TNFα-Induced LDL Cholesterol Accumulation Involve Elevated LDLR Cell Surface Levels and SR-B1 Downregulation in Human Arterial Endothelial Cells.

Excess lipid droplets are frequently observed in arterial endothelial cells at sites of advanced atherosclerotic plaques. Here, the role of tumor necrosis factor alpha (TNFα) in modulating the low-density lipoprotein (LDL) content in confluent primary human aortic endothelial cells (pHAECs) was investigated. TNFα promoted an up to 2 folds increase in cellular cholesterol, which was resistant to ACAT inhibition. The cholesterol increase was associated with increased 125I-LDL surface binding. Using the non-hydrolysable label, Dil, TNFα could induce a massive increase in Dil-LDL by over 200 folds. The elevated intracellular Dil-LDL was blocked with excess unlabeled LDL and PCSK9, but not oxidized LDL (oxLDL), or apolipoprotein (apoE) depletion. Moreover, the TNFα-induced increase of LDL-derived lipids was elevated through lysosome inhibition. Using specific LDLR antibody, the Dil-LDL accumulation was reduced by over 99%. The effects of TNFα included an LDLR cell surface increase of 138%, and very large increases in ICAM-1 total and surface proteins, respectively. In contrast, that of scavenger receptor B1 (SR-B1) was reduced. Additionally, LDLR antibody bound rapidly in TNFα-treated cells by about 30 folds, inducing a migrating shift in the LDLR protein. The effect of TNFα on Dil-LDL accumulation was inhibited by the antioxidant tetramethythiourea (TMTU) dose-dependently, but not by inhibitors against NF-κB, stress kinases, ASK1, JNK, p38, or apoptosis caspases. Grown on Transwell inserts, TNFα did not enhance apical to basolateral LDL cholesterol or Dil release. It is concluded that TNFα promotes LDLR functions through combined increase at the cell surface and SR-B1 downregulation.

The Modulation of PCSK9 and LDLR by Supercritical CO2 Extracts of Mentha longifolia and Isolated Piperitone Oxide, an In Vitro Study.

In the present study the ability of supercritical carbon dioxide (SCO2) extracts of M. longifolia L. leaves to modulate low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9) expression was evaluated in cultured human hepatoma cell lines Huh7 and HepG2. Two SCO2 extracts, one oil (ML-SCO2) and a semisolid (MW-SCO2), were subjected to detailed chemical characterization by mono- and bidimensional nuclear magnetic resonance (1D, 2D-NMR), gas chromatography coupled with mass spectrometry (GC-MS) and liquid chromatography coupled with mass spectrometry (LC-MS). Chemical analysis revealed significant amounts of fatty acids, phytosterols and terpenoids. ML-SCO2 was able to induce LDLR expression at a dose of 60 µg/mL in HuH7 and HepG2 cell lines. Furthermore, ML-SCO2 reduced PCSK9 secretion in a concentration-dependent manner in both cell lines. Piperitone oxide, the most abundant compound of the volatile constituent of ML-SCO2 (27% w/w), was isolated and tested for the same targets, showing a very effective reduction of PCSK9 expression. The overall results revealed the opportunity to obtain a new nutraceutical ingredient with a high amount of phytosterols and terpenoids using the SCO2 extraction of M. longifolia L., a very well-known botanical species used as food. Furthermore, for the first time we report the high activity of piperitone oxide in the reduction of PCSK9 expression.

Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression.

Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo-/-) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo-/- mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species.

A Review of the Efficacy and Tolerability of Bempedoic Acid in the Treatment of Hypercholesterolemia.

Despite the widespread use of statins and ezetimibe to decrease low-density lipoprotein cholesterol (LDL-C) levels and associated atherosclerotic cardiovascular disease (ASCVD), many patients do not achieve adequate LDL-C lowering as per the recommended American College of Cardiology (ACC)/American Heart Association (AHA) and European Society of Cardiology (ESC)/European Atherosclerosis Society (EAS) guidelines and demonstrate residual cardiovascular risk. The introduction of proprotein convertase subtilisin/kexin type 9 (PCSK-9) inhibitors in 2015 was a promising addition to hypercholesterolemia therapies, but their cost and subcutaneous administration has limited their use, and therefore, new affordable and patient friendly treatment strategies are crucial to help reduce ASCVD risk. Bempedoic acid, a drug currently under investigation, is a small molecule that has been shown to upregulate LDL receptors, decrease LDL-C, and reduce atherosclerotic plaque formation in hypercholesterolemic patients. Furthermore, bempedoic acid is a prodrug that becomes activated by an enzyme expressed primarily in the liver, allowing it to avoid the potential myotoxicity associated with statin therapy. The purpose of this review is to summarize the major clinical studies evaluating bempedoic acid and describe its potential addition to currently approved lipid-lowering therapies.

miR-500 promotes cell proliferation by directly targetting LRP1B in prostate cancer.

Accumulating evidence suggests that miRNAs play a crucial role in the development of prostate cancer (PC); however, the role of miR-500 in PC remains poorly understood. The data presented here reveal abnormal increases in miR-500 expression in PC tissues and cell lines. Suppression of miR-500 expression significantly inhibited the proliferation of PC-3 and LnCap cells and was negatively regulative with low-density lipoprotein receptor-related protein 1B (LRP1B). Increased cell cycle arrest at the G1 stage and decreased protein expression of cyclinD1 and CDK2 was observed in response to miR-500 knockdown in PC-3 and LnCap cells, in combination with LRP1B overexpression. LRP1B was identified as a target of miR-500 and was significantly decreased in PC tissues. Taken together, these findings demonstrate that miR-500 plays an important role in the proliferation of PC cells via the inhibition of LRP1B expression.

Bempedoic Acid (ETC-1002): A Current Review.

Although statins are first-line therapy for low-density lipoprotein cholesterol (LDL-C) reduction, many individuals on maximally tolerated statin therapy have elevated LDL-C. Bempedoic acid (ETC-1002) is a novel once-daily LDL-C-lowering agent in phase 3 clinical trials. In phase 1 and 2 studies, ETC-1002 was efficacious in lowering LDL-C when used as monotherapy and when added to statin and/or ezetimibe and was well tolerated in patients with statin intolerance. ETC-1002 also improved cardiometabolic risk factors. Ongoing phase 3 studies of ETC-1002 are evaluating its long-term efficacy and safety, and effects on cardiovascular events. This article discusses current evidence and future directions for ETC-1002.

A Novel Role of Id1 in Regulating Oscillatory Shear Stress-Mediated Lipid Uptake in Endothelial Cells.

Inhibitor of DNA binding 1 (Id1) has been shown to be involved in adipogenesis and metabolism, which may contribute to atherosclerotic progression. However, it remains unclear how Id1 regulates endothelial cell functions and atherosclerosis in response to oscillatory shear stress. The current study aims to evaluate the effects of oscillatory shear stress on LDL uptake by endothelial cells and to delineate the roles of Id1 in this process. Using an in vivo ligation model of ApoE-/- mice and applying low and oscillatory shear stress (OSS) in vitro, we found that OSS can effectively promote lipid uptake. In vivo en face staining results showed that OSS down-regulated Id1 expression. In vitro, OSS activated Id1 transiently but eventually inhibited its expression with time. Overexpression of Id1 can abolish OSS-mediated lipid uptake in ECs. In addition, Id1 overexpression and knockdown experiments demonstrated that Id1 can regulate LDLR expression to influence lipid uptake. Immunoprecipitation and subcellular localization results further suggested that Id1 can combine with sterol regulatory element-binding protein1 (SREBP1), which may be related to the Id1-mediated LDLR down-expression. Our study shows a biomechanical role of Id1 in endothelial cells' uptake of lipid by down-regulating LDLR, which could help understand how oscillatory flow affects atherosclerotic development.

A Novel Type 2 Diabetes Mouse Model of Combined Diabetic Kidney Disease and Atherosclerosis.

Diabetic kidney disease and atherosclerotic disease are major causes of morbidity and mortality associated with type 2 diabetes (T2D), and diabetic kidney disease is a major cardiovascular risk factor. The black and tan, brachyury (BTBR) mouse strain with leptin deficiency (Lepob) has emerged as one of the best models of human diabetic kidney disease. However, no T2D mouse model of combined diabetic kidney disease and atherosclerosis exists. Our goal was to generate such a model. To this end, the low-density lipoprotein (LDL) receptor was targeted for degradation via inducible degrader of the LDL receptor (IDOL) overexpression, using liver-targeted adenoassociated virus serotype DJ/8 (AAV-DJ/8) in BTBR wild-type and BTBR Lepob mice. Liver-targeted IDOL-AAV-DJ/8 increased plasma LDL cholesterol compared with the control enhanced green fluorescent protein AAV-DJ/8. IDOL-induced dyslipidemia caused formation of atherosclerotic lesions of an intermediate stage, which contained both macrophages and smooth muscle cells. BTBR Lepob mice exhibited diabetic kidney disease. IDOL-induced dyslipidemia worsened albuminuria and glomerular macrophage accumulation but had no effect on mesangial expansion or podocyte numbers. Thus, by inducing hepatic degradation of the LDL receptor, we generated a T2D model of combined kidney disease and atherosclerosis. This model provides a new tool to study mechanisms, interactions, and treatment strategies of kidney disease and atherosclerosis in T2D.

Malignant Phenotypes in Metastatic Melanoma are Governed by SR-BI and its Association with Glycosylation and STAT5 Activation.

Metastatic melanoma is hallmarked by elevated glycolytic flux and alterations in cholesterol homeostasis. The contribution of cholesterol transporting receptors for the maintenance of a migratory and invasive phenotype is not well defined. Here, the scavenger receptor class B type I (SCARB1/SR-BI), a high-density lipoprotein (HDL) receptor, was identified as an estimator of melanoma progression in patients. We further aimed to identify the SR-BI-controlled gene expression signature and its related cellular phenotypes. On the basis of whole transcriptome analysis, it was found that SR-BI knockdown, but not functional inhibition of its cholesterol-transporting capacity, perturbed the metastasis-associated epithelial-to-mesenchymal transition (EMT) phenotype. Furthermore, SR-BI knockdown was accompanied by decreased migration and invasion of melanoma cells and reduced xenograft tumor growth. STAT5 is an important mediator of the EMT process and loss of SR-BI resulted in decreased glycosylation, reduced DNA binding, and target gene expression of STAT5. When human metastatic melanoma clinical specimens were analyzed for the abundance of SR-BI and STAT5 protein, a positive correlation was found. Finally, a novel SR-BI-regulated gene profile was determined, which discriminates metastatic from nonmetastatic melanoma specimens indicating that SR-BI drives gene expression contributing to growth at metastatic sites. Overall, these results demonstrate that SR-BI is a highly expressed receptor in human metastatic melanoma and is crucial for the maintenance of the metastatic phenotype.Implications: High SR-BI expression in melanoma is linked with increased cellular glycosylation and hence is essential for a metastasis-specific expression signature. Mol Cancer Res; 16(1); 135-46. ©2017 AACR.

Adiponectin controls the apoptosis and the expression of tight junction proteins in brain endothelial cells through AdipoR1 under beta amyloid toxicity.

Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by excessive beta amyloid (Aß) deposition in brain, leading to blood-brain barrier (BBB) disruption. The mechanisms of BBB disruption in AD are still unclear, despite considerable research. The adipokine adiponectin is known to regulate various metabolic functions and reduce inflammation. Though adiponectin receptors have been reported in the brain, its role in the central nervous system has not been fully characterized. In the present study, we investigate whether adiponectin contributes to the tight junction integrity and cell death of brain endothelial cells under Aß-induced toxicity conditions. We measured the expression of adiponectin receptors (AdipoR1 and AdipoR2) and the alteration of tight junction proteins in in vivo 5xFAD mouse brain. Moreover, we examined the production of reactive oxygen species (ROS) and the loss of tight junction proteins such as Claudin 5, ZO-1, and inflammatory signaling in in vitro brain endothelial cells (bEnd.3 cells) under Aß toxicity. Our results showed that Acrp30 (a globular form of adiponectin) reduces the expression of proinflammatory cytokines and the expression of RAGE as Aß transporters into brain. Moreover, we found that Acrp 30 attenuated the apoptosis and the tight junction disruption through AdipoR1-mediated NF-κB pathway in Aß-exposed bEnd.3 cells. Thus, we suggest that adiponectin is an attractive therapeutic target for treating BBB breakdown in AD brain.

Atorvastatin and lovastatin, but not pravastatin, increased cellular complex formation between PCSK9 and the LDL receptor in human hepatocyte-like C3A cells.

Statins are the first-line treatment for hypercholesterolemic patients. Herein, the effects of three statins on complex formation between proprotein convertase subtilisin-kexin 9 (PCSK9) and the low density lipoprotein receptor (LDLR), a critical step for the PCSK9-dependent degradation of LDLR in the lysosome, were examined. Human hepatocyte-like C3A cells grown in control (containing 10% fetal bovine serum) or MITO+ (supplemented with BD™ MITO + serum extender) medium were also treated with atorvastatin (Atorv), lovastatin (Lov), or pravastatin (Prav) for 24 h. RNA and protein expression studies and determinations of PCSK9/LDLR complex formation were performed. As expected, the statins increased the expression of PCSK9 and LDLR independently of the medium employed. Interestingly, Atov and Lov caused increases in PCSK9/LDLR complex formation, whereas Prav decreased complex formation when compared to cells treated without drugs. These results may explain why Prav works better for statin intolerant patients than other statins such as Atorv and Lov.

A Genomic DNA Reporter Screen Identifies Squalene Synthase Inhibitors That Act Cooperatively with Statins to Upregulate the Low-Density Lipoprotein Receptor.

Hypercholesterolemia remains one of the leading risk factors for the development of cardiovascular disease. Many large double-blind studies have demonstrated that lowering low-density lipoprotein (LDL) cholesterol using a statin can reduce the risk of having a cardiovascular event by approximately 30%. However, despite the success of statins, some patient populations are unable to lower their LDL cholesterol to meet the targeted lipid levels, due to compliance or potency issues. This is especially true for patients with heterozygous familial hypercholesterolemia who may require additional upregulation of the low-density lipoprotein receptor (LDLR) to reduce LDL cholesterol levels below those achievable with maximal dosing of statins. Here we identify a series of small molecules from a genomic DNA reporter screen that upregulate the LDLR in mouse and human liver cell lines at nanomolar potencies (EC50 = 39 nM). Structure-activity relationship studies carried out on the lead compound, OX03771 [(E)-N,N-dimethyl-3-(4-styrylphenoxy)propan-1-amine], led to the identification of compound OX03050 [(E)-3-(4-styrylphenoxy)propan-1-ol], which had similar potency (EC50 = 26 nM) but a much-improved pharmacokinetic profile and showed in vivo efficacy. Compounds OX03050 and OX03771 were found to inhibit squalene synthase, the first committed step in cholesterol biosynthesis. These squalene synthase inhibitors were shown to act cooperatively with statins to increase LDLR expression in vitro. Overall, we demonstrated here a novel series of small molecules with the potential to be further developed to treat patients either alone or in combination with statins.

Knockdown of microRNA-17-5p ameliorates atherosclerotic lesions in ApoE-/- mice and restores the expression of very low density lipoprotein receptor.

OBJECTIVE: To propose and verify a hypothesis that miR-17-5p knockdown may mitigate atherosclerotic lesions using atherosclerotic ApoE-/- mice as serum microRNA-17-5p (miR-17-5p) is elevated in patients with atherosclerosis. RESULTS: The level of miR-17-5p was higher while the level of very low density lipoprotein receptor (VLDLR), a predicted target of miR-17-5p, was lower in the peripheral blood lymphocytes (PBLs) of atherosclerosis patients as compared with control PBLs. ApoE-/- mice fed with a high-cholesterol diet displayed marked atherosclerotic vascular lesions, which were ameliorated after treatment with antagomiR-17-5p. Moreover, the decreased VLDLR in atherosclerotic mice was partly restored when miR-17-5p was antagonized. Further, luciferase assay confirmed VLDLR as a direct target of miR-17-5p in vascular smooth muscle cells (VSMCs). In addition, the elevated expression of proprotein convertase subtilisin kexin 9 (PCSK9), a secreted protease that binds to and promotes VLDLR degradation, in the atherosclerotic mice was suppressed by antagomiR-17-5p. CONCLUSIONS: A novel interaction between miR-17-5p and VLDLR is revealed and suggests that miR-17-5p may be a potential therapeutic target for AS.

Nuclear Localized LSR: A Novel Regulator of Breast Cancer Behavior and Tumorigenesis.

Lipolysis-stimulated lipoprotein receptor (LSR) has been found in the plasma membrane and is believed to function in lipoprotein endocytosis and tight junctions. Given the impact of cellular metabolism and junction signaling pathways on tumor phenotypes and patient outcome, it is important to understand how LSR cellular localization mediates its functions. We conducted localization studies, evaluated DNA binding, and examined the effects of nuclear LSR in cells, xenografts, and clinical specimens. We found LSR within the membrane, cytoplasm, and the nucleus of breast cancer cells representing multiple intrinsic subtypes. Chromatin immunoprecipitation (ChIP) showed direct binding of LSR to DNA, and sequence analysis identified putative functional motifs and post-translational modifications of the LSR protein. While neither overexpression of transcript variants, nor pharmacologic manipulation of post-translational modification significantly altered localization, inhibition of nuclear export enhanced nuclear localization, suggesting a mechanism for nuclear retention. Coimmunoprecipitation and proximal ligation assays indicated LSR-pericentrin interactions, presenting potential mechanisms for nuclear-localized LSR. The clinical significance of LSR was evaluated using data from over 1,100 primary breast tumors, which showed high LSR levels in basal-like tumors and tumors from African-Americans. In tumors histosections, nuclear localization was significantly associated with poor outcomes. Finally, in vivo xenograft studies revealed that basal-like breast cancer cells that overexpress LSR exhibited both membrane and nuclear localization, and developed tumors with 100% penetrance, while control cells lacking LSR developed no tumors. These results show that nuclear LSR alters gene expression and may promote aggressive cancer phenotypes. IMPLICATIONS: LSR functions in the promotion of aggressive breast cancer phenotypes and poor patient outcome via differential subcellular localization to alter cell signaling, bioenergetics, and gene expression. Mol Cancer Res; 15(2); 165-78. ©2016 AACR.

PCSK9 inhibition-mediated reduction in Lp(a) with evolocumab: an analysis of 10 clinical trials and the LDL receptor's role.

Lipoprotein (a) [Lp(a)] is independently associated with CVD risk. Evolocumab, a monoclonal antibody (mAb) to proprotein convertase subtilisin/kexin type 9 (PCSK9), decreases Lp(a). The potential mechanisms were assessed. A pooled analysis of Lp(a) and LDL cholesterol (LDL-C) in 3,278 patients from 10 clinical trials (eight phase 2/3; two extensions) was conducted. Within each parent study, biweekly and monthly doses of evolocumab statistically significantly reduced Lp(a) at week 12 versus control (P < 0.001 within each study); pooled median (quartile 1, quartile 3) percent reductions were 24.7% (40.0, 3.6) and 21.7% (39.9, 4.2), respectively. Reductions were maintained through week 52 of the open-label extension, and correlated with LDL-C reductions [with and without correction for Lp(a)-cholesterol] at both time points (P < 0.0001). The effect of LDL and LDL receptor (LDLR) availability on Lp(a) cell-association was measured in HepG2 cells: cell-associated LDL fluorescence was reversed by unlabeled LDL and Lp(a). Lp(a) cell-association was reduced by coincubation with LDL and PCSK9 and reversed by adding PCSK9 mAb. These studies support that reductions in Lp(a) with PCSK9 inhibition are partly due to increased LDLR-mediated uptake. In most situations, Lp(a) appears to compete poorly with LDL for LDLR binding and internalization, but when LDLR expression is increased with evolocumab, particularly in the setting of low circulating LDL, Lp(a) is reduced.

Kaempferol stimulates gene expression of low-density lipoprotein receptor through activation of Sp1 in cultured hepatocytes.

A high level of plasma low-density lipoprotein (LDL) cholesterol is considered a risk factor for atherosclerosis. Because the hepatic LDL receptor (LDLR) is essential for clearing plasma LDL cholesterol, activation of LDLR is a promising therapeutic target for patients with atherosclerotic disease. Here we demonstrated how the flavonoid kaempferol stimulated the gene expression and activity of LDLR in HepG2 cells. The kaempferol-mediated stimulation of LDLR gene expression was completely inhibited by knockdown of Sp1 gene expression. Treatment of HepG2 cells with kaempferol stimulated the recruitment of Sp1 to the promoter region of the LDLR gene, as well as the phosphorylation of Sp1 on Thr-453 and Thr-739. Moreover, these kaempferol-mediated processes were inhibited in the presence of U0126, an ERK pathway inhibitor. These results suggest that kaempferol may increase the activity of Sp1 through stimulation of Sp1 phosphorylation by ERK1/2 and subsequent induction of LDLR expression and activity.

Resveratrol exerts a biphasic effect on apolipoprotein M.

BACKGROUND AND PURPOSE: Resveratrol exerts a range of beneficial actions in several areas of pathophysiology, including vascular biology. Here, we have investigated the effects of resveratrol on apolipoprotein M (apoM), a carrier and modulator of sphingosine 1-phosphate (S1P), a vasoactive lipid mediator. EXPERIMENTAL APPROACH: We used a hepatoma cell line (HepG2), human primary hepatocytes and C57BL/6 mice. We measured apoM, S1P and related enzymes, LDL receptors and sirtuin1 activity, using Western blotting, RT-PCR and enzyme assays. We also used si-RNA to knock-down sirtuin1 in HepG2 cells. KEY RESULTS: In cultures of HepG2 cells, resveratrol (1-10 µM) increased intracellular apoM and S1P. High concentrations of resveratrol (100 µM) decreased extracellular (in the culture medium) apoM, whereas moderate concentrations of resveratrol (1-10 µM) increased extracellular apoM. High concentrations of resveratrol also increased LDL receptor expression, while all concentrations of resveratrol activated the histone deacetylase sirtuin1. In cultures of human primary hepatocytes, resveratrol, at all concentrations, increased both intra- and extracellular apoM. When wild-type mice were fed a resveratrol-containing chow (0.3% w/w) for 2 weeks, both the plasma and hepatic apoM and S1P levels were increased. However, the resveratrol diet did not affect hepatic LDL receptor levels in this in vivo study. CONCLUSIONS AND IMPLICATIONS: Resveratrol increased intra- and extracellular levels of apoM, along with intracellular S1P levels, while a high concentration of resveratrol reduced extracellular apoM. The present findings suggest that resveratrol has novel effects on the metabolic kinetics of S1P, a multi-functional bioactive phospholipid.

Follicle-Stimulating Hormone Induces Postmenopausal Dyslipidemia Through Inhibiting Hepatic Cholesterol Metabolism.

CONTEXT: The elevated low-density-lipoprotein cholesterol (LDL-C) in menopausal women is associated with higher risks of cardiovascular diseases. OBJECTIVE: The aim of this study is to investigate the influence and mechanism by which high postmenopausal FSH levels affect lipid profiles. METHODS: The serum FSH and lipid levels were examined in 400 Chinese postmenopausal women. The FSH receptor (FSHR) expression was identified in liver and HepG2 cells by PCR and Western blotting. The effects of FSH on lipid metabolism were confirmed in an ovariectomized mouse model by using GnRH agonist with or without additional FSH to mimic different FSH status. LDL receptor (LDLR), a necessary factor for clearance of LDL-C through endocytosis, was examined by PCR and Western blotting. RESULTS: The postmenopausal women with higher serum FSH (≥78.3 IU/L at baseline) had higher serum total cholesterol and LDL-C levels than those women with FSH levels of 40-78.3 IU/L (P < .01). The improvements of total cholesterol and LDL-C levels were more significant in higher FSH women group after treatment with hormone replacement therapy. It was only in the women whose FSH levels were reduced more than 30% after hormone replacement therapy who showed significant improvement of lipid levels. Ovariectomized mice had high serum FSH and lipids levels and reduced hepatic LDLR expression. In HepG2 cells, FSH inhibited the LDLR in a dose- and time-dependent manner, and the FSHR knockdown with specific siRNA reversed the lower LDLR induced by FSH. CONCLUSIONS: FSH may interact with its receptors in hepatocytes and reduce LDLR levels, which subsequently attenuates the endocytosis of LDL-C, resulting in an elevated circulating LDL-C level.

The Liver Clock Controls Cholesterol Homeostasis through Trib1 Protein-mediated Regulation of PCSK9/Low Density Lipoprotein Receptor (LDLR) Axis.

Disruption of the body clock has been recognized as a risk factor for cardiovascular disease. How the circadian pacemaker interacts with the genetic factors associated with plasma lipid traits remains poorly understood. Recent genome-wide association studies have identified an expanding list of genetic variants that influence plasma cholesterol and triglyceride levels. Here we analyzed circadian regulation of lipid-associated candidate genes in the liver and identified two distinct groups exhibiting rhythmic and non-rhythmic patterns of expression during light-dark cycles. Liver-specific inactivation of Bmal1 led to elevated plasma LDL/VLDL cholesterol levels as a consequence of the disruption of the PCSK9/LDL receptor regulatory axis. Ablation of the liver clock perturbed diurnal regulation of lipid-associated genes in the liver and markedly reduced the expression of the non-rhythmically expressed gene Trib1. Adenovirus-mediated rescue of Trib1 expression lowered plasma PCSK9 levels, increased LDL receptor protein expression, and restored plasma cholesterol homeostasis in mice lacking a functional liver clock. These results illustrate an unexpected mechanism through which the biological clock regulates cholesterol homeostasis through its regulation of non-rhythmic genes in the liver.

A novel peroxisome proliferator response element modulates hepatic low-density lipoprotein receptor gene transcription in response to PPARδ activation.

The hepatic expression of low-density lipoprotein (LDL) receptor (LDLR) gene is regulated primarily at the transcriptional level by a sterol-regulatory element (SRE) in its proximal promoter region which is the site of action of SRE-binding protein 2 (SREBP2). However whether additional cis-regulatory elements contribute to LDLR transcription has not been fully explored. We investigated the function of a putative peroxisome proliferator-activated receptor (PPAR)-response element (PPRE) sequence motif located at -768 to -752 bases upstream of the transcription start site of human LDLR gene in response to PPARδ activation. Promoter luciferase reporter analyses showed that treating HepG2 cells with PPARδ agonist L165041 markedly increased the activity of a full-length LDLR promoter construct (pLDLR-1192) without any effects on the shorter promoter reporter pLDLR-234 that contains only the core regulatory elements SRE-1 and SP1 sites. Importantly, mutation of the PPRE sequence greatly attenuated the induction of the full-length LDLR promoter activity by L165041 without affecting rosuvastatin (RSV)-mediated transactivation. EMSA and ChIP assay further confirmed the binding of PPARδ to the LDLR-PPRE site. Treating HepG2 cells with L165041 elevated the mRNA and protein expressions of LDLR without affecting the LDLR mRNA decay rate. The induction of LDLR expression by PPARδ agonist was further observed in liver tissue of mice and hamsters treated with L165041. Altogether, our studies identify a novel PPRE-mediated regulatory mechanism for LDLR transcription and suggest that combined treatment of statin with PPARδ agonists may have advantageous effects on LDLR expression.