12.5 mg/kg/day) consumption, decreased the extent of oxidized-LDL (Ox-LDL) uptake by 17% and the cellular cholesterol biosynthesis rate, by 34%, and stimulated HDL-induced macrophage cholesterol efflux, by 19%. Most important, a significant reduction in macrophage cholesterol mass (by 24%), and in the atherosclerotic lesion area (by 30%) was noted. We thus conclude that liposomal glutathione possesses anti-oxidative and anti-atherogenic properties towards lipoproteins and macrophages, leading to attenuation of atherosclerosis development.

Altered cholesterol metabolism in APP695-transfected neuroblastoma cells

Cholesterol has been implicated to play an important role in the generation of Aβ peptides, which are the main component of β-amyloid plaques in the brains of patients suffering from Alzheimer's disease (AD). Epidemiological data implicate that lowering cholesterol levels has beneficial effects on the extent of β-amyloid pathology. Thus therapeutic intervention using cholesterol lowering drugs like statins seems to be a promising approach. A couple of studies, in vitro or in vivo by the use of AD transgenic mouse models, focused on the manipulation of cholesterol levels and the resulting effects on Aβ generation. In contrast, there is not much known about the effect of the amyloid precursor protein (APP) on cholesterol levels. In the present report, we transfected human neuroblastoma cells with human APP695 and compared cellular cholesterol levels with the respective levels in Mock-transfected control cells. Furthermore, we determined the levels of diverse cholesterol precursors and metabolites using gas chromatography-mass spectrometry (GC-MS). Significant differences in the levels of the respective cholesterol precursors were observed, whereas inhibition of γ-secretase activity by the γ-secretase inhibitor DAPT did not have a significant effect on cellular cholesterol metabolism.

Macrophage NADPH oxidase activation, impaired cholesterol fluxes, and increased cholesterol biosynthesis in diabetic mice: A stimulatory role for d-glucose

Diabetes is clearly associated with accelerated atherosclerosis development, but molecular mechanisms involved in diabetes-induced atherosclerosis remain to be clarified. The aim of this study was to identify cellular mechanisms involved in diabetes-induced macrophage foam cell formation, the hallmark of early atherogenesis. Mouse peritoneal macrophages (MPMs) isolated from Balb-C streptozotocin-induced diabetic mice, exhibited significantly higher total peroxides, lipid peroxides and paraoxonase 2 (PON2) activity by 290%, 61% and 55%, respectively, compared to non-diabetic mice. In vitro studies revealed that glucose-induced oxidative stress was obtained by d-glucose, but not by l-glucose and it involved activation of the NADPH oxidase complex, and up-regulation of the macrophage PON2. Next, MPMs isolated from Balb-C diabetic mice, compared to control Balb-C mice, demonstrated increased cholesterol content by 4.2-fold associated with increased cholesterol biosynthesis and increased uptake of oxidized LDL (Ox-LDL) by 5.9-fold and 31%, respectively. These effects on cellular cholesterol metabolism were associated with up-regulation of the scavenger receptors for Ox-LDL (CD-36 and SR-A), and of HMG-CoA reductase (cholesterol biosynthesis rate limiting enzyme). Finally, using pravastatin (inhibitor of HMG-CoA reductase) and the antioxidant Vitamin E, we have shown that d-glucose-induced macrophage oxidative stress is secondary to its stimulatory effect on macrophage cholesterol biosynthesis. In conclusion, macrophages from diabetic mice demonstrate increased oxidative stress associated with activation of NADPH oxidase and up-regulation of cellular PON2, as well as increased macrophages cholesterol uptake and biosynthesis (increased expression of CD-36 and HMG-CoA reductase). The above mechanisms in diabetic mice could be the result of the effect of high d-glucose on macrophages.

Changes in cellular cholesterol metabolism are associated with fetal programming of atherosclerosis

WITH FETAL PROGRAMMING OF ATHEROSCLEROSIS NIMA GOHARKHAY, KARINA VILLARREAL, SERGIO RODRIGUEZ, AMANDA MARETH, KENDRA STISSER, ESTHER TAMAYO, PHYLLIS GAMBLE, MICHEL MAKHLOUF, GARLAND D. ANDERSON, MONICA LONGO, GEORGE SAADE, University of Texas Medical Branch, Obstetrics & Gynecology/Maternal Fetal Medicine, Galveston, Texas, University of Texas Medical Branch, Obstetrics & Gynecology/ Gynecologic Oncology, Galveston, Texas OBJECTIVE: We previously described a model of fetal programming of atherosclerosis in the offspring of hypercholesterolemic ApoE knockout mice. The objective of this study was to determine the mechanisms affected by inutero programming, focusing on transcriptional control, synthesis and cellular uptake of cholesterol in the liver. STUDY DESIGN: ApoE knockout (KO) and wild-type (WT) mice were crossbred to obtain 4 groups of offspring: KO, WT, heterozygous born to ApoE deficient mothers (KOMat) and heterozygous born to WT mothers (KOPat). All animals were sacrificed at eight months of age. Real-time RT-PCR was performed to detect mRNA expression levels for the LDL receptor (LDLR), HMG-CoA synthase, liver X receptor (LXR), SREBP cleavage activator protein (SCAP), peroxisome proliferator-activator receptor (PPAR)-gamma and PPAR-delta in the liver. LDLR protein levels were determined by Western blot. RESULTS: SCAP levels were significantly elevated in KOMat offspring as compared to both KOPat and WT animals (p=0.03). HMG-CoA synthase expression was also higher in the KOMat versus the other groups. In KOMat animals HMG-CoA synthase and SCAP mRNA levels were highly correlated (p!0.0001, r=0.81). PPAR-g and PPAR-d levels were somewhat higher in KOMat mice, while LXR and LDLR expression did not vary significantly. No substantial gender differences were noted. CONCLUSION: Elevated SCAP levels stimulate HMG-CoA synthase expression in the liver of offspring born to hypercholesterolemic mothers compared with genomically similar offspring born to normal mothers. This may underlie the mechanism by which in-utero exposure to high cholesterol levels alters fetal programming of cholesterol synthesis.

Human Immunodeficiency Virus Impairs Reverse Cholesterol Transport from Macrophages

Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1–infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings have implications for pathogenesis of both HIV disease and atherosclerosis, because they reveal the role of cholesterol efflux impairment in HIV infectivity and suggest a possible mechanism by which HIV infection of macrophages may contribute to increased risk of atherosclerosis in HIV-infected patients.

ABCA1 mediates high-affinity uptake of 25-hydroxycholesterol by membrane vesicles and rapid efflux of oxysterol by intact cells

ATP Binding Cassette (ABC) transporter, ABCA1, plays a pivotal role in reverse cholesterol transport by mediating the cellular efflux of phospholipid and cholesterol. Studies using intact cells strongly suggest that ABCA1 acts as a phospholipid floppase, but there has been no direct demonstration that the protein is a primary active sterol transporter. Using membrane vesicles from insect Sf21 cells, we found that ABCA1 mediated ATP-dependent uptake of [(3)H]25-hydroxycholesterol with an apparent K(m) of 0.7 muM. Consistent with this high apparent affinity, expression of ABCA1 in human embryonic kidney cells both increased rapid efflux of 25-hydroxcholesterol and prevented oxysterol-mediated repression of low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase mRNAs. Comparison of wild-type and ABCA1(-/-) murine fibroblasts indicates that 25-hydroxycholesterol is effluxed approximately 5-fold more rapidly by wild-type cells. In addition, the rate of efflux from the wild-type but not the ABCA1(-/-) fibroblasts is increased a further twofold by inducers of ABCA1 expression. Thus under the experimental conditions employed, endogenous ABCA1 is a major contributor to 25-hydroxycholesterol efflux from wild-type fibroblasts. Evidence from in vitro studies indicates that oxysterols are potent inducers of genes involved in cellular cholesterol efflux and metabolism, including the ABCA1 gene, and repressors of genes involved in cholesterol synthesis or uptake. Our observations raise the possibility that efflux of oxysterols by ABCA1 could contribute to a homeostatic mechanism, which both attenuates oxysterol-induced expression of its cognate gene and alleviates repression of genes encoding proteins, such as HMG-CoA reductase and LDL receptor.

Changes in Cholesterol Metabolism are Associated With PS1 and PS2 Gene Regulation in SK-N-BE

Several lines of evidence suggest that the cholesterol content of neuronal membranes influences amyloid precursor protein (APP) processing; however, its role in transcriptional regulation of the cofactors for gamma-secretase, the key enzyme for the production of the Abeta peptide, is poorly understood. This study investigates whether the changes in cellular cholesterol metabolism modulate the expression of genes involved in the gamma-secretase complex function. The abundance of mRNA transcripts for presenilin 1 and 2 (PS1 and PS2), APP, and nicastrin were evaluated in neuroblastoma cells exposed either to serum-depleted medium or to low-density lipoproteins (LDL). Cholesterol esterification was markedly inhibited by mevinolin and U18666A, but was not significantly affected by any other of the tested treatments. gamma-Secretase genes and cofactors were not co-regulated and were not influenced by statin inhibition of cholesterol synthesis. Nicastrin and the APP isoforms showed constitutive expression. In the absence of exogenous lipids, cell PS1 and PS2 expression was induced by LDL and by lysosomal sequestration of cholesterol. However, a different pattern of induction of presenilin gene expression was observed in the latter condition, suggesting that lysosomal cholesterol levels are strong inducers of PS2 transcription. Taken together, these results indicate that lipid metabolism has a complex influence on gamma-secretase transcriptional pathways and, in particular, exogenous cholesterol and compartmentalization in neuroblastoma cells play a relevant role in regulating the transcription of presenilins, while modulation of the cholesterol biosynthesis pathway seems to exert a minor influence on the expression of gamma-secretase genes and cofactors.

Replication of Coxiella burnetii Is Inhibited in CHO K‐1 Cells Treated with Inhibitors of Cholesterol Metabolism

Coxiella burnetii directs the synthesis of a large parasitophorous vacuole (PV) that is required for its replication. While some lysosomal characteristics of the PV have been described, the origin and composition of the PV membrane remain largely undefined. Cholesterol is an essential component of mammalian membranes where it lends mechanical stability and serves as a platform for signaling proteins. Using infected Chinese hamster ovary cells as a model, we examined whether cholesterol is trafficked to the C. burnetii PV membrane and the effects of inhibitors of cholesterol metabolism on C. burnetii replication. When infected cells were stained with filipin, a fluorescent polyene antifungal agent that binds cholesterol, obvious staining of PV was observed indicating the PV membrane is cholesterol-rich. Furthermore, replication of C. burnetii was significantly inhibited in cells treated with the cholesterol metabolism inhibitors lovastatin, ketoconazole, imipramine, progesterone, and U18666A. These data suggest that cholesterol is an important component of the C. burnetii PV membrane and that normal cellular cholesterol metabolism is required for optimal C. burnetii replication.

Coxiella burnetii inhabits a cholesterol-rich vacuole and influences cellular cholesterol metabolism

Coxiella burnetii directs the synthesis of a large parasitophorous vacuole (PV) required for replication. While some lysosomal characteristics of the PV have been described, the origin and composition of the PV membrane is largely undefined. Cholesterol is an essential component of mammalian cell membranes where it plays important regulatory and structural roles. Here we investigated the role of host cholesterol in biogenesis and maintenance of the C. burnetii PV in Vero cells. The C. burnetii PV membrane stained with filipin and was positive for the lipid raft protein flotillin-1, suggesting PV membranes are enriched in cholesterol and contain lipid raft microdomains. C. burnetii infection increased host cell cholesterol content by 1.75-fold with a coincident upregulation of host genes involved in cholesterol metabolism. Treatment with U18666A, lovastatin, or 25-hydroxycholesterol, pharmacological agents that inhibit cholesterol uptake and/or biosynthesis, altered PV morphology and partially inhibited C. burnetii replication. Complete inhibition of C. burnetii PV development and replication was observed when infected cells were treated with imipramine or ketoconazole, inhibitors of cholesterol uptake and biosynthesis respectively. We conclude that C. burnetii infection perturbs host cell cholesterol metabolism and that free access to host cholesterol stores is required for optimal C. burnetii replication.

Silencing of the mutant SCAP allele accounts for restoration of a normal phenotype in CT60 cells selected for NPC1 expression

The sterol regulatory element binding protein (SREBP)/SREBP cleavage-activating protein (SCAP) complex regulates the transcription of numerous genes involved in cellular cholesterol metabolism. The CHO mutant, CT60, and its parental cell line, 25RA, possess a gain-of-function mutation in one allele of the SCAP gene that renders the cells resistant to sterol-mediated suppression of cholesterol synthesis and uptake. In addition, CT60 cells do not express a functional Niemann-Pick type C1 (NPC1) protein, which leads to lysosomal accumulation of free cholesterol. Correction of the NPC1 defect by expression of a yeast artificial chromosome (YAC) containing the NPC1 genetic interval restored normal mobilization of cholesterol from the lysosomal compartment. Unexpectedly, the YAC-containing cell lines have overall cellular cholesterol concentrations that are comparable to wild-type levels, despite the assumed presence of the SCAP mutation. This phenotypic change results from a reduction in endogenous sterol synthesis, LDL receptor message, and HMG-CoA reductase message. Genetic analysis of the SCAP gene revealed that the YAC-expressing CT60 cells have normal regulation of these sentinel cholesterogenic genes as a result of selective silencing of the mutant SCAP allele, which appears to be independent of functional NPC1 expression.

Alzheimer's disease amyloid-beta peptide modulates apolipoprotein E isoform specific receptor binding

The major protein component of the extracellular deposits in Alzheimer's disease (AD) is a 4 kDa peptide termed amyloid-beta (Abeta). This peptide is known to bind apolipoprotein E (apoE), a key mediator of lipoprotein transport, in an isoform specific manner. Whilst these isoform specific effects on apoE are well recognized, the functional significance of this interaction is poorly understood. Here, we investigated the influence of Abeta on apoE-mediated lipoprotein binding to cells using fluorescently tagged lipoprotein-like emulsions. Using this approach, we demonstrate that Abeta enhanced the normally poor binding of apoE2 lipoprotein-like particles to fibroblasts in culture, whilst markedly reducing the binding of apoE3 and apoE4. This suggests that the action of apoE isoforms on cellular lipoprotein or cholesterol metabolism is differentially modulated by Abeta. This also suggests that Abeta may also compromise apoE function in the Alzheimer disease affected brain.

Overexpression of OSBP-related protein 2 (ORP2) induces changes in cellular cholesterol metabolism and enhances endocytosis

ORP2 [OSBP (oxysterol-binding protein)-related protein 2] belongs to the 12-member mammalian ORP gene/protein family. We characterize in the present study the effects of inducible ORP2 overexpression on cellular cholesterol metabolism in HeLa cells and compare the results with those obtained for CHO cells (Chinese-hamster ovary cells) that express ORP2 constitutively. In both cell systems, the prominent phenotype is enhancement of [14C]cholesterol efflux to all extracellular acceptors, which results in a reduction of cellular free cholesterol. No change was observed in the plasma membrane cholesterol content or distribution between raft and non-raft domains upon ORP2 expression. However, elevated HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase activity and LDL (low-density lipoprotein) receptor expression, as well as enhanced transport of newly synthesized cholesterol to a cyclodextrin-accessible pool, suggest that the ORP2 expression stimulates transport of cholesterol out of the endoplasmic reticulum. In contrast with ORP2/CHO cells, the inducible ORP2/HeLa cells do not show down-regulation of cholesterol esterification, suggesting that this effect represents an adaptive response to long-term cholesterol depletion in the CHO cell model. Finally, we provide evidence that ORP2 binds PtdIns(3,4,5)P(3) and enhances endocytosis, phenomena that are probably interconnected. Our results suggest a function of ORP2 in both cholesterol trafficking and control of endocytic membrane transport.

Plant Stanol and Sterol Esters in the Control of Blood Cholesterol Levels: Mechanism and Safety Aspects

Incorporation of plant stanol esters into margarine is among the first examples of a functional food with proven low-density lipoprotein (LDL) cholesterol-lowering effectiveness. Recently, there have been many studies on the effects of plant stanols/sterols on cholesterol metabolism. It has been found that the serum LDL cholesterol-lowering effect of plant stanols/sterols originates from reduced intestinal cholesterol absorption, a process in which changes in micellar composition are thought to play a major role. However, recent findings suggest that there is an additional process in which plant stanols/sterols actively influence cellular cholesterol metabolism within intestinal enterocytes. Furthermore, in response to the reduced supply of exogenous cholesterol, receptor-mediated lipoprotein cholesterol uptake is probably enhanced, as shown by increased LDL receptor expression. At recommended intakes of about 2 to 2.5 g/day, products enriched with plant stanol/sterol esters lower plasma LDL cholesterol levels by 10% to 14% without any reported side effects. Thus, plant stanols/sterols can be considered to be effective and safe cholesterol-lowering functional food ingredients.

Proprotein convertase subtilisin kexin 9: the third locus implicated in autosomal dominant hypercholesterolemia

Autosomal dominant hypercholesterolemia is a genetic disease in which patients have elevated LDL cholesterol levels and premature atherosclerosis. Mutations in the LDL receptor and its ligand apolipoprotein B are causative for autosomal dominant hypercholesterolemia, and the study of this pathway has been crucial to understanding LDL metabolism and receptor-mediated endocytosis in general. Recently, families were identified with a clinical diagnosis of autosomal dominant hypercholesterolemia, but without linkage to the LDL receptor or apolipoprotein B genes. Identification and study of the causative genes in these families should provide additional insights into LDL metabolism. Recent microarray studies and database searches identified a novel member of the proprotein convertase family called proprotein convertase subtilisin kexin 9 (PCSK9). A role for PCSK9 in cholesterol metabolism was proposed from the expression studies and confirmed by the discovery that PCSK9 missense mutations were associated with a form of autosomal dominant hypercholesterolemia, Hchola3. The cellular role for PCSK9 and the mechanism behind its mutations are under study, and a role for PCSK9 in regulating LDL receptor protein levels has been demonstrated. PCSK9 is the third locus implicated in autosomal dominant hypercholesterolemia (Hchola3), and it appears to play an important role in cellular cholesterol metabolism. Understanding the function of PCSK9 will be important for broadening our knowledge of LDL metabolism and may aid in the development of novel hypocholesterolemic agents.

The effect of sterol regulatory element-binding protein 2 polymorphism on the serum lipid in northern Chinese subjects

Sterol regulatory element-binding protein 2 (SREBP-2) is an important nuclear transcription factor in the regulation of cellular cholesterol metabolism. To determine allele frequency of the 1784G>C polymorphism at the SREBP-2 locus and investigate the relationship between this polymorphism and serum lipid levels in Chinese people, we selected 486 individuals (118 men and 368 women) from the Xicheng District of Beijing. The subjects were divided into four groups: hypercholesterolemic subjects, hypertriglyceridemic subjects, combined hyperlipidemic subjects, and normal subjects. Serum lipid profiles were measured in all subjects, and 1784G>C was analyzed using polymerase chain reaction-restriction fragment length polymorphism analysis. There was no significant difference in genotype frequencies or allele frequencies of this polymorphism between the hyperlipidemic and control groups. The serum total cholesterol (TC) and LDL cholesterol (LDL-C) levels of the individuals carrying the C allele were higher than the noncarriers in both males and females in the hypercholesterolemic group, but statistical significance was only observed in females. The results of this study indicate that the SREBP-2 polymorphism is related to elevated concentrations of serum TC and LDL-C in hypercholesterolemic subjects. Further work is necessary to confirm the role of 1784G>C in the development of hyperlipidemia.

Diet induced regulation of genes involved in cholesterol metabolism in rat liver parenchymal and Kupffer cells

Feeding rodents atherogenic diets enriched in cholesterol or cholic acid changes hepatic cholesterol metabolism. In the present study, the effect of an atherogenic diet enriched in cholesterol and cholic acid on cellular hepatic cholesterol metabolism was studied. Gene and protein expression analysis was performed on parenchymal, endothelial, and Kupffer cells isolated from rats fed a chow or atherogenic diet using quantitative real-time PCR and immunoblotting, respectively. The atherogenic diet raised the serum cholesterol concentration 11-fold, mostly in the VLDL fraction, and led to heavy lipid loading of rat liver parenchymal and Kupffer cells. Only moderate changes in the expression of genes involved in cholesterol metabolism were observed in parenchymal cells on the diet, while PPAR delta expression was 6.8-fold decreased. Kupffer cells, however, showed a highly adaptive response with a 2- to 9-fold induction of SR-BI, ABCA1, and ABCG5/G8, and an 82-fold induction in CYP7A1 mRNA expression, respectively. Heavy lipid loading of parenchymal cells leads to moderate gene expression changes, while Kupffer cells respond in a highly adaptive fashion by stimulating the expression of genes involved in cholesterol metabolism and transport.

Regulation of luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acid expression in the rat ovary: relationship to cholesterol metabolism.

Down-regulation of LH/human chorionic gonadotropin (hCG) receptor (LHR) mRNA in the ovary after the preovulatory LH surge or the administration of a pharmacological dose of LH/hCG occurs through a posttranscriptional mechanism. A LHR mRNA-binding protein was identified as the LHR mRNA destabilizing factor, and its identity was established as mevalonate kinase (Mvk). In the present study, we determined that, in the pseudopregnant rat ovary, LHR mRNA levels began to fall 4 h after hCG injection, at which time Mvk protein levels were elevated, and this elevation was preceded by an increase in Mvk mRNA levels. When the cytosolic fractions of hCG-treated ovaries were subjected to RNA EMSA, an increase in LHR mRNA-LHR mRNA-binding protein complex formation was observed, in parallel with the increase of Mvk expression. We also found that hCG coordinately up-regulated the expression of Mvk and other sterol-responsive elements containing cholesterol biosynthesis enzymes, such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, and farnesyl pyrophosphate synthase. This up-regulation was transient, but the hCG-induced ovarian cholesterol depletion lasted for more than 24 h. Taken together, our results suggest that, in the ovary, LH/hCG up-regulates the expression of cholesterol biosynthesis enzymes and lipoprotein receptors to replenish cellular cholesterol, and the up-regulation of Mvk leads to a down-regulation of LHR and suppresses the LH/hCG signal cascade transiently. Thus Mvk, an enzyme involved in cholesterol biosynthesis, serves as a link between LHR mRNA expression and cellular cholesterol metabolism.

Separation of Lipid Transport Functions by Mutations in the Extracellular Domain of Scavenger Receptor Class B, Type I

Scavenger receptor class B, type I (SR-BI) shows a variety of effects on cellular cholesterol metabolism, including increased selective uptake of high density lipoprotein (HDL) cholesteryl ester, stimulation of free cholesterol (FC) efflux from cells to HDL and phospholipid vesicles, and changes in the distribution of plasma membrane FC as evidenced by increased susceptibility to exogenous cholesterol oxidase. Previous studies showed that these multiple effects require the extracellular domain of SR-BI, but not the transmembrane and cytoplasmic domains. To test whether 1) the extracellular domain of SR-BI mediates multiple activities by virtue of discrete functional subdomains, or 2) the multiple activities are, in fact, secondary to and driven by changes in cholesterol flux, the extracellular domain of SR-BI was subjected to insertional mutagenesis by strategically placing an epitope tag into nine sites. These experiments identified four classes of mutants with disruptions at different levels of function. Class 4 mutants showed a clear separation of function between HDL binding, HDL cholesteryl ester uptake, and HDL-dependent FC efflux on one hand and FC efflux to small unilamellar vesicles and an increased cholesterol oxidase-sensitive pool of membrane FC on the other. Selective disruption of the latter two functions provides evidence for multiple functional subdomains in the extracellular receptor domain. Furthermore, these findings uncover a difference in the SR-BI-mediated efflux pathways for FC transfer to HDL acceptors versus phospholipid vesicles. The loss of the cholesterol oxidase-sensitive FC pool and FC efflux to small unilamellar vesicle acceptors in Class 4 mutants suggests that these activities may be mechanistically related.