Upregulation Of LDL Receptor Research Articles

Hepatitis C Virus Stimulates Low-Density Lipoprotein Receptor Expression To Facilitate Viral Propagation

Lipids play a crucial role in multiple aspects of hepatitis C virus (HCV) life cycle. HCV modulates host lipid metabolism to enrich the intracellular milieu with lipids to facilitate its proliferation. However, very little is known about the influence of HCV on lipid uptake from bloodstream. Low-density lipoprotein receptor (LDLR) is involved in uptake of cholesterol rich low-density lipoprotein (LDL) particles from the bloodstream. The association of HCV particles with lipoproteins implicates their role in HCV entry; however, the precise role of LDLR in HCV entry still remains controversial. Here, we investigate the effect of HCV infection on LDLR expression and the underlying mechanism(s) involved. We demonstrate that HCV stimulates LDLR expression in both HCV-infected Huh7 cells and in liver tissue from chronic hepatitis C patients. Fluorescence activated cell sorting and immunofluorescence analysis revealed enhanced cell surface and total expression of LDLR in HCV-infected cells. Increased LDLR expression resulted in the enhanced uptake of lipoprotein particles by HCV-infected cells. Analysis of LDLR gene promoter identified a pivotal role of sterol-regulatory element binding proteins (SREBPs), in the HCV-mediated stimulation of LDLR transcription. In addition, HCV negatively modulated the expression of proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein that facilitates LDLR degradation. Ectopic expression of wild-type PCSK9 or gain-of-function PCSK9 mutant negatively affected HCV replication. Overall, our results demonstrate that HCV regulates LDLR expression at transcriptional and posttranslational level via SREBPs and PCSK9 to promote lipid uptake and facilitate viral proliferation. HCV modulates host lipid metabolism to promote enrichment of lipids in intracellular environment, which are essential in multiple aspects of HCV life cycle. However, very little is known about the influence of HCV on lipid uptake from the bloodstream. LDLR is involved in uptake of cholesterol rich lipid particles from bloodstream. In this study, we investigated the effect of HCV on LDLR expression and the underlying mechanism triggered by the virus to modulate LDLR expression. Our observations suggest that HCV upregulates LDLR expression at both the protein and the transcript levels and that this upregulation likely contributes toward the uptake of serum lipids by infected hepatocytes. Abrogation of HCV-mediated upregulation of LDLR inhibits serum lipid uptake and thereby perturbs HCV replication. Overall, our findings highlight the importance of serum lipid uptake by infected hepatocytes in HCV life cycle.

Berberine metabolites could induce low density lipoprotein receptor up-regulation to exert lipid-lowering effects in human hepatoma cells

Berberine (BBR) is an isoquinoline alkaloid isolated from several Chinese herbal medicines, such as Coptis chinensis, Berberis aristata, and Coptis japonica. It exhibits a lipid-lowering effect by up-regulating the hepatic low density lipoprotein receptor (LDLR) expression. However, the plasma concentration of BBR is very low after oral administration for the reason that BBR is poorly absorbed and rapidly metabolized. Therefore, it is hard to explain the pharmacological effects of BBR in vivo. Here, RT-PCR, Western blotting and Oil Red O staining were used to investigate the effects of four BBR metabolites on LDLR expression and lipid accumulation in human hepatoma Hep G2 cells. Our results suggested that BBR increased the LDLR mRNA and protein levels in a time- and dose-dependent manner. Four metabolites of BBR, jatrorrhizine, columbamine, berberrubine and demethyleneberberine, were found to be able to up-regulate LDLR mRNA and protein expression. Moreover, almost all the metabolites had potent effects on inhibiting cellular lipid accumulation. These results suggest that both BBR and its metabolites exhibit lipid-lowering effects by up-regulating LDLR expression, and BBR and its metabolites might be the in vivo active forms of BBR produced after oral administration. This study provides information to help us understand the mechanisms underlying the hypolipidemic effects of BBR in vivo.

Abstract 19083: Macrophage Expression of Pcsk9 Influences Atherosclerosis Development

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that regulates low-density lipoprotein (LDL) cholesterol levels by binding and degrading hepatic LDL receptor (LDLR), thus contributing to atherosclerosis. Little is known of PCSK9 effect in macrophages and whether this contributes to the development of the atheroma. First, we demonstrated that murine peritoneal macrophages (MPM) express endogenous (m) PCSK9 both at the mRNA and protein level. Then, to test the effect of human (h) PCSK9 on atherosclerosis we developed transgenic mice expressing hPCSK9 from multiple tissues and cells, including macrophages. As it occurs in hepatocytes, hPCSK9 reduced LDLR levels in MPM. The effect on LDLR was more evident in resident than in elicited MPM, and was completely abolished after culturing MPM in medium containing FBS or LPDS, that cause LDLR upregulation. Co-immunoprecipitation experiments showed a direct interaction of hPCSK9 with LDL-R. In addition, we found that PCSK9 also interacts with LRP-1, a member of the LDL-R gene family involved in modulation of inflammatory responses and cell survival. It has been recently reported that PCSK9 reduces LRP1 levels in transduced renal HEK293T cells. However, we found that the interaction of hPCSK9 with LRP1 did not affect either total or membrane levels of the receptor. Surprisingly, analyses of spleen lysates from hPCSK9 transgenics revealed an increase in the percentage of Ly6C hi and CD11b positive cells compared with WT. To study the effect of macrophage PCSK9 in the atheroma, bone marrow from hPCSK9/apoE -/- or apoE -/- mice was transplanted into apoE -/- recipients. Low levels of hPCSK9 were detected in serum of hPCSK9/apoE -/- marrow recipients, but no effects on cholesterol were noted. Although lesion size was not different, lesion composition analyses showed a higher percentage of Ly6C hi positive cells in recipients of hPCSK9/apoE -/- marrow compared to controls (7.4±1.5% vs. 5.6±1.1%, respectively, p <0.05). Our results show for first time that PCSK9 expression by macrophages directly influences atherosclerotic plaque composition in the absence of changes in serum cholesterol levels, suggesting a direct effect of macrophage PCSK9 in inflammation and plaque development.

Cholesterol-induced mammary tumorigenesis is enhanced by adiponectin deficiency: role of LDL receptor upregulation

Adiponectin is an adipokine that can suppress the proliferation of various human carcinoma cells. Although its anti-tumor activities have been suggested by many clinical investigations and animal studies, the underlying mechanisms are not fully characterized. In MMTV-polyomavirus middle T antigen (MMTV-PyVT) transgenic mice models, reduced- or complete loss-of-adiponectin expression promotes mammary tumor development. The present study demonstrated that while tumor development in control MMTV-PyVT mice is associated with a progressively decreased circulating cholesterol concentration, adiponectin deficient MMTV-PyVT mice showed significantly elevated total- and low density lipoprotein (LDL)-cholesterol levels. Cholesterol contents in tumors derived from adiponectin deficient mice were dramatically augmented. High fat high cholesterol diet further accelerated the tumor development in adiponectin deficient PyVT mice. The protein levels of LDL receptor (LDLR) were found to be upregulated in adiponectin-deficient tumor cells. In human breast carcinoma cells, treatment with LDL-cholesterol or overexpressing LDLR elevates nuclear beta-catenin activity and facilitates tumor cell proliferation. On the other hand, adiponectin decreased LDLR protein expression in breast cancer cells and inhibited LDL-cholesterol-induced tumor cell proliferation. Both in vivo and in vitro evidence demonstrated a stimulatory effect of adiponectin on autophagy process, which mediated the down-regulation of LDLR. Adiponectin-induced reduction of LDLR was blocked by treatment with a specific inhibitor of autophagy, 3-methyladenine. In conclusion, the study demonstrates that adiponectin elicits tumor suppressive effects by modulating cholesterol homeostasis and LDLR expression in breast cancer cells, which is at least in part attributed to its role in promoting autophagic flux.

Essential Oil ofPinus koraiensisExerts Antiobesic and Hypolipidemic Activity via Inhibition of Peroxisome Proliferator-Activated Receptors Gamma Signaling

Our group previously reported that essential oil of Pinus koraiensis (EOPK) exerts antihyperlipidemic effects via upregulation of low-density lipoprotein receptor and inhibition of acyl-coenzyme A. In the present study, we investigated the antiobesity and hypolipidemic mechanism of EOPK using in vitro 3T3-L1 cells and in vivo HFD-fed rats. EOPK markedly suppressed fat accumulation and intracellular triglyceride associated with downregulation of adipogenic transcription factor expression, including PPARγ and CEBPα in the differentiated 3T3-L1 adipocytes. Additionally, EOPK attenuated the expression levels of FABP and GPDH as target genes of PPARγ during adipocyte differentiation. Furthermore, PPARγ inhibitor GW9662 enhanced the decreased expression of FABP and PPARγ and fat accumulation induced by EOPK. To confirm the in vitro activity of EOPK, animal study was performed by administering normal diet, HFD, and/or EOPK at the dose of 100 or 200 mg/kg for 6 weeks. Consistently, EOPK significantly suppressed body weight gain, serum triglyceride, total cholesterol, LDL cholesterol, and AI value and increased HDL cholesterol in a dose-dependent manner. Immunohistochemistry revealed that EOPK treatment abrogated the expression of PPARγ in the liver tissue sections of EOPK-treated rats. Taken together, our findings suggest that EOPK has the antiobesic and hypolipidemic potential via inhibition of PPARγ-related signaling.

Hepatic Gene Expression Patterns Following Trauma-Hemorrhage

The aim of this study was to examine the role of estrogen on hepatic gene expression profiles at an early time point following trauma-hemorrhage in rats. Groups of injured and sham controls receiving estrogen or vehicle were killed 2 h after injury and resuscitation, and liver tissue was harvested. Complementary RNA was synthesized from each RNA sample and hybridized to microarrays. A large number of genes were differentially expressed at the 2-h time point in injured animals with or without estrogen treatment. The upregulation or downregulation of a cohort of 14 of these genes was validated by reverse transcription-polymerase chain reaction. This large-scale microarray analysis shows that at the 2-h time point, there is marked alteration in hepatic gene expression following trauma-hemorrhage. However, estrogen treatment attenuated these changes in injured animals. Pathway analysis demonstrated predominant changes in the expression of genes involved in metabolism, immunity, and apoptosis. Upregulation of low-density lipoprotein receptor, protein phosphatase 1, regulatory subunit 3C, ring-finger protein 11, pyroglutamyl-peptidase I, bactericidal/permeability-increasing protein, integrin, αD, BCL2-like 11, leukemia inhibitory factor receptor, ATPase, Cu transporting, α polypeptide, and Mk1 protein was found in estrogen-treated trauma-hemorrhaged animals. Thus, estrogen produces hepatoprotection following trauma-hemorrhage likely via antiapoptosis and improving/restoring metabolism and immunity pathways.

Deregulation of lipid metabolism pathway genes in nasopharyngeal carcinoma cells.

Nasopharyngeal carcinoma (NPC) is a unique tumour of epithelial origin with a distinct geographical distribution, closely associated with the Epstein-Barr virus (EBV). EBV-encoded RNAs (EBERs) are small non-polyadenylated RNAs that are abundantly expressed in latent EBV-infected NPC cells. To study the role of EBERs in NPC, we established stable expression of EBERs in HK1, an EBV-negative NPC cell line. Cells expressing EBERs consistently exhibited an increased growth rate. However, EBERs did not confer resistance towards cisplatin-induced apoptosis or promote migration or invasion ability in the cells tested. Using microarray gene expression profiling, we identified potential candidate genes that were deregulated in NPC cells expressing EBERs. Gene Ontology analysis of the data set revealed that EBERs upregulate the cellular lipid metabolic process. Upregulation of low-density lipoprotein receptor (LDLR) and fatty acid synthase (FASN) was observed in EBER-expressing cells. NPC cells exhibited LDL-dependent cell proliferation. In addition, a polyphenolic flavonoid compound, quercetin, known to inhibit FASN, was found to inhibit proliferation of NPC cells.

Synthesis and structure–activity relationship of berberine analogues in LDLR up-regulation and AMPK activation

Currently there is no approved medicine for the treatment of metabolic syndrome. A series of new derivatives of berberine (BBR) or pseudoberberine (1) was synthesized and evaluated for their activity on AMP-activated protein kinase (AMPK) activation and up-regulatory low-density-lipoprotein receptor (LDLR) gene expression, respectively. In addition, the four major metabolites of BBR in vivo were also examined for their activity on AMPK in order to further understand the chemical mechanisms responsible for its glucose-lowering efficacy. Among those BBR analogues, compound 1 exhibited the potential effect on AMPK activation and LDLR up-regulation as compared with BBR. The results suggested that compound 1 might be a multiple-target agent for the treatment of metabolic syndrome, and thus was selected as a promising drug candidate for further development.

Beyond reasonable doubt: Who is the culprit in lipotoxicity in NAFLD/NASH?

Abstract BACKGROUND & AIMS: Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH. METHODS: Alms1 mutant (foz/foz) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice. RESULTS: Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis. CONCLUSIONS: In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.

Suppression of endothelial cell adhesion by XJP-1, a new phenolic compound derived from banana peel

The adhesion of monocytes to activated vascular endothelial cells is a critical event in the initiation of atherosclerosis. Adhesion is mediated by oxidized low-density lipoprotein (ox-LDL) which up-regulates inflammatory markers on endothelial cells. Here we report that (±) 7, 8-dihydroxy-3-methyl-isochromanone-4 (XJP-1), an inhibitor of ox-LDL-induced adhesion of monocytes to endothelial cells blocks cellular functions which are associated with adhesion. We show that XJP-1 down-regulates ox-LDL-induced over-expression of adhesion molecules (ICAM-1 and VCAM-1) in a dose-dependent manner in human umbilical vein endothelial cells (HUVECs), attenuates ox-LDL-induced up-regulation of low-density lipoprotein receptor (LOX)-1, decreases generation of reactive oxygen species (ROS), blocks translocation of nuclear factor-kappa B (NF-κB) activity, and prevents activation of c-Jun N-terminal kinase (JNK)/p38 pathways in endothelial cells. These findings suggest that XJP-1 may attenuate ox-LDL-induced endothelial adhesion of monocytes by blocking expression of adhesion molecules through suppressing ROS/NF-κB, JNK and p38 pathways.

Modified methylenedioxyphenol analogs lower LDL cholesterol through induction of LDL receptor expression

Although statin therapy is a cornerstone of current low density lipoprotein (LDL)-lowering strategies, there is a need for additional therapies to incrementally lower plasma LDL cholesterol. In this study, we investigated the effect of several methylenedioxyphenol derivatives in regulating LDL cholesterol through induction of LDL receptor (LDLR). INV-403, a modified methylenedioxyphenol derivative, increased LDLR mRNA and protein expression in HepG2 cells in a dose- and time-dependent fashion. These effects were apparent even under conditions of HMG-CoA reductase inhibition. Electrophoresis migration shift assays demonstrated that INV-403 activates SREBP2 but not SREBP1c, with immunoblot analysis showing an increased expression of the mature form of SREBP2. Knockdown of SREBP2 reduced the effect of INV-403 on LDLR expression. The activation of SREBP2 by INV-403 is partly mediated by Akt/GSK3β pathways through inhibition of phosphorylation-dependent degradation by ubiquitin-proteosome pathway. Treatment of C57Bl/6j mice with INV-403 for two weeks increased hepatic SREBP2 levels (mature form) and upregulated LDLR with concomitant lowering of plasma LDL levels. Transient expression of a LDLR promoter-reporter construct, a SRE-mutant LDLR promoter construct, and a SRE-only construct in HepG2 cells revealed an effect predominantly through a SRE-dependent mechanism. INV-403 lowered plasma LDL cholesterol levels through LDLR upregulation. These results indicate a role for small molecule approaches other than statins for lowering LDL cholesterol.

Abstract 9763: Discovery of a New Role of Human Resistin in Hepatocyte Low-Density Lipoprotein (LDL) Receptor Suppression Mediated by PCSK9

Background: Serum levels of the adipose tissue-derived signalling protein, resistin, are increased in human obesity and are positively correlated with atherosclerotic cardiovascular disease (ASCVD). However, the function of resistin in humans has been enigmatic. An elevation in serum low-density lipoprotein (LDL) levels is both necessary and sufficient for ASCVD development in humans. However, few physiological factors have been identified that directly affect LDL levels through its main receptor, the LDL receptor (LDLR). In this study, we wished to determine if human resistin plays a role in regulating the uptake of atherogenic LDL in human hepatocytes. Methods: Human hepatocytes (HepG2 and primary) were treated for 24 hours with: (1.) purified human resistin at various concentrations, with and without PCSK9 siRNA, and with and without lovastatin; and (2.) obese human serum with elevated resistin levels or serum from which resistin was removed via antibody-immunoprecipitation. The effect of the treatments on cellular LDL receptor (LDLR) and PCSK9 mRNA and protein levels were determined via real-time PCR and Western blotting, respectively. Results: Resistin, at physiological levels observed in human obesity (50 ng/mL), downregulated hepatocyte LDLR expression substantially by 40%. A key mechanism by which human resistin inhibited hepatocyte LDLR was via increased expression of the recently identified protease, PCSK9, which enhances intracellular LDLR lysosomal degradation. To support this notion, we showed that the addition of resistin reversed the marked over 100% elevation in LDLR expression induced with PCSK9 siRNA treatment. The quantitatively important role of human resistin in LDLR expression was demonstrated by antibody-immunoprecipitation removal of resistin in obese human serum, which increased serum stimulation of LDLR markedly by 80%. Furthermore, resistin diminished statin-mediated upregulation of LDLR by 70%, implicating resistin in the relative ineffectiveness of statins in selective target populations. Conclusions: These results reveal for the first time that resistin is potentially a highly attractive therapeutic target in ameliorating elevated serum LDL, and, thereby, ASCVD, in obese humans.

Upregulation of Low-Density Lipoprotein Receptor After Exposure to Transforming Growth Factor-β2in Tenon's Capsule Fibroblasts from Patients with Glaucoma

The aim of this study was to investigate the dynamic alteration of a low-density lipoprotein receptor (LDLr) and its binding and uptake of LDL after exposure to transforming growth factor-β(2) (TGF-β(2)) in Tenon's capsule fibroblasts from glaucoma patients. Tenon's capsule fibroblasts obtained from patients who had undergone selective glaucoma surgery were cultured and stimulated with different concentrations (0.1-10 ng/mL) of TGF-β(2) for 1, 3, 5, and 7 days. Expression of LDLr mRNA was detected with relative quantification real-time reverse transcription-polymerase chain reaction, and expression of the corresponding protein was demonstrated with western blot. The extent of binding and uptake of 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI)-labeled LDL by GTFs was also checked. After exposure to TGF-β(2), LDLr was upregulated at the mRNA and protein levels in a concentration-dependent and/or time-dependent manner with a peak at a concentration of 5 ng/mL TGF-β(2). Binding and uptake of LDL were time dependent, reaching saturation at the first 6 h. This study shows that LDLr has a dose- and/or time-effect relationship with TGF-β(2) in Tenon's capsule fibroblasts from glaucoma patients. The results suggest that LDLr may play an important role in wound healing and scar formation in the activated Tenon's capsule fibroblasts from patients with glaucoma.

An LXR Agonist Promotes Glioblastoma Cell Death through Inhibition of an EGFR/AKT/SREBP-1/LDLR–Dependent Pathway

Glioblastoma (GBM) is the most common malignant primary brain tumor of adults and one of the most lethal of all cancers. Epidermal growth factor receptor (EGFR) mutations (EGFRvIII) and phosphoinositide 3-kinase (PI3K) hyperactivation are common in GBM, promoting tumor growth and survival, including through sterol regulatory element-binding protein 1 (SREBP-1)-dependent lipogenesis. The role of cholesterol metabolism in GBM pathogenesis, its association with EGFR/PI3K signaling, and its potential therapeutic targetability are unknown. In our investigation, studies of GBM cell lines, xenograft models, and GBM clinical samples, including those from patients treated with the EGFR tyrosine kinase inhibitor lapatinib, uncovered an EGFRvIII-activated, PI3K/SREBP-1-dependent tumor survival pathway through the low-density lipoprotein receptor (LDLR). Targeting LDLR with the liver X receptor (LXR) agonist GW3965 caused inducible degrader of LDLR (IDOL)-mediated LDLR degradation and increased expression of the ABCA1 cholesterol efflux transporter, potently promoting tumor cell death in an in vivo GBM model. These results show that EGFRvIII can promote tumor survival through PI3K/SREBP-1-dependent upregulation of LDLR and suggest a role for LXR agonists in the treatment of GBM patients.

164 Discovery of a new role of human resistin in hepatocyte low-density lipoprotein (LDL) receptor suppression mediated by PCSK9

Serum levels of the adipose tissue adipokine, resistin, are increased in human obesity and are positively correlated with atherosclerotic cardiovascular disease (ASCVD). However, the function of resistin in humans has been enigmatic. In this study, we wished to determine if human resistin plays a role in regulating the uptake of atherogenic low-density lipoproteins (LDL) in human hepatocytes. Human hepatocytes (HepG2 and primary) were treated for 24 hours with: (1) purified human resistin at various concentrations, with and without lovastatin; and (2) obese human serum with elevated resistin levels or serum from which resistin was removed via antibody-immunoprecipitation. The effect of the treatments on cellular LDL receptor (LDLR) and PCSK9 mRNA and protein levels were determined via real-time PCR and Western blotting, respectively. Resistin, at physiological levels observed in human obesity, downregulated hepatocyte LDLR expression substantially by 40%. A key mechanism by which human resistin inhibited hepatocyte LDLR was via increased expression of the recently identified protease, PCSK9, which enhances intracellular LDLR lysosomal degradation. The quantitatively important role of human resistin in LDLR expression was demonstrated by antibody-immunoprecipitation removal of resistin in obese human serum, which decreased serum stimulation of LDLR markedly by 80%. Furthermore, resistin diminished statin-mediated upregulation of LDLR by 70%, implicating resistin in the relative ineffectiveness of statins in selective target populations. These results reveal for the first time that resistin is a highly attractive therapeutic target in ameliorating elevated serum LDL, and, thereby, ASCVD, in obese humans.

Epigallocatechin gallate changes mRNA expression level of genes involved in cholesterol metabolism in hepatocytes

Catechins, compounds derived from green tea, have been shown to improve cholesterol metabolism in animal studies, but the molecular mechanisms underlying this function have not been fully understood. We performed DNA microarray analysis in order to clarify the effects of epigallocatechin gallate (EGCG), the dominant catechin in green tea, on cholesterol metabolism in HepG2 hepatocytes. This revealed that the expression levels of several genes related to cholesterol metabolism, including the LDL receptor, were changed by EGCG treatment. Using a real-time PCR technique, we confirmed that EGCG treatment up-regulated mRNA expression level of the LDL receptor. Moreover, EGCG decreased extracellular apoB levels. These findings indicated that EGCG improves cholesterol metabolism through the up-regulation of LDL receptor and also reduces extracellular apoB levels.

Hepatic Free Cholesterol Accumulates in Obese, Diabetic Mice and Causes Nonalcoholic Steatohepatitis

Type 2 diabetes and nonalcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to the pathogenesis of NASH. Alms1 mutant (foz/foz) and wild-type NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice. Hepatic cholesterol accumulation was attributed to up-regulation of low-density lipoprotein receptor via activation of sterol regulatory element binding protein 2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and low-density lipoprotein receptor and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol levels exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis. In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.

Squalene Synthase Inhibitor Lapaquistat Acetate

Elevated serum cholesterol level is an important risk factor for cardiovascular disease,1 and cholesterol lowering by 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors or statins has been shown to reduce cardiovascular events.2 The reduction in cardiovascular risks with statin therapy, whether for primary or secondary prevention, correlates almost linearly with the reduction in serum cholesterol levels.3 Indeed, for every 30 mg/dL decrease in low-density lipoprotein-cholesterol (LDL-C), there is a concomitant 30% decrease in cardiovascular events.2 For patients presenting with acute coronary syndrome,4 or in patients with stable coronary artery disease,5 intensive lipid-lowering therapy provides even greater clinical benefits, implying that the lower the LDL-C, the better the outcome is, especially for high-risk patients. Because most of the US adult population with coronary heart disease has serum LDL-C levels between 135 and 145 mg/dL,6 to achieve an Adult Treatment Panel III/National Cholesterol Education Panel guideline for LDL-C target goal of <100 mg/dL with an optional goal of <70 mg/dL, most high-risk patients with coronary heart disease will likely require at least a 40% to 50% reduction in LDL-C.2 Article see p 1974 Depending on the dose used, most statins can reduce serum cholesterol by 30% to 58%.7 This is compared with bile acid sequestrants or intestinal cholesterol absorption inhibitors, which tend to lower serum cholesterol by only 15% to 18%. Thus, it is not surprising that statins have emerged as the principal cholesterol-lowering therapy for patients at risk for cardiovascular disease. However, most statins, with the exception of the more potent statins, such as atorvastatin and rosuvastatin, will require near-maximum dosages to achieve a 40% to 50% LDL-C reduction. This is because the majority of statins' LDL-C–lowering efficacy occurs at the starting dose with only a modest 4% to 6% further LDL-C reduction with …

Plasma lipoproteins are required for both basal and stress-induced adrenal glucocorticoid synthesis and protection against endotoxemia in mice

Lipoprotein-associated cholesterol has been suggested to make a significant contribution to adrenal steroidogenesis in vivo. To determine whether lipoproteins indeed contribute to optimal adrenal steroidogenesis in mice, in the current study we have determined the effect of relative lipoprotein deficiency on adrenal steroidogenesis in C57BL/6 wild-type mice. Feeding C57BL/6 mice the lipid-lowering drug probucol (0.25% wt/wt) for 2 wk induced a 90% decrease in plasma high-density lipoprotein (HDL) cholesterol levels and a 77% reduction in low-density lipoprotein (LDL) cholesterol levels. Neutral lipid stores were depleted upon probucol treatment specifically in the glucocorticoid-producing zona fasciculata of the adrenal, leading to a 44% decreased plasma corticosterone level under basal conditions. Exposure to lipopolysaccharide (LPS) induced a 37% increase in the adrenal uptake of HDL cholesteryl esters. Probucol-treated mice could induce only a relatively minor corticosterone response upon a LPS challenge compared with controls, which coincided with an approximately twofold increased hepatic expression level of interleukin-6 and tumor necrosis factor (TNF)α and an 89% higher TNFα response in plasma. Furthermore, a compensatory two- to fivefold upregulation of LDL receptor (cholesterol uptake) and HMG-CoA reductase (cholesterol synthesis) expression was noticed in the adrenals of probucol-treated mice. In conclusion, we have shown that lipoprotein deficiency in mice as a result of probucol feeding is associated with decreased adrenal cortex cholesterol levels, a lower basal and stress-induced plasma glucocorticoid level, and an increased susceptibility to LPS-induced inflammation. Therefore, it is suggested that plasma lipoproteins are required for optimal adrenal steroidogenesis and protection against endotoxemia in mice.

Curcumin inhibits cholesterol uptake in Caco-2 cells by down-regulation of NPC1L1 expression

BackgroundCurcumin is a polyphenol and the one of the principle curcuminoids of the spice turmeric. Its antioxidant, anti-cancer and anti-inflammatory effects have been intensively studied. Previous in vivo studies showed that administration of curcumin also decreased cholesterol levels in the blood, and the effects were considered to be related to upregulation of LDL receptor. However, since plasma cholesterol levels are also influenced by the uptake of cholesterol in the gut, which is mediated by a specific transporter Niemann-Pick Cl-like 1 (NPC1L1) protein, the present study is to investigate whether curcumin affects cholesterol uptake in the intestinal Caco-2 cells.MethodsCaco-2 cells were cultured to confluence. The micelles composed of bile salt, monoolein, and 14C-cholesterol were prepared. We first incubated the cells with the micelles in the presence and absence of ezetimibe, the specific inhibitor of NPC1L1, to see whether the uptake of the cholesterol in the cells was mediated by NPC1L1. We then pretreated the cells with curcumin at different concentrations for 24 h followed by examination of the changes of cholesterol uptake in these curcumin-treated cells. Finally we determined whether curcumin affects the expression of NPC1L1 by both Western blot analysis and qPCR quantification.ResultsWe found that the uptake of radioactive cholesterol in Caco-2 cells was inhibited by ezetimibe in a dose-dependent manner. The results indicate that the uptake of cholesterol in this study was mediated by NPC1L1. We then pretreated the cells with 25-100 μM curcumin for 24 h and found that such a treatment dose-dependently inhibited cholesterol uptake with 40% inhibition obtained by 100 μM curcumin. In addition, we found that the curcumin-induced inhibition of cholesterol uptake was associated with significant decrease in the levels of NPC1L1 protein and NPC1L1 mRNA, as analyzed by Western blot and qPCR, respectively.ConclusionCurcumin inhibits cholesterol uptake through suppression of NPC1L1 expression in the intestinal cells.