Hepatic Cholesterol Uptake Research Articles

Anlotinib affects systemic lipid metabolism and induces lipid accumulation in human lung cancer cells

BackgroundAnlotinib has demonstrated encouraging clinical outcomes in the treatment of lung cancer, soft tissue sarcoma and thyroid carcinoma. Several clinical studies have shown a relationship between anlotinib treatment and the occurrence of hyperlipidemia. The fundamental mechanisms, however, are still largely unclear. Here, the effect of anlotinib on lipid metabolism in an animal model and human cancer cells was evaluated and the role of lipid metabolism in the antitumor efficacy of anlotinib was investigated.MethodsThe C57BL/6 J mouse model as well as A549 and H460 human lung cancer cell lines were used to examine the impact of anlotinib on lipid metabolism both in vivo and in vitro. Levels of triglycerides, high-density lipoprotein, low-density lipoprotein (LDL), and total cholesterol in serum or cell samples were determined using assay kits. The expression levels of crucial genes and proteins involved in lipid metabolism were measured by quantitative RT-PCR and Western blotting. Furthermore, exogenous LDL and knockdown of low-density lipoprotein receptor (LDLR) were used in H460 cells to investigate the relevance of lipid metabolism in the anticancer efficacy of anlotinib.ResultsAnlotinib caused hyperlipidemia in C57BL/6 J mice, possibly by downregulating hepatic LDLR-mediated uptake of LDL cholesterol. AMP-activated protein kinase and mammalian target of rapamycin inhibition may also be involved. Additionally, anlotinib enhanced sterol response element binding protein 1/2 nuclear accumulation as well as upregulated LDLR expression in A549 and H460 cells, which may be attributable to intracellular lipid accumulation. Knockdown of LDLR reduced intracellular cholesterol content, but interestingly, anlotinib significantly improved intracellular cholesterol accumulation in LDLR-knockdown cells. Both exogenous LDL and LDLR knockdown decreased the sensitivity of cells to anlotinib.ConclusionsAnlotinib modulates host lipid metabolism through multiple pathways. Anlotinib also exerts a significant impact on lipid metabolism in cancer cells by regulating key transcription factors and metabolic enzymes. In addition, these findings suggest lipid metabolism is implicated in anlotinib sensitivity.

Modifying pH-sensitive PCSK9/LDLR interactions as a strategy to enhance hepatic cell uptake of low-density lipoprotein cholesterol (LDL-C).

LDL-receptor (LDLR)-mediated uptake of LDL-C into hepatocytes is impaired by lysosomal degradation of LDLR, which is promoted by proprotein convertase subtilisin/kexin type 9 (PCSK9). Cell surface binding of PCSK9 to LDLR produces a complex that translocates to an endosome, where the acidic pH strengthens the binding affinity of PCSK9 to LDLR, preventing LDLR recycling to the cell membrane. We present a new approach to inhibit PCSK9-mediated LDLR degradation, namely, targeting the PCSK9/LDLR interface with a PCSK9-antagonist, designated Flag-PCSK9PH, which prevents access of WT PCSK9 to LDLR. In HepG2 cells, Flag-PCSK9PH, a truncated version (residues 53-451) of human WT PCSK9, strongly bound LDLR at the neutral pH of the cell surface but dissociated from it in the endosome (acidic pH), allowing LDLR to exit the lysosomes intact and recycle to the cell membrane. Flag-PCSK9PH thus significantly enhanced cell-surface LDLR levels and the ability of LDLR to take up extracellular LDL-C.

USP2-Related Cellular Signaling and Consequent Pathophysiological Outcomes.

Ubiquitin specific protease (USP) 2 is a multifunctional deubiquitinating enzyme. USP2 modulates cell cycle progression, and therefore carcinogenesis, via the deubiquitination of cyclins and Aurora-A. Other tumorigenic molecules, including epidermal growth factor and fatty acid synthase, are also targets for USP2. USP2 additionally prevents p53 signaling. On the other hand, USP2 functions as a key component of the CLOCK/BMAL1 complex and participates in rhythmic gene expression in the suprachiasmatic nucleus and liver. USP2 variants influence energy metabolism by controlling hepatic gluconeogenesis, hepatic cholesterol uptake, adipose tissue inflammation, and subsequent systemic insulin sensitivity. USP2 also has the potential to promote surface expression of ion channels in renal and intestinal epithelial cells. In addition to modifying the production of cytokines in immune cells, USP2 also modulates the signaling molecules that are involved in cytokine signaling in the target cells. Usp2 knockout mice exhibit changes in locomotion and male fertility, which suggest roles for USP2 in the central nervous system and male genital tract, respectively. In this review, we summarize the cellular events with USP2 contributions and list the signaling molecules that are upstream or downstream of USP2. Additionally, we describe phenotypic differences found in the in vitro and in vivo experimental models.

High folic acid intake increases methylation-dependent expression of Lsr and dysregulates hepatic cholesterol homeostasis

Food fortification with folic acid and increased use of vitamin supplements have raised concerns about high folic acid intake. We previously showed that high folic acid intake was associated with hepatic degeneration, decreased levels of methylenetetrahydrofolate reductase (MTHFR), lower methylation potential, and perturbations of lipid metabolism. MTHFR synthesizes the folate derivative for methylation reactions. In this study, we assessed the possibility that high folic acid diets, fed to wild-type and Mthfr+/− mice, could alter DNA methylation and/or deregulate hepatic cholesterol homeostasis. Digital restriction enzyme analysis of methylation in liver revealed DNA hypomethylation of a CpG in the lipolysis-stimulated lipoprotein receptor (Lsr) gene, which is involved in hepatic uptake of cholesterol. Pyrosequencing confirmed this methylation change and identified hypomethylation of several neighboring CpG dinucleotides. Lsr expression was increased and correlated negatively with DNA methylation and plasma cholesterol. A putative binding site for E2F1 was identified. ChIP-qPCR confirmed reduced E2F1 binding when methylation at this site was altered, suggesting that it could be involved in increasing Lsr expression. Expression of genes in cholesterol synthesis, transport or turnover (Abcg5, Abcg8, Abcc2, Cyp46a1, and Hmgcs1) was perturbed by high folic acid intake. We also observed increased hepatic cholesterol and increased expression of genes such as Sirt1, which might be involved in a rescue response to restore cholesterol homeostasis. Our work suggests that high folic acid consumption disturbs cholesterol homeostasis in liver. This finding may have particular relevance for MTHFR-deficient individuals, who represent ~10% of many populations.

Two weeks of western diet disrupts liver molecular markers of cholesterol metabolism in rats

BackgroundThe present study was designed to test the hypothesis that in the liver, excessive fat accumulation impairs cholesterol metabolism mainly by altering the low-density lipoprotein-receptor (LDL-R) pathway.MethodYoung male Wistar rats were fed standard (SD), high fat (HFD; 60% kcal) or Western (WD; 40% fat + 35% sucrose (17.5% fructose)) diets for 2 or 6 weeks.ResultsWeight gain (~ 40 g) was observed only following 6 weeks of the obesogenic diets (P < 0.01). Compared to the 2-week treatment, obesogenic diets tripled fat pad weight (~ 20 vs 7 g) after 6 weeks. Hepatic triglyceride (TG) levels were greater in response to both the WD and HFD compared to the SD (P < 0.01) at 2 and 6 weeks and their concentrations were greater (P < 0.05) in WD than HFD at 2 weeks. Plasma total cholesterol levels were higher (P < 0.05) in animals submitted to WD. After 2 and 6 weeks, liver expression of LDL-R, proprotein convertase subtilisin/kexin 9 (PCSKk9) and sterol regulatory element binding protein 2 (SREBP2), involved in LDL-cholesterol uptake, was lower in animals submitted to WD than in others treated with HFD or SD (P < 0.01). Similarly, low-density lipoprotein-receptor-related protein 1 (LRP1) and acyl-CoA cholesterol acyltransferase-2 (ACAT-2) mRNA levels were lower (P < 0.01) among WD compared to SD-fed rats. Expression of the gene coding the main regulator of endogenous cholesterol synthesis, 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR) was reduced in response to WD compared to SD and HFD at 2 (P < 0.001) and 6 (P < 0.05) weeks. Being enriched in fructose, the WD strongly promoted the expression of carbohydrate-response element binding protein (ChREBP) and acetyl-CoA carboxylase (ACC), two key regulators of de novo lipogenesis.ConclusionThese results show that the WD promptly increased TG levels in the liver by potentiating fat storage. This impaired the pathway of hepatic cholesterol uptake via the LDL-R axis, promoting a rapid increase in plasma total cholesterol levels. These results indicate that liver fat content is a factor involved in the regulation of plasma cholesterol.

Long-term effects of maternal resveratrol intake during lactation on cholesterol metabolism in male rat offspring

Resveratrol (RSV) can protect against non-communicable diseases by improving cholesterol metabolism. However, it is unclear that effects of maternal RSV intake on health of adult offspring. In this study, we examined effects of maternal RSV intake during lactation on cholesterol metabolism in adult male rat offspring. Female Wistar rats were fed a control diet (CON) supplemented with or without RSV (20 mg/kg body weight/day) during their lactation period. Male offspring were weaned onto a standard diet and maintained on this diet for 36 weeks. As a result, plasma cholesterol level significantly decreased in RSV offspring compared to CON offspring. Furthermore, a decrease in hepatic 3-hydroxy-3-methylglutaryl-CoA reductase level and an increase in hepatic LDL-receptor level were observed in the RSV offspring. These results indicate that maternal RSV intake causes long-term decrease in plasma cholesterol level in the offspring through suppression of hepatic cholesterol biosynthesis and promotion of hepatic cholesterol uptake.

SCARB1 rs5888 gene polymorphisms in coronary heart disease: A systematic review and a meta-analysis

BackgroundStudies have suggested that high-density lipoprotein (HDL) stimulates scavenger receptor class B type 1 (SR-B1) to promote hepatic uptake of cholesterol. SR-B1 is encoded by scavenger receptor class B member 1 (SCARB1) gene in human. A rare mutation in SCARB1 gene has been associated with coronary heart disease (CHD). A polymorphism rs5888 of SCARB1 gene has been linked to CHD risk in humans. ObjectivesThe objective was to investigate the relationship between the SCARB1 gene polymorphism rs5888 and risk of CHD. MethodsWe searched databases of case-control studies and cohort studies on rs5888 polymorphism of SCARB1 gene and risk of CHD. Two reviewers independently screened literature, extracted data, and estimated potential bias of included studies. The quality of the studies was evaluated by recommendation of Newcastle-Ottawa Scale (NOS). Meta-analysis was performed with Stata 12.0 software. ResultsSeven studies including 6360 subjects (cases: 2456, controls: 3904) were included in the final data combination. Meta-analysis showed T allele had a lower risk of CHD as compared to C allele in allele model (T vs. C: OR = 0.87, 95% CI: 0.70 to 1.09, P = 0.229). Moreover, we found that T allele or TT/TC had a lower risk of CHD as compared to C/CC in male in allele model (T vs. C: OR = 0.79, 95% CI: 0.61 to 1.01). However, no significant association was observed in women in all allele models. ConclusionsOur findings suggested that polymorphism rs5888 had negative association with CHD, especially in male. However, the conclusion needs further verification with high quality studies with larger sample size and rigorous designs.

Rare SCARB1 mutations associate with high-density lipoprotein cholesterol but not with coronary artery disease.

AimsScavenger receptor Class B Type 1 (SR-BI) is a major receptor for high-density lipoprotein (HDL) that promotes hepatic uptake of cholesterol from HDL. A rare mutation p.P376L, in the gene encoding SR-BI, SCARB1, was recently reported to associate with elevated HDL cholesterol (HDL-C) and increased risk of coronary artery disease (CAD), suggesting that increased HDL-C caused by SR-BI impairment might be an independent marker of cardiovascular risk. We tested the hypothesis that alleles in or close to SCARB1 that associate with elevated levels of HDL-C also associate with increased risk of CAD in the relatively homogeneous population of Iceland.Methods and resultsUsing a large resource of whole-genome sequenced Icelanders, we identified thirteen SCARB1 coding mutations that we examined for association with HDL-C (n = 136 672). Three rare SCARB1 mutations, encoding p.G319V, p.V111M, and p.V32M (combined allelic frequency = 0.2%) associate with elevated levels of HDL-C (p.G319V: β = 11.1 mg/dL, P = 8.0 × 10−7; p.V111M: β = 8.3 mg/dL, P = 1.1 × 10−6; p.V32M: β = 10.2 mg/dL, P = 8.1 × 10−4). These mutations do not associate with CAD (36 886 cases/306 268 controls) (odds ratio = 0.90, 95% confidence interval 0.67–1.22, P = 0.49), despite effects on HDL-C comparable to that reported for p.P376L, both in terms of direction and magnitude. Furthermore, HDL-C raising alleles of three common SCARB1 non-coding variants, including one previously unreported (rs61941676-C: β = 1.25 mg/dL, P = 1.7 × 10−18), and of one low frequency coding variant (p.V135I) that independently associate with higher HDL-C, do not confer increased risk of CAD.ConclusionElevated HDL-C due to genetically compromised SR-BI function is not a marker of CAD risk.

Malaria severity: Possible influence of the E670G PCSK9 polymorphism: A preliminary case-control study in Malian children.

AimProprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) is a hepatic secretory protein which promotes the degradation of low-density lipoprotein receptors leading to reduced hepatic uptake of plasma cholesterol. Non-synonymous single-nucleotide polymorphisms in its gene have been linked to hypo- or hyper- cholesterolemia, depending on whether they decrease or increase PCSK9 activity, respectively. Since the proliferation and the infectivity of Plasmodium spp. partially depend on cholesterol from the host, we hypothesize that these PCSK9 genetic polymorphisms could influence the course of malaria infection in individuals who carry them. Here we examined the frequency distribution of one dominant (C679X) and two recessive (A443T, I474V) hypocholesterolemic polymorphisms as well as that of one recessive hypercholesterolemic polymorphism (E670G) among healthy and malaria-infected Malian children.MethodsDried blood spots were collected in Bandiagara, Mali, from 752 age, residence and ethnicity-matched children: 253 healthy controls, 246 uncomplicated malaria patients and 253 severe malaria patients. Their genomic DNA was extracted and genotyped for the above PCSK9 polymorphisms using Taqman assays. Associations of genotype distributions and allele frequencies with malaria were evaluated.ResultsThe minor allele frequency of the A443T, I474V, E670G, and C679X polymorphisms in the study population sample was 0.12, 0.20, 0.26, and 0.02, respectively. For each polymorphism, the genotype distribution among the three health conditions was statistically insignificant, but for the hypercholesterolemic E670G polymorphism, a trend towards association of the minor allele with malaria severity was observed (P = 0.035). The association proved to be stronger when allele frequencies between healthy controls and severe malaria cases were compared (Odd Ratio: 1.34; 95% Confidence Intervals: 1.04–1.83); P = 0.031).ConclusionsCarriers of the minor allele of the E670G PCSK9 polymorphism might be more susceptible to severe malaria. Further investigation of the cholesterol regulating function of PCSK9 in the pathophysiology of malaria is needed.

Structure-Specific Effects of Short-Chain Fatty Acids on Plasma Cholesterol Concentration in Male Syrian Hamsters.

Previous studies have shown that short-chain fatty acids (SCFAs) are capable of decreasing plasma cholesterol. However, the relative plasma-cholesterol-lowering activity of individual SCFAs and the underlying mechanisms by which SCFAs decrease plasma cholesterol remain largely unknown. The present study was done to compare the plasma-cholesterol-lowering potencies of four common SCFAs with 2-5 carbons and to investigate their interactions with gene expressions of key regulatory factors involved in cholesterol metabolism. For 6 weeks, five groups of male Golden hamsters were fed either a control high-cholesterol diet (HCD) or one of the four experimental HCDs containing 0.5 mol of acetate (Ac), propionate (Pr), butyrate (Bu), or valerate (Va) per kilogram of the diet. The results showed that Ac, Pr, and Bu significantly reduced plasma total cholesterol (TC) by 24, 18, and 17% (P < 0.05), respectively. All four SCFAs could decrease non-HDL cholesterol (non-HDL-C) and the non-HDL-C/HDL-C ratio. The addition of Ac, Pr, or Bu into the diet significantly promoted fecal excretion of bile acids by 121, 113, or 120% (P < 0.05), respectively, and upregulated the gene expressions of sterol-regulatory-element-binding protein 2 (SREBP2), low-density-lipoprotein receptor (LDLR), and cholesterol 7α-hydroxylase (CYP7A1) in the liver. It was concluded that SCFAs with 2-4 carbons (Ac, Pr, and Bu) are more hypocholesterolemic than Va, which has 5 carbons, via enhancing fecal excretion of bile acids and promoting the hepatic uptake of cholesterol from the blood.

Demystifying HDL Cholesterol-A "Human Knockout" to the Rescue?

The substantial cost associated with failure of a therapeutic approach in a large outcomes-based clinical trial, as has occurred recently for multiple compounds targeting HDL cholesterol metabolism, has led to intense interest in improved target selection. Careful study of humans who carry mutations that inactivate a putative drug target leverages naturally occurring genetic variation to study the impact of lifelong deficiency of a specific protein. As such, human genetics research can provide an opportunity to confirm the physiologic relevance of a target in humans before large-scale investments in drug development. Naturally occurring human DNA sequence variation can provide the foundation for important insights into disease biology and novel therapeutic strategies. Within the field of lipid metabolism, the rapid clinical development of proprotein convertase subtilisin kexin type 9 (PCSK9) inhibitors to reduce LDL cholesterol provides proof-of-concept for the potential of this approach. Following the initial 2003 discovery of a gain-of-function mutation in the gene PCSK9 (proprotein convertase subtilisin/kexin type 9)3 associated with significantly increased LDL cholesterol, a 2005 report noted that about 2% of black individuals harbor an inactivating (premature stop) mutation in the PCSK9 gene. Carriers of this mutation benefitted from lifelong lower LDL cholesterol concentrations and inborn protection from coronary disease without detectable toxicity (1). Further, the identification of healthy individuals with 2 inactivating mutations, effectively “human knockouts” for PCSK9, further increased optimism that the protein could be targeted without off-target effects. These observations provided additional support for the hypothesis that pharmacologic inhibition of PCSK9 might similarly lower LDL cholesterol and decrease vascular risk. Translational research studies demonstrated that PCSK9 plays a previously unrecognized role in preventing hepatic uptake of LDL cholesterol by accelerating the catabolism of LDL receptors. These findings led to a pharmaceutical arms race to develop PCSK9 inhibitors using monoclonal antibody or RNA interference approaches. …

Endoplasmic Reticulum Stress and Ca2+ Depletion Differentially Modulate the Sterol Regulatory Protein PCSK9 to Control Lipid Metabolism

Accumulating evidence implicates endoplasmic reticulum (ER) stress as a mediator of impaired lipid metabolism, thereby contributing to fatty liver disease and atherosclerosis. Previous studies demonstrated that ER stress can activate the sterol regulatory element-binding protein-2 (SREBP2), an ER-localized transcription factor that directly up-regulates sterol regulatory genes, including PCSK9 Given that PCSK9 contributes to atherosclerosis by targeting low density lipoprotein (LDL) receptor (LDLR) degradation, this study investigates a novel mechanism by which ER stress plays a role in lipid metabolism by examining its ability to modulate PCSK9 expression. Herein, we demonstrate the existence of two independent effects of ER stress on PCSK9 expression and secretion. In cultured HuH7 and HepG2 cells, agents or conditions that cause ER Ca2+ depletion, including thapsigargin, induced SREBP2-dependent up-regulation of PCSK9 expression. In contrast, a significant reduction in the secreted form of PCSK9 protein was observed in the media from both thapsigargin- and tunicamycin (TM)-treated HuH7 cells, mouse primary hepatocytes, and in the plasma of TM-treated C57BL/6 mice. Furthermore, TM significantly increased hepatic LDLR expression and reduced plasma LDL concentrations in mice. Based on these findings, we propose a model in which ER Ca2+ depletion promotes the activation of SREBP2 and subsequent transcription of PCSK9. However, conditions that cause ER stress regardless of their ability to dysregulate ER Ca2+ inhibit PCSK9 secretion, thereby reducing PCSK9-mediated LDLR degradation and promoting LDLR-dependent hepatic cholesterol uptake. Taken together, our studies provide evidence that the retention of PCSK9 in the ER may serve as a potential strategy for lowering LDL cholesterol levels.

Deletion of liver-specific STAT5 gene alters the expression of bile acid metabolism genes and reduces liver damage in lithogenic diet-fed mice

Signal transducers and activators of transcription 5 (STAT5) mediates growth hormone signals, which may control hepatic cholesterol uptake and bile acid metabolism. Deregulation of liver cholesterol homeostasis and bile acid metabolism may cause liver damage and cholesterol gallstone development. The purpose of this study was to understand the role of local STAT5 signaling in cholesterol and bile acid metabolism using liver-specific STAT5 knock-out (STAT5 LKO) mice on a normal diet and a cholesterol- and bile acid-containing lithogenic diet. STAT5 LKO mice showed significant down-regulation of STAT5 and insulin-like growth factor-1 genes. STAT5 gene deletion had a minor effect on cholesterol metabolism, as evidenced by a minor change in circulating cholesterol levels and no changes in expression of hepatic low-density lipoprotein receptor and cholesterol synthesis genes in STAT5 LKO mice. In contrast, bile acid synthesis and uptake genes were profoundly down-regulated and bile acid detoxification genes were up-regulated in STAT5 LKO mice. In STAT5 fl/fl mice, a lithogenic diet induced liver damage, as evidenced by moderate increases in liver ballooning, inflammation and fibrosis. However, STAT5 deletion ameliorated the degree of liver damage induced by the lithogenic diet. In STAT5 LKO mice, a lithogenic diet did not alter the incidence or severity of cholesterol gallstones. In conclusion, local STAT5 signaling does not have a significant role in cholesterol metabolism. In contrast, hepatic STAT5 signaling has significant roles in regulating transcription of genes for synthesis, transport and detoxification of bile acids, but it has only a minor role in bile acid metabolism.

Novel Role for Matrix Metalloproteinase 9 in Modulation of Cholesterol Metabolism.

BackgroundThe development of atherosclerosis is strongly linked to disorders of cholesterol metabolism. Matrix metalloproteinases (MMPs) are dysregulated in patients and animal models with atherosclerosis. Whether systemic MMP activity influences cholesterol metabolism is unknown.Methods and ResultsWe examined MMP‐9–deficient (Mmp9 −/−) mice and found them to have abnormal lipid gene transcriptional responses to dietary cholesterol supplementation. As opposed to Mmp9 +/+ (wild‐type) mice, Mmp9 −/− mice failed to decrease the hepatic expression of sterol regulatory element binding protein 2 pathway genes, which control hepatic cholesterol biosynthesis and uptake. Furthermore, Mmp9 −/− mice failed to increase the expression of genes encoding the rate‐limiting enzymes in biliary cholesterol excretion (eg, Cyp7a and Cyp27a). In contrast, MMP‐9 deficiency did not impair intestinal cholesterol absorption, as shown by the 14C‐cholesterol and 3H‐sitostanol absorption assay. Similar to our earlier study on Mmp2 −/− mice, we observed that Mmp9 −/− mice had elevated plasma secreted phospholipase A2 activity. Pharmacological inhibition of systemic circulating secreted phospholipase A2 activity (with varespladib) partially normalized the hepatic transcriptional responses to dietary cholesterol in Mmp9 −/− mice. Functional studies with mice deficient in other MMPs suggested an important role for the MMP system, as a whole, in modulation of cholesterol metabolism.ConclusionsOur results show that MMP‐9 modulates cholesterol metabolism, at least in part, through a novel MMP‐9–plasma secreted phospholipase A2 axis that affects the hepatic transcriptional responses to dietary cholesterol. Furthermore, the data suggest that dysregulation of the MMP system can result in metabolic disorder, which could lead to atherosclerosis and coronary heart disease.

Genistein increased hepatic cholesterol uptake via a JNK mediated activation of SREBP‐2 and LDLR expression

Genistein has been implicated for its anti‐atherogenic effects. To understand the anti‐atherogenic mechanism of action genistein was investigated its impact on hepatic cholesterol uptake, the expression of LDLR which is the receptor for LDL‐cholesterol, and related signaling pathways in human hepatoma HepG2 cells. the The addition of 40 μM genistein increased cholesterol uptake into HepG2cells. Genistein increased mRNA and protein levels of LDLR in a time‐dependent manner. The addition of cholesterol up to 5 μg/mL for 24 hours did not significantly affect the effect of genistein towards LDLR protein expression. Genistein increased the transcriptional activity of LDLR promoter containing reporter gene (pLDLR‐luc, −805 to +50). But sterol‐regulatory element (SRE) deletion mutant construct was failed to be activated by genistein. Genistein increased the nuclear fraction of SREBP‐2 and DNA binding activity of SREBP‐2 to LDLR promoter as assessed by chromatin immunoprecipitation assay (CHIP). Genistein phosphorylated JNKs. JNK inhibitor (SP600126) abolished genistein‐stimulated levels of LDLR and nuclear SREBP‐2. To minimize the effects of c‐Jun, a transcription factor activated by JNK signals, a truncated LDLR luciferase construct that was contained SRE but lacked the c‐jun putative binding site was constructed. Genistein was still able to boost the transcriptional activity of the truncated LDLR construct. In conclusion, our data supports that genistein may have the anti‐atherogenic effects by increasing hepatic LDL cholesterol uptake via activating JNK signals and SREBP‐2 processing, which is followed by up‐regulation of LDLR.

Combinations of phytomedicines with different lipid lowering activity for dyslipidemia management: The available clinical data

BackgroundCardiovascular diseases are the primary cause of death and the leading cause of disability in industrialized countries. Dyslipidemia is a major independent and reversible risk factor for these diseases: it is estimated that a reduction of 1 mmol/l (38 mg/dl) of LDL cholesterol is associated with a risk of developing a cardiovascular complication reduced by 25%, a reduction potentially achieved by life-style improvement associated to adequate dietary supplementation with bioactive substances. AimThe aim of this review is to focus on the major phytochemical nutraceuticals combinations supported by clinical trials that have demonstrated positive effects in the treatment of dyslipidemia. Main textThere are many nutraceuticals with significant lipid-lowering properties: most of them are used in association with a low dosage, because that permits to reduce the risk of side effects and theoretically to improve efficacy. In fact, natural products with different synergetic lipid-lowering could be combined: they can reduce the absorption of lipids from the bowel and/or increase their excretion (soluble fibers, plant sterols, probiotics), enhance the hepatic uptake of cholesterol (berberine, soybean proteins), inhibit Hydroxy-Methil-Gglutaryl Coenzyme A reductase enzyme and consequently the hepatic synthesis of cholesterol (monacolins, policosanols, allicin, soybean proteins, bergamot); furthermore some products are able to reduce the oxidation of the LDL and increase the thermogenesis and lipid metabolism (chlorogenic acid). ConclusionRational combinations of nutraceuticals with different lipid-lowering activities, whether associated with an appropriate lifestyle, should provide an alternative to drug treatment in patients in primary cardiovascular disease prevention with mildly added cardiovascular risk and in some statin-intolerant patients.

Abstract 17627: Is the Post-menopausal Rise in PCSK9 Levels Due to Loss of the Estrogen Responsive Anti-atherogenic Protein, Heat Shock Protein 27? Insights Into a Potential New PCSK9 Lowering Therapy

Introduction: Previously we reported that elevated serum levels of Heat Shock Protein 27 represent a predictive biomarker of reduced cardiovascular clinical events. Moreover, attenuation of experimental atherogenesis, characterized by reduced cholesterol levels in both the artery wall and serum, can be achieved by augmenting serum HSP27 levels (e.g., with recombinant HSP27). PCSK9 plays a central role in regulating the abundance of hepatic LDL-receptors, and hence serum and plaque cholesterol levels. Interestingly, PCSK9 levels rise with menopause (Dallas Heart Study) and we previously published that HSP27 levels are markedly attenuated post-ovariectomy. Hence, we hypothesize that HSP27 augments hepatic cholesterol uptake via reductions in PCSK9 levels. Methods / Results: 1. In vitro studies: HepG2 (liver) cells were maintained in culture for 24 hours with or without rHSP27. The hepatic levels of apoB100 did not change with rHSP27 treatment. ApoB100 containing lipoproteins were reduced in the cell culture media with rHSP27 treatment (Western Blot: p=0.008; ELISA: p=0.0025). rHSP27 treatment reduced hepatic PCSK9 protein levels (Western Blot: p=0.019), increased LDL receptor protein levels (Western Blot: p=0.001) and increased (fluorescent) LDL uptake (p=0.022). Levels of hepatic SREBP2 and HMG-CoA reductase proteins were unaltered with rHSP27 treatment. 2. In vivo studies: Female ApoE-/- mice fed high fat diet and injected weekly with rHSP27 or PBS for 8 weeks. Serum total cholesterol and plasma PCSK9 levels were ~20-40% lower in the rHSP27 treated vs. control mice. Mice treated with rHSP27 showed reductions in aortic lesion area of ~40% for en face and ~35% for cross-sectional sinus tissue sections. Conclusions: These findings suggest that HSP27, an endogenous estrogen responsive protein, can lower extracellular cholesterol levels by reducing PCSK9 expression. Hence there is promise for the development of HSP27 “replacement” therapy for the prevention and modification of atherosclerosis via reductions in PCSK9 expression. Current clinical studies examining the relationship between HSP27 and PCSK9 levels in women undergoing prophylactic ovariectomy for BRAC1/2 (i.e., the “Angelina Effect”) are ongoing.

Increased plasma cholesterol esterification by LCAT reduces diet-induced atherosclerosis in SR-BI knockout mice

LCAT, a plasma enzyme that esterifies cholesterol, has been proposed to play an antiatherogenic role, but animal and epidemiologic studies have yielded conflicting results. To gain insight into LCAT and the role of free cholesterol (FC) in atherosclerosis, we examined the effect of LCAT over- and underexpression in diet-induced atherosclerosis in scavenger receptor class B member I-deficient [Scarab(-/-)] mice, which have a secondary defect in cholesterol esterification. Scarab(-/-)×LCAT-null [Lcat(-/-)] mice had a decrease in HDL-cholesterol and a high plasma ratio of FC/total cholesterol (TC) (0.88 ± 0.033) and a marked increase in VLDL-cholesterol (VLDL-C) on a high-fat diet. Scarab(-/-)×LCAT-transgenic (Tg) mice had lower levels of VLDL-C and a normal plasma FC/TC ratio (0.28 ± 0.005). Plasma from Scarab(-/-)×LCAT-Tg mice also showed an increase in cholesterol esterification during in vitro cholesterol efflux, but increased esterification did not appear to affect the overall rate of cholesterol efflux or hepatic uptake of cholesterol. Scarab(-/-)×LCAT-Tg mice also displayed a 51% decrease in aortic sinus atherosclerosis compared with Scarab(-/-) mice (P < 0.05). In summary, we demonstrate that increased cholesterol esterification by LCAT is atheroprotective, most likely through its ability to increase HDL levels and decrease pro-atherogenic apoB-containing lipoprotein particles.

Adverse Signaling of Scavenger Receptor Class B1 and PGC1s in Alcoholic Hepatosteatosis and Steatohepatitis and Protection by Betaine in Rat

Because scavenger receptor class B type 1 is the cholesterol uptake liver receptor, whereas peroxisome proliferator-activated receptor γ coactivator-1β (PGC-1β) and PGC-1α are critical for lipid synthesis and degradation, we investigated the roles of these signaling molecules in the actions of ethanol-polyunsaturated fatty acids and betaine on hepatosteatosis and steatohepatitis. Ethanol-polyunsaturated fatty acid treatment caused the following: i) hepatosteatosis, as evidenced by increased liver cholesterol and triglycerides, lipid score, and decreased serum adiponectin; ii) marked inhibition of scavenger receptor class B type 1 glycosylation, its plasma membrane localization, and its hepatic cholesterol uptake function; and iii) moderate steatohepatitis, as evidenced by histopathological characteristics, increased liver tumor necrosis factor α and IL-6, decreased glutathione, and elevated serum alanine aminotransferase. These actions of ethanol involved up-regulated PGC-1β, sterol regulatory element-binding proteins 1c and 2, acetyl-CoA carboxylase, and HMG-CoA reductase mRNAs/proteins and inactive non-phosphorylated AMP kinase; and down-regulated silence regulator gene 1 and PGC-1α mRNA/proteins and hepatic fatty acid oxidation. Betaine markedly blunted all these actions of ethanol on hepatosteatosis and steatohepatitis. Therefore, we conclude that ethanol-mediated impaired post-translational modification, trafficking, and function of scavenger receptor class B type 1 may account for alcoholic hyperlipidemia. Up-regulation of PGC-1β and lipid synthetic genes and down-regulation of silence regulator gene 1, PGC-1α, adiponectin, and lipid degradation genes account for alcoholic hepatosteatosis. Induction of proinflammatory cytokines and depletion of endogenous antioxidant, glutathione, account for alcoholic steatohepatitis. We suggest betaine as a potential therapeutic agent because it effectively protects against adverse actions of ethanol.

Effects and Potential Mechanisms of Pioglitazone on Lipid Metabolism in Obese Diabetic KKAy Mice.

This study aimed to analyze the effects and potential mechanisms of pioglitazone on triglyceride and cholesterol metabolism in obese diabetic KKAy mice. Pioglitazone was orally administered to KKAy mice over 30 days. Compared to C57BL/6J mice, KKAy mice developed obvious insulin resistance, hepatic steatosis, and hyperlipidemia. Pioglitazone treatment resulted in deteriorated microvesicular steatosis and elevated hepatic triglyceride levels, though plasma triglyceride and free fatty acid levels were reduced by the treatment, compared to nontreated KKAy mice. Plasma alanine aminotransferase activities were also significantly increased. Additionally, pioglitazone increased plasma concentrations of total cholesterol, HDL-cholesterol, and LDL-cholesterol but decreased hepatic cholesterol. Gene expression profiling revealed that pioglitazone stimulated hepatic peroxisome proliferator-activated receptor gamma hyperactivity, and induced the upregulation of adipocyte-specific and lipogenesis-related genes but downregulated of genes involved in triglyceride lipolysis and fatty acid β-oxidation. Pioglitazone also regulated the genes expression of hepatic cholesterol uptake and excretion, such as low density lipoprotein receptor (LDL-R) and scavenger receptor type-BI (SR-BI). These results suggested that pioglitazone could induce excessive hepatic triglyceride accumulation, thus aggravating liver steatosis and lesions in KKAy mice. Furthermore, pioglitazone may suppress the clearance of serum cholesterol from the liver predominantly through inhibition of LDL-R and SR-BI expression, thus increasing the plasma cholesterol.