Cholesterol Homeostasis In Liver Research Articles

Liver-Specific Bmal1 Depletion Reverses the Beneficial Effects of Nobiletin on Liver Cholesterol Homeostasis in Mice Fed with High-Fat Diet.

Nobiletin (NOB), a naturally occurring small-molecule compound abundant in citrus peels, has displayed potential lipid-lowering and circadian-enhancing properties in preclinical studies. However, the requirement of specific clock genes for the beneficial effects of NOB is not well understood. In the current study, mice with a liver-specific deletion of the core clock component, Bmal1-Bmal1LKO-were fed a high-fat diet (HFD) ad libitum for eight weeks, while NOB (200 mg/kg) was administered by daily oral gavage from the fifth week and throughout the last four weeks. NOB decreased liver triglyceride (TG) alongside the decreasing mRNA levels of de novo lipogenesis (DNL) genes in both Bmal1flox/flox and Bmal1LKO mice. NOB increased serum very low-density lipoprotein (VLDL) levels in Bmal1LKO mice, which was consistent with higher liver Shp and lower Mttp mRNA expression levels, the key genes that facilitate VLDL assembly and secretion. NOB decreased liver and serum cholesterol levels in the Bmal1flox/flox mice, consistent with lower Hmgcr and higher Cyp7a1, Cyp8b1, Gata4 and Abcg5 mRNA levels in the liver. In contrast, in the Bmal1LKO mice, NOB increased Hmgcr mRNA levels and had no effect on the above-mentioned genes related to bile acid synthesis and cholesterol excretion, which might contribute to the elevation of liver and serum cholesterol levels in NOB-treated Bmal1LKO mice. NOB inhibited hepatic DNL and decreased liver TG levels in HFD-fed mice independently of liver Bmal1, whereas liver-specific Bmal1 depletion reversed the beneficial effects of NOB on liver cholesterol homeostasis. The complex interactions between NOB, the circadian clock and lipid metabolism in the liver warrant further research.

Roux-en-Y gastric bypass induces hepatic transcriptomic signatures and plasma metabolite changes indicative of improved cholesterol homeostasis

Roux-en-Y gastric bypass (RYGB), the most effective surgical procedure for weight loss, decreases obesity and ameliorates comorbidities, such as non-alcoholic fatty liver (NAFLD) and cardiovascular (CVD) diseases. Cholesterol is a major CVD risk factor and modulator of NAFLD development, and the liver tightly controls its metabolism. How RYGB surgery modulates systemic and hepatic cholesterol metabolism is still unclear. We studied the hepatic transcriptome of 26 patients with obesity but not diabetes before and 1 year after undergoing RYGB. In parallel, we measured quantitative changes in plasma cholesterol metabolites and bile acids (BAs). RYGB surgery improved systemic cholesterol metabolism and increased plasma total and primary BA levels. Transcriptomic analysis revealed specific alterations in the liver after RYGB, with the downregulation of a module of genes implicated in inflammation and the upregulation of three modules, one associated with BA metabolism. A dedicated analysis of hepatic genes related to cholesterol homeostasis pointed towards increased biliary cholesterol elimination after RYGB, associated with enhancement of the alternate, but not the classical, BA synthesis pathway. In parallel, alterations in the expression of genes involved in cholesterol uptake and intracellular trafficking indicate improved hepatic free cholesterol handling. Finally, RYGB decreased plasma markers of cholesterol synthesis, which correlated with an improvement in liver disease status after surgery. Our results identify specific regulatory effects of RYGB on inflammation and cholesterol metabolism. RYGB alters the hepatic transcriptome signature, likely improving liver cholesterol homeostasis. These gene regulatory effects are reflected by systemic post-surgery changes of cholesterol-related metabolites, corroborating the beneficial effects of RYGB on both hepatic and systemic cholesterol homeostasis. Roux-en-Y gastric bypass (RYGB) is a widely used bariatric surgery procedure with proven efficacy in body weight management, combatting cardiovascular disease (CVD) and non-alcoholic fatty liver disease (NAFLD). RYGB exerts many beneficial metabolic effects, by lowering plasma cholesterol and improving atherogenic dyslipidemia. Using a cohort of patients undergoing RYGB, studied before and 1 year after surgery, we analyzed how RYGB modulates hepatic and systemic cholesterol and bile acid metabolism. The results of our study provide important insights on the regulation of cholesterol homeostasis after RYGB and open avenues that could guide future monitoring and treatment strategies targeting CVD and NAFLD in obesity.

Selected Soybean Varieties Regulate Hepatic LDL-Cholesterol Homeostasis Depending on Their Glycinin:β-Conglycinin Ratio.

Clinical studies indicate that the consumption of soybean protein might reduce cholesterol and LDL levels preventing the development of atherosclerotic cardiovascular diseases. However, soybean variety can influence soybean protein profile and therefore affect soybean protein health-promoting properties. This study investigated the composition and effects of nineteen soybean varieties digested under simulated gastrointestinal conditions on hepatic cholesterol metabolism and LDL oxidation in vitro. Soybean varieties exhibited a differential protein hydrolysis during gastrointestinal digestion. Soybean varieties could be classified according to their composition (high/low glycinin:β-conglycinin ratio) and capacity to inhibit HMGCR (IC50 from 59 to 229 µg protein mL-1). According to multivariate analyses, five soybean varieties were selected. These soybean varieties produced different peptide profiles and differently reduced cholesterol concentration (43-55%) by inhibiting HMGCR in fatty-acid-stimulated HepG2 hepatocytes. Selected digested soybean varieties inhibited cholesterol esterification, triglyceride production, VLDL secretion, and LDL recycling by reducing ANGPTL3 and PCSK9 and synchronously increasing LDLR expression. In addition, selected soybean varieties hindered LDL oxidation, reducing the formation of lipid peroxidation early (conjugated dienes) and end products (malondialdehyde and 4-hydroxynonenal). The changes in HMGCR expression, cholesterol esterification, triglyceride accumulation, ANGPTL3 release, and malondialdehyde formation during LDL oxidation were significantly (p < 0.05) correlated with the glycinin:β-conglycinin ratio. Soybean varieties with lower glycinin:β-conglycinin exhibited a better potential in regulating cholesterol and LDL homeostasis in vitro. Consumption of soybean flour with a greater proportion of β-conglycinin may, consequently, improve the potential of the food ingredient to maintain healthy liver cholesterol homeostasis and cardiovascular function.

Differential cholesterol uptake in liver cells: A role for PCSK9.

The clearance of low-density lipoprotein (LDL) particles from the circulation is regulated by the LDL receptor (LDLR) and proprotein convertase subtilisin/kexin 9 (PCSK9) interaction. Its disruption reduces blood cholesterol levels and delays atherosclerosis progression. Whether other members of the LDLR superfamily are in vivo targets of PCSK9has been poorly explored. The aim of this work was to study the interaction between PCSK9 and members of the LDLR superfamily in the regulation of liver cholesterol homeostasis in an in vivo low-density lipoprotein receptor related protein 5 (LRP5) deficient mice model challenged with high-fat diet. Our results show that Wt and Lrp5-/- mice fed a hypercholesterolemic diet (HC) have increased cholesterol ester accumulation and decreased liver LDLR and LRP5gene and protein expression. Very low-density lipoprotein receptor (VLDLR), LRP6, LRP2, and LRP1 expression levels were analyzed in liver samples and show that they do not participate in Lrp5-/- liver cholesterol uptake. Immunoprecipitation experiments show that LRP5 forms a complex with PCSK9 in liver-specific fat-storing stellate cells but not in structural HepG2 cells. Hepatic stellate cells silenced for LRP5 and/or PCSK9 expression and challenged with lipids show reduced cholesterol ester accumulation, indicating that both proteins are involved in lipid processing in the liver. Our results indicate that cholesterol esters accumulate in livers of Wt mice in a LDLR-family-members dependent manner as VLDLR, LRP2, and LRP6show increased expression in HC mice. However, this increase is lost in livers of Lrp5-/- mice, where scavenger receptors are involved in cholesterol uptake. PCSK9 expression is strongly downregulated in mice livers after HC feeding. However PCSK9 and LRP5 bind in the cytoplasm of fat storing liver cells, indicating that this PCSK9-LRP5 interaction is cell-type specific and that both proteins contribute to lipid uptake.

HOMEOSTASIS OF CHOLESTEROL IN THE BODY, MECHANISMS OF ITS DISORDER, ROLE OF 7α-HYDROXYLASE

It has been shown that in humans and animals, CS homeostasis occurs in accordance with the same formula: (СSendog + CSexog) – (CSexcr + BAexcr) = 0. But in herbivores, the positive balance of CS in the body is not occurs; a person creates it by consuming animal products, high-calorie foods and substrates for the synthesis of cholesterol. Like herbivores, it does not need constant intake of cholesterol from the food, the release of which from the body in the form of bile acids is also limited by 7α-hydroxylase activity. This point of view is supported by the fact that a person's daily need for cholesterol can be met by one of its biosynthesis. The paper provides information on the role of liver cholesterol homeostasis and the transformation of cholesterol into bile acids (BA) in the disturbance of homeostasis. Hydroxylation is the only process that irreversibly removes CS from membranes and LP-complexes. For the formation of BA, 60–80 % of the total cholesterol synthesized daily in the body is spent. The rate of the 7α-hydroxylase reaction is affected by the presence of hypercholesterolemia: in experimental animals and in patients with HCS, the rate of the 7α-hydroxylase reaction of the liver is sharply reduced, the rate of conversion of cholesterol to BA and its removal from the body is reduced. A role in atherogenesis of a decrease in the rate of 7α-hydroxylation with aging in humans has been shown. The fact is underlined that the release of cholesterol in the form of bile acids reflects the rate of its synthesis in the body. The formation of BA and the excretion of cholesterol is inhibited by insufficient intake of vitamin C and unsaturated fatty acids into the human body. It is concluded that it is necessary to search for and synthesize substances that enhance the conversion of cholesterol into bile acids and its excretion from the body. The combination of stimulators of bile acid synthesis and blockers of HMG-CoA reductase can significantly increase the effectiveness of treatment of patients with atherosclerosis.

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.

Perfluorooctanoic acid activates multiple nuclear receptor pathways and skews expression of genes regulating cholesterol homeostasis in liver of humanized PPARα mice fed an American diet

Humans are exposed to per- and polyfluoroalkyl substances (PFAS) in their drinking water, food, air, dust, and by direct use of consumer products. Increased concentrations of serum total cholesterol and low density lipoprotein cholesterol are among the endpoints best supported by epidemiology. The objectives of this study were to generate a new model for examining PFAS-induced dyslipidemia and to conduct molecular studies to better define mechanism(s) of action. We tested the hypothesis that perfluorooctanoic acid (PFOA) exposure at a human-relevant level dysregulates expression of genes controlling cholesterol homeostasis in livers of mice expressing human PPARα (hPPARα). Female and male hPPARα and PPARα null mice were fed a diet based on the “What we eat in America” analysis and exposed to PFOA in drinking water (8 μM) for 6 weeks. This resulted in a serum PFOA concentration of 48 μg/ml. PFOA increased liver mass, which was associated with histologically-evident lipid accumulation. Pooled analyses of serum lipoprotein cholesterol suggest that PFOA increased serum cholesterol, particularly in male mice. PFOA induced PPARα and constitutive androstane receptor target gene expression in liver. Expression of genes in four pathways regulating cholesterol homeostasis were also measured. PFOA decreased expression of Hmgcr in a PPARα-dependent manner. PFOA decreased expression of Ldlr and Cyp7a1 in a PPARα-independent manner. Apob expression was not changed. Sex differences were evident. This novel study design (hPPARα mice, American diet, long term exposure) generated new insight on the effects of PFOA on cholesterol regulation in the liver and the role of hPPARα.

PAR2 controls cholesterol homeostasis and lipid metabolism in nonalcoholic fatty liver disease

ObjectiveIncreases in hepatic and plasma cholesterol occur in patients with nonalcoholic fatty liver disease (NAFLD), although the reason for this is not well understood. We investigated whether Protease-Activated Receptor 2 (PAR2) plays a role in cholesterol and lipid homeostasis in NAFLD. MethodsHuman liver biopsies (n = 108) were quantified for PAR2 expression from NAFLD cases randomly selected and stratified by liver fibrosis stage, the primary predictor for clinical outcomes, while controlling for age, gender, and BMI between fibrosis groups. Demographic data and laboratory studies on plasma samples were obtained within 6 months of liver biopsy. Wild-type and PAR2-KO (C57BL/6 F2rl1−/−) mice were fed either normal or high fat diet for 16 weeks and plasma and liver assayed for lipids and soluble metabolites. ResultsSeverity of NAFLD and plasma cholesterol levels significantly correlated with hepatocyte PAR2 expression in NAFLD patients. Conversely, PAR2 deficiency in mice resulted in reduced expression of key hepatic genes involved in cholesterol synthesis, a 50% drop in plasma and total liver cholesterol, and induced a reverse cholesterol transport system that culminated in 25% higher fecal bile acid output. PAR2-deficient mice exhibited enhanced fatty acid β-oxidation with a ketogenic shift and an unexpected increase in liver glycogenesis. Mechanistic studies identified Gi-Jnk1/2 as key downstream effectors of protease-activated PAR2 in the regulation of lipid and cholesterol homeostasis in liver. ConclusionsThese data indicate that PAR2 may be a new target for the suppression of plasma cholesterol and hepatic fat accumulation in NAFLD and related metabolic conditions.

Hepatic cholesterol accumulation ascribed to the activation of ileum Fxr-Fgf15 pathway inhibiting hepatic Cyp7a1 in high-fat diet-induced obesity rats

AimsHigh-fat diet (HFD)-induced obesity resulting in cholesterol accumulation is one of the common pathogenic factors for lipids metabolic disorders. However, the potential mechanisms about cholesterol accumulation during obesity are still not clearly identified. Bile acids (BAs) as the natural ligands of farnesoid x receptor (Fxr) are demonstrated that can regulate the relevant enzymes and transporters at transcriptional level to determine the cholesterol homeostasis. Here, we explored the underlying mechanisms of hepatic cholesterol accumulation in HFD-induced obesity rats via the BAs-Fxr-enzymes/transporters signaling pathways. Materials and methodsBAs and cholesterol levels as well as mRNA expressions of enzymes, transporters and nuclear receptors involving in cholesterol homeostasis in liver and ileum tissue were evaluated in 4-week HFD-induced obesity rats. Key findingsHFD promoted BAs intestine passive absorption to increase the concentrations of BAs especially the chenodeoxycholic acids (CDCAs) in ileum of HFD-induced obesity rats. The increased CDCAs concentrations activated Fxr-Fgf15 pathway in ileum to result in the mRNA expression of Cyp7a1 in liver down-regulation, which inhibited cholesterol metabolizing into primary BAs to contribute to the cholesterol level increase in liver tissue in HFD-induced obesity rats. SignificanceThe hepatic cholesterol accumulation should be ascribed to the activation of ileum Fxr-Fgf15 pathway by the increased BAs passive absorption into ileal enterocytes under the condition of rats fed with HFD, which inhibited hepatic Cyp7a1 gene transcription to reduce metabolic elimination of cholesterol. Moreover, these findings are expected to provide a cue for the treatment of cholesterol metabolism disorders in obesity patient.

PKCβ: Expanding role in hepatic adaptation of cholesterol homeostasis to dietary fat/cholesterol.

Cholesterol homeostasis relies on an intricate network of cellular processes whose deregulation in response to Western type high-fat/cholesterol diets can lead to several life-threatening pathologies. Significant advances have been made in resolving the molecular identity and regulatory function of transcription factors sensitive to fat, cholesterol, or bile acids, but whether body senses the presence of both fat and cholesterol simultaneously is not known. Assessing the impact of a high-fat/cholesterol load, rather than an individual component alone, on cholesterol homeostasis is more physiologically relevant because Western diets deliver both fat and cholesterol at the same time. Moreover, dietary fat and dietary cholesterol are reported to act synergistically to impair liver cholesterol homeostasis. A key insight into the role of protein kinase C-β (PKCβ) in hepatic adaptation to high-fat/cholesterol diets was gained recently through the use of knockout mice. The emerging evidence indicates that PKCβ is an important regulator of cholesterol homeostasis that ensures normal adaptation to high-fat/cholesterol intake. Consistent with this function, high-fat/cholesterol diets induce PKCβ expression and signaling in the intestine and liver, while systemic PKCβ deficiency promotes accumulation of cholesterol in the liver and bile. PKCβ disruption results in profound dysregulation of hepatic cholesterol and bile homeostasis and imparts sensitivity to cholesterol gallstone formation. The available results support involvement of a two-pronged mechanism by which intestine and liver PKCβ signaling converge on liver ERK1/2 to dictate diet-induced cholesterol and bile acid homeostasis. Collectively, PKCβ is an integrator of dietary fat/cholesterol signal and mediates changes to cholesterol homeostasis.

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.

Human apolipoprotein E allele and docosahexaenoic acid intake modulate peripheral cholesterol homeostasis in mice

Carrying at least one apolipoprotein E ε4 allele (E4+) is the main genetic risk factor for Alzheimer's disease (AD). Epidemiological studies support that consuming fatty fish rich in docosahexaenoic acid (DHA; 22:6ω3) is protective against development of AD. However, this protective effect seems not to hold in E4+. The involvement of APOE genotype on the relationship between DHA intake and cognitive decline could be mediated through cholesterol. Many studies show a link between cholesterol metabolism and AD progression. In this study, we investigated whether cholesterol metabolism is improved in E3+ and E4+ mice consuming a diet rich in DHA. Plasma cholesterol was 36% lower in E4+ mice compared to E3+ mice fed the control diet (P=.02), and in the liver, there was a significant genotype effect where cholesterol levels were 18% lower in E4+ mice than E3+ mice. The low-density lipoprotein receptor was overexpressed in the liver of E4+ mice. Plasma cholesterol levels were 33% lower after the DHA diet (P=.02) in E3+ mice only, and there was a significant diet effect where cholesterol level was 67% lower in the liver of mice fed DHA. Mice fed the DHA diet also had 62% less lipolysis stimulated lipoprotein receptor expression in the liver compared to mice fed the control diet (P<.0001), but there was no genotype effect. These findings suggest that plasma and liver cholesterol homeostasis and the receptors regulating uptake of cholesterol in the liver are modulated differently and independently by APOE allele and DHA intake.

Sitosterolemia: diagnosis, investigation, and management.

Sitosterolemia is a rare autosomal recessively inherited disease caused by mutations affecting ABCG5 or ABCG8, which are located on human chromosome band 2p21. Around 100 cases have been reported in the literature. Sitosterolemic patients typically exhibit a 30-fold to 100-fold increase in plasma concentrations of plant sterols. The clinical manifestations include xanthomas, premature atherosclerosis, hemolytic anemia, and macrothrombocytopenia. It is noteworthy that abnormal hematological parameters may be the only clinical feature of sitosterolemic patients, suggesting that sitosterolemia may be more frequent than previously thought. Severe accumulation of plant sterols in mouse models of sitosterolemia induced complex cardiac lesions, anemia, and macrothrombocytopenia, disrupted adrenal and liver cholesterol homeostasis, and caused infertility and hypertriglyceridemia. It remains unclear whether all disease traits are present in sitosterolemic patients. The drug ezetimibe appears to be effective in reducing plasma plant sterol levels, promotes xanthoma regression, and improves the cardiovascular and hematological signs in sitosterolemic patients.

Disodium ascorbyl phytostanol phosphate (FM-VP4), a modified phytostanol, is a highly active hypocholesterolaemic agent that affects the enterohepatic circulation of both cholesterol and bile acids in mice

Disodium ascorbyl phytostanol phosphate (FM-VP4) is a synthetic compound derived from sitostanol and campestanol that has proved to be efficient as a cholesterol-lowering therapy in mice and human subjects. However, the mechanism of action of FM-VP4 remains unknown. The present study tests the ability of FM-VP4 to alter intestinal and liver cholesterol homeostasis in mice. Female C57BL/6J mice were fed either a control chow or a 2 % FM-VP4-enriched diet for 4 weeks. FM-VP4 reduced the in vivo net intestinal cholesterol absorption and plasma and liver cholesterol concentrations by 2.2-, 1.5- and 1.6-fold, respectively, compared with control mice. Furthermore, FM-VP4 also showed an impact on bile acid homeostasis. In FM-VP4 mice, liver and intestinal bile acid content was increased by 1.3- and 2.3-fold, respectively, whereas faecal bile acid output was 3.3-fold lower. FM-VP4 also increased the intestinal absorption of orally administered [3H]taurocholic acid to small intestine in vivo. Inhibition of intestinal cholesterol absorption by FM-VP4 was not mediated via transcriptional increases in intestine liver X receptor (LXR)-alpha, adenosine triphosphate-binding cassette transporter (ABC)-A1, ABCG5/G8 nor to decreases in intestinal Niemann-Pick C1-like 1 (NPC1L1) expression. In contrast, FM-VP4 up-regulated liver LXRalpha, ABCA1, ABCG5, scavenger receptor class BI (SR-BI) and hydroxymethylglutaryl coenzyme A reductase (HMGCoA-R) gene expression, whereas it down-regulated several farnesoid X receptor (FXR)-target genes such as cytochrome P450 family 7 subfamily A polypeptide 1 (CYP7A1) and Na+/taurocholate co-transporter polypeptide (NTCP). In conclusion, FM-VP4 reduced intestinal cholesterol absorption, plasma and liver cholesterol and affected bile acid homeostasis by inducing bile acid intestinal reabsorption and changed the liver expression of genes that play an essential role in cholesterol homeostasis. This is the first phytosterol or stanol that affects bile acid metabolism and lowers plasma cholesterol levels in normocholesterolaemic mice.

Hepatic cell-specific ATP-binding cassette (ABC) transporter profiling identifies putative novel candidates for lipid homeostasis in mice

Background ABC-transporters play an important role in lipid trafficking. Therefore, hepatic expression patterns of ABC-transporters involved in the regulation of cholesterol metabolism were evaluated. Methods and results RT-PCR analysis showed that the mRNA expression of 38 ABC-transporters detected in livers of C57Bl/6 mice varied greatly. Although most ABC-transporters were ubiquitously expressed, some members displayed very restricted expression patterns, e.g. ABCA6, A8, B1, B8, B10, B11, C3, D2, and G5/G8 were exclusively (>99%) expressed in parenchymal cells. Interestingly, another 13 ABC-transporters, including ABCA4, A5, A9, A13, B2, B9, C1, C5, D3, D4, F2, G1, and G4 were primarily expressed in Kupffer cells. Although Kupffer cells only contribute to 2.5% of the total liver protein, these 13 genes did contain 9–27% of the total liver expression. Western-type diet feeding (0.25% cholesterol, 15% fat) induced the expression of several primarily Kupffer cell expressed genes, including ABCA5, B9, D3, and D4 (2 to 3-fold higher), whereas the other ABC-transporters were not significantly changed. Conclusions Our findings underscore the importance of cellular localization for studying the regulation of key ABC-transporters in liver cholesterol homeostasis. Furthermore, several novel ABC-transporters, including ABCA5, B9, D3, and D4 were identified as putative novel candidates involved in liver macrophage cholesterol homeostasis.

Clofibrate causes an upregulation of PPAR-α target genes but does not alter expression of SREBP target genes in liver and adipose tissue of pigs

This study investigated the effect of clofibrate treatment on expression of target genes of peroxisome proliferator-activated receptor (PPAR)-alpha and various genes of the lipid metabolism in liver and adipose tissue of pigs. An experiment with 18 pigs was performed in which pigs were fed either a control diet or the same diet supplemented with 5 g clofibrate/kg for 28 days. Pigs treated with clofibrate had heavier livers, moderately increased mRNA concentrations of various PPAR-alpha target genes in liver and adipose tissue, a higher concentration of 3-hydroxybutyrate, and markedly lower concentrations of triglycerides and cholesterol in plasma and lipoproteins than control pigs (P < 0.05). mRNA concentrations of sterol regulatory element-binding proteins (SREBP)-1 and -2, insulin-induced genes (Insig)-1 and Insig-2, and the SREBP target genes acetyl-CoA carboxylase, 3-methyl-3-hydroxyglutaryl-CoA reductase, and low-density lipoprotein receptor in liver and adipose tissue and mRNA concentrations of apolipoproteins A-I, A-II, and C-III in the liver were not different between both groups of pigs. In conclusion, this study shows that clofibrate treatment activates PPAR-alpha in liver and adipose tissue and has a strong hypotriglyceridemic and hypocholesterolemic effect in pigs. The finding that mRNA concentrations of some proteins responsible for the hypolipidemic action of fibrates in humans were not altered suggests that there were certain differences in the mode of action compared with humans. It is also shown that PPAR-alpha activation by clofibrate does not affect hepatic expression of SREBP target genes involved in synthesis of triglycerides and cholesterol homeostasis in liver and adipose tissue of pigs.

Liver X Receptor Agonists Suppress Vascular Smooth Muscle Cell Proliferation and Inhibit Neointima Formation in Balloon-Injured Rat Carotid Arteries

The liver X receptors alpha and beta (LXRalpha and LXRbeta) are important regulators of cholesterol homeostasis in liver and macrophages. Synthetic LXR ligands prevent the development of atherosclerosis in murine models; however, the potential functional relevance of LXRs in vascular smooth muscle cells (VSMCs) has not been investigated. In the present study, we demonstrate that LXRs are expressed and functional in primary human coronary artery VSMCs (CASMCs). LXR ligands inhibited mitogen-induced VSMC proliferation and G1-->S phase progression of the cell cycle. Inhibition of G1 exit by LXR ligands was accompanied by a dose-dependent inhibition of retinoblastoma protein (Rb) phosphorylation, which functions as the key switch for G1-->S cell cycle progression. LXR ligands suppressed mitogen-induced degradation of the cyclin-dependent kinase inhibitor p27Kip1, attenuated cyclin D1 and cyclin A expression, and inhibited the expression of S phase-regulatory minichromosome maintenance protein 6. Stabilization of p27kip1 by LXR ligands was mediated by supressing the transcriptional activation of the S phase kinase-associated protein 2 (Skp2), an F-box protein that targets p27Kip1 for degradation. Inhibition of Rb phosphorylation and G1-->S cell cycle progression by LXR ligands was reversed in VSMCs overexpressing Skp2, indicating that Skp2 as an upstream regulator of p27Kip1 degradation plays a central role in LXR ligand-mediated inhibition of VSMC proliferation. Furthermore, adenovirus-mediated overexpression of the S phase transcription factor E2F, which is released after Rb phosphorylation, reversed the inhibitory effect of LXR ligands on VSMC proliferation and S phase gene expression, suggesting that the primary mechanisms by which LXR ligands inhibit VSMC proliferation occur upstream of Rb phosphorylation. Finally, neointima formation in a model of rat carotid artery balloon injury was significantly attenuated after treatment with the LXR ligand T1317 compared with vehicle-treated animals. These data demonstrate that LXR ligands inhibit VSMC proliferation and neointima formation after balloon injury and suggest that LXR ligands may constitute a novel therapy for proliferative vascular diseases. The full text of this article is available online at http://circres.ahajournals.org.

Effects of peroxisome proliferator-activated receptor α activation on pathways contributing to cholesterol homeostasis in rat hepatocytes

Peroxisome proliferator-activated receptor alpha (PPARα) activation by fibrates controls expression of several genes involved in hepatic cholesterol metabolism. Other genes could be indirectly controlled in response to changes in cellular cholesterol availability. To further understand how fibrates may affect cholesterol synthesis, we investigated in parallel the changes in the metabolic pathways contributing to cholesterol homeostasis in liver. Ciprofibrate increased HMG-CoA reductase and FPP synthase mRNA levels in rat hepatocytes, together with cholesterogenesis from [ 14C] acetate and [ 3H] mevalonate. The up-regulation observed in fenofibrate- and WY-14,643-treated mice was abolished in PPARα-null mice, showing an essential role of PPARα. Among the three sterol regulatory element-binding protein (SREBP) mRNA species, only SREBP-1c level was significantly increased. In ciprofibrate-treated hepatocytes, cholesterol efflux was decreased, in parallel with cholesteryl ester storage and bile acids synthesis. As expected, AOX expression was strongly induced, supporting evidence of the peroxisome proliferation. Taken together, these results show that fibrates can cause cholesterol depletion in hepatocytes, possibly in part as a consequence of an important requirement of cholesterol for peroxisome proliferation, and increase cholesterogenesis by a compensatory phenomenon afterwards. Such cholesterogenesis regulation could occur in vivo, in species responsive to the peroxisome proliferative effect of PPARα ligands.

Modulation of endoplasmic reticulum-bound cholesterol regulatory enzymes by iron/ascorbate-mediated lipid peroxidation

Mammalian sterol regulatory enzymes are integral membrane proteins of the endoplasmic reticulum. They play a critical role in liver cholesterol homeostasis and the maintenance of overall cholesterol balance in different species. Because lipid peroxidation has been implicated in hepatic dysfunction and atherosclerosis, we hypothesized that its occurrence could alter the composition and properties of the bilayer lipid environment, and thereby affect the functions of these membrane proteins. Preincubation of rat liver microsomes with iron (Fe)/ascorbate (50 μM/200 μM), known to induce peroxidation, resulted in a significant inhibition of (i) the rate-limiting enzyme in cholesterol biosynthesis, HMG-CoA reductase (46%, p < .01), (ii) the crucial enzyme controlling the conversion of cholesterol in bile acids, cholesterol 7α-hydroxylase (48%, p < .001), and (iii)the central enzyme for cholesterol esterification: Acyl-CoA:cholesterol acyltransferase (ACAT, 80%, p < .0001). The disturbances of these key enzymes took place concomitantly with the high production of malondialdehyde (350%, p < .007) and the loss of polyunsaturated fatty acids (36.19 ± 1.06% vs. 44.24 ± 0.41 in controls, p < .0008). While α-tocopherol simultaneously neutralized lipid peroxidation, preserved microsomal fatty acid status, and restored ACAT activity, it was not effective in preventing Fe/ascorbate-induced inactivation of both HMG-CoA reductase (44%, p < .01) and cholesterol 7α-hydroxylase (71%, p < .0001). These results indicate that Fe/ascorbate alters the activity of the rate-determining steps in liver cholesterol metabolism, either directly or via lipid peroxidation, capable of modifying their membrane environment. The present data also suggest that the three regulatory enzymes respond differently when exposed to Fe/ascorbate or antioxidants, which may be due to dissimilar mechanisms.

Reduced hepatic sterol carrier protein-2 expression in the streptozotocin treated diabetic rat

While a strong relationship between the hypercholesterolemia of diabetes and premature atherosclerosis is established, the etiology for the elevation in serum cholesterol in this disease is unknown. To determine whether diabetic hypercholesterolemia may be related to alterations in hepatic cholesterol transport capacity, sterol carrier protein-2 (SCP2) expression was examined in rats treated with streptozotocin (SZT). Furthermore, this study examined whether 17β-estradiol and insulin confer a protective effect on liver cholesterol homeostasis by maintaining hepatic SCP2 levels. SCP2 protein and mRNA expression were examined 13 days following SZT-induced diabetes onset and in diabetic rats treated with estradiol (1 cm silastic implant) or insulin (12 units/day). Data indicate that SCP2 protein levels were significantly reduced in the diabetic animals and that SCP2 protein expression in the liver was inversely related to the level of serum cholesterol in the diabetic animals. In contrast, SCP2 mRNA levels examined by slot blot, ribonuclease protection assay, and Northern blot analysis were significantly elevated. Both insulin and estradiol were able to enhance the expression of SCP2 protein in the liver following SZT treatment. The results of this investigation clearly indicate that hepatic SCP2 protein levels are significantly altered in the diabetic state suggesting that cholesterol transport capacity is reduced in the SZT-treated diabetic rat. The inverse relationship between serum cholesterol and hepatic SCP2 protein content suggests that the reduction in this protein may be a contributing factor in diabetic hypercholesterolemia.