EP4 emerges as a novel regulator of bile acid synthesis and its activation protects against hypercholesterolemia

Prostaglandin E receptor subtype 4 (EP4) knockout mice develops spontaneous hypercholesterolemia but the detailed mechanisms by which EP4 affects cholesterol homeostasis remains unexplored. We sought to determine the cause of hypercholesterolemia in EP4 knockout mice, focusing on the role of EP4 in regulating the synthesis and elimination of cholesterol. Deficiency of EP4 significantly decreased total bile acid levels in the liver by 26.2% and the fecal bile acid content by 27.6% as compared to wild type littermates, indicating that the absence of EP4 decreased hepatic bile acid synthesis and their subsequent excretion in stools. EP4 deficiency negatively regulate bile acid synthesis through repression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK)-mediated cholesterol 7α-hydroxylase (CYP7A1) expression and that the hypercholesterolemia in EP4 knockout mice is due to a defect in cholesterol conversion into bile acids. Deficiency of EP4 also increased de novo cholesterol synthesis and altered cholesterol fluxes in and out of the liver. Treating high fat diet-challenged mice with the pharmacological EP4 agonist, CAY10580 (200?μg/kg body weight/day i.p) for three weeks effectively prevented diet-induced hypercholesterolemia, enhanced endogenous bile acid synthesis and their fecal excretion. In summary, EP4 plays a critical role in maintaining cholesterol homeostasis by regulating the synthesis and elimination of bile acids. Activation of EP4 serves as an effective novel strategy to promote cholesterol disposal in the forms of bile acids in order to lower plasma cholesterol levels.     

Effects of dietary fat on cholesterol movement between tissues in CBA/J and C57BR/cdJ mice.

Differences in dietary fats cause differences in cholesterol metabolism in mice. CBA/J mice are resistant to diet-induced hypercholesterolemia and atherosclerosis; they adjust hepatic hydroxymethyl-glutaryl-CoA reductase activity (HMGR) to maintain homeostasis; C57BR/cdJ mice are susceptible, but young animals are thought to maintain homeostasis by changing fecal excretion of sterols. Compartmental modelling of movement of [4-14C]cholesterol was used to analyze movement of cholesterol between serum and liver, heart, and carcass in mice fed 40 en% fat, polyunsaturated to saturated fatty acid ratio (P/S) = 0.24 (US74) or 30 en% fat, P/S = 1 (MOD). Dietary effects were quite pronounced, while strain effects were more subdued. The C57/cdJ animals appear to regulate the overall cholesterol balance by reducing synthesis, as do the CBA/J animals, even though synthesis is not reduced to the same degree as in the CBA/J animals. Both diet and strain influence the whole-animal turnover rate, with slower turnover occurring for C57BR/cdJ animals and animals fed the US74 diet.     

Cholesterol gallstones in alloxan-diabetic mice

Normal and alloxan-diabetic male mice (Crj-ICR) were fed a diet containing 0.5% cholesterol for 5 and 10 weeks, and gallbladder bile was analyzed for cholesterol, phospholipids and bile acids, feces for sterols and bile acids, and plasma and liver for cholesterol, phospholipids, and triglycerides. Normal mice developed no gallstones but the diabetic mice developed cholesterol gallstones with an incidence of 70% by 5 weeks and 80% by 10 weeks after feeding of the cholesterol diet. Diabetic mice fed the ordinary diet also developed stones (23%) by 10 weeks. In the diabetic mice, the gallbladder was enlarged about threefold, and biliary lipid concentration, diet intake, and fecal excretion of sterols and bile acids increased but body weight decreased. Cholic acid and beta-muricholic acid comprised over 40% each of the total biliary bile acids in normal mice, but cholic acid increased to about 80% and beta-muricholic acid decreased to a few percent in the diabetic mice. Fecal excretion of bile acids increased after cholesterol feeding in both normal and diabetic mice, but the increased bile acid in the normal animals was beta-muricholic acid and that in the diabetic mice was deoxycholic acid. The mice that developed gallstones showed a marked increase in biliary cholesterol value and decreases in gallbladder bile and bile acid concentration, but no difference in biliary and fecal bile acid composition, bile acid synthesis, fecal sterols, or plasma and liver lipid levels. Cholesterol absorption was increased in the diabetic mice when examined by plasma 14C/3H ratio and fecal 14C-labeled sterol excretion after a single oral administration of [14C]cholesterol and a simultaneous intravenous injection of [3H]cholesterol. These data led to the conclusion that cholesterol gallstones developed in alloxan-diabetic mice fed excess cholesterol, due to the hyperphagia and the enhancement of cholesterol absorption caused by increases in the synthesis and secretion of cholic acid.     

Hepatocyte-specific lysosomal acid lipase deficiency protects mice from diet-induced obesity but promotes hepatic inflammation

Lysosomal acid lipase (LAL) hydrolyzes cholesteryl esters (CE) and triglycerides (TG) to generate fatty acids (FA) and cholesterol. LAL deficiency (LAL-D) in both humans and mice leads to hepatomegaly, hypercholesterolemia, and shortened life span. Despite its essential role in lysosomal neutral lipid catabolism, the cell type-specific contribution of LAL to disease progression is still elusive. To investigate the role of LAL in the liver in more detail and to exclude the contribution of LAL in macrophages, we generated hepatocyte-specific LAL-deficient mice (Liv-Lipa^−/−) and fed them either chow or high fat/high cholesterol diets (HF/HCD). Comparable to systemic LAL-D, Liv-Lipa^−/− mice were resistant to diet-induced obesity independent of food intake, movement, and energy expenditure. Reduced body weight gain was mainly due to reduced white adipose tissue depots. Furthermore, Liv-Lipa^−/− mice exhibited improved glucose clearance during glucose and insulin tolerance tests compared to control mice. Analysis of hepatic lipid content revealed a massive reduction of TG, whereas CE concentrations were markedly increased, leading to CE crystal formation in the livers of Liv-Lipa^−/− mice. Elevated plasma transaminase activities, increased pro-inflammatory cytokines and chemokines as well as hepatic macrophage infiltration indicated liver inflammation. Our data provide evidence that hepatocyte-specific LAL deficiency is sufficient to alter whole-body lipid and energy homeostasis in mice. We conclude that hepatic LAL plays a pivotal role by preventing liver damage and maintaining lipid and energy homeostasis, especially during high lipid availability.     

Alteration of biliary ursodeoxycholic acid in guinea pig during early stages of cholestyramine feeding

The effects of cholestyramine feeding on biliary ursodeoxycholic acid, fecal excretion of bile acids and neutral sterols on cholesterol 7α-hydroxylase and hepatic HMG-CoA reductase were examined in the guinea pig. In the bile there was a 57% decrease in the concentration of ursodeoxycholic acid while an increase was observed in the concentration of chenodeoxycholic acid. Cholestyramine feeding for ten days resulted in a decrease in plasma cholesterol levels and an increase in both hepatic HMG-CoA reductase and cholesterol 7α-hydroxylase activities. The fecal excretion of both bile acids and neutral sterols was significantly increased.     

Hepatic lysosomal acid lipase drives the autophagy-lysosomal response and alleviates cholesterol metabolic disorder in ApoE deficient mice

Lysosomal acid lipase (LAL)-dependent lipolysis degrades cholesteryl ester (CE) and triglyceride in the lysosome. LAL deficiency in human and mice leads to hypercholesterolemia, hepatic CE deposition, and atherosclerosis. Despite its hepatocyte-specific deficiency leads to CE accumulation, the regulation of LAL in cholesterol metabolic disease remains elusive. For the in vitro study, the target gene Lipa was transfected with recombinant shRNA or lentiviral vector in Hepa1-6 cells. It was found that LAL silencing in cells affected lysosomal function by reducing LAL activity and proteolytic activity, and altered the expression of genes related to cholesterol metabolism and autophagy, leading to cholesterol accumulation; whereas LAL overexpression improved the above effects. To explore the impacts of hepatic LAL on cholesterol metabolic disease in vivo, apolipoprotein E deficient (ApoE^−/−) mice were intravenously injected with lentivirus to achieve hepatic LAL overexpression and fed a Western diet for 16 weeks. The results showed that hepatic LAL overexpression significantly reduced plasma lipid levels, alleviated inflammation and oxidative status in plasma and liver, and attenuated hepatic steatosis and fibrosis in ApoE^−/− mice. Mechanically, hepatic LAL promoted cholesterol transport and biliary excretion by increasing liver X receptor alpha (LXRα) and its downstream genes, and modulated the compliance of the autophagy-lysosomal pathway. Our data provide the original evidence of the validity of hepatic LAL in controlling cholesterol metabolism and liver homeostasis, suggesting that targeting hepatic LAL may provide a promising approach to rescue cholesterol metabolic disorders, such as hypercholesterolemia and liver disease.     

Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis

Mice lacking the nuclear bile acid receptor FXR/BAR developed normally and were outwardly identical to wild-type littermates. FXR/BAR null mice were distinguished from wild-type mice by elevated serum bile acid, cholesterol, and triglycerides, increased hepatic cholesterol and triglycerides, and a proatherogenic serum lipoprotein profile. FXR/BAR null mice also had reduced bile acid pools and reduced fecal bile acid excretion due to decreased expression of the major hepatic canalicular bile acid transport protein. Bile acid repression and induction of cholesterol 7alpha-hydroxylase and the ileal bile acid binding protein, respectively, did not occur in FXR/BAR null mice, establishing the regulatory role of FXR/BAR for the expression of these genes in vivo. These data demonstrate that FXR/BAR is critical for bile acid and lipid homeostasis by virtue of its role as an intracellular bile acid sensor.     

Lactobacillus plantarum LRCC 5273 isolated from Kimchi ameliorates diet-induced hypercholesterolemia in C57BL/6 mice

ABSTRACT

This study was designed to select potent cholesterol-lowering probiotic strains on HepG2 cell and investigate the effect of selected strain, Lactobacillus plantarum LRCC 5273 and LRCC 5279 in hypercholesterolemic mice. In the results, LP5273 group showed significantly reduced total and LDL cholesterol compared to HCD group. In addition to significantly up-regulated hepatic mRNA expression of LXR-α and CYP7A1, intestinal LXR-α and ABCG5 were significantly up-regulated in LP5273 group. With activation of hepatic and intestinal LXR-α and its target genes, fecal cholesterol and bile acid excretion were increased in LP5273 fed mice. These results suggest that LP5273 ameliorates hypercholesterolemia in mice through the activation of hepatic and intestinal LXR-α, resulting in enhancement of fecal cholesterol and bile acids excretion in the small intestine. The results of present study suggest mechanistic evidences for hypocholesterolemic effects of L. plantarum spp., and may contribute to future researches for prevention of hypercholesterolemia and cardiovascular disease.     

Hypocholesterolemic effects of fatty acid bile acid conjugates (FABACs) in mice

Fatty acid bile acid conjugates (FABACs) prevent and dissolve cholesterol gallstones and prevent diet induced fatty liver, in mice. The present studies aimed to test their hypocholesterolemic effects in mice. Gallstone susceptible (C57L/J) mice, on high fat (HFD) or regular diet (RD), were treated with the conjugate of cholic acid with arachidic acid (FABAC; Aramchol). FABAC reduced the elevated plasma cholesterol levels induced by the HFD. In C57L/J mice, FABAC reduced plasma cholesterol by 50% (p < 0.001). In mice fed HFD, hepatic cholesterol synthesis was reduced, whereas CYP7A1 activity and expression were increased by FABAC. The ratio of fecal bile acids/neutral sterols was increased, as was the total fecal sterol excretion. In conclusion, FABACs markedly reduce elevated plasma cholesterol in mice by reducing the hepatic synthesis of cholesterol, in conjunction with an increase of its catabolism and excretion from the body.     

Disruption of the murine protein kinase Cbeta gene promotes gallstone formation and alters biliary lipid and hepatic cholesterol metabolism

The protein kinase C (PKC) family of Ca(2+) and/or lipid-activated serine-threonine protein kinases is implicated in the pathogenesis of obesity and insulin resistance. We recently reported that protein kinase Cβ (PKCβ), a calcium-, diacylglycerol-, and phospholipid-dependent kinase, is critical for maintaining whole body triglyceride homeostasis. We now report that PKCβ deficiency has profound effects on murine hepatic cholesterol metabolism, including hypersensitivity to diet-induced gallstone formation. The incidence of gallstones increased from 9% in control mice to 95% in PKCβ(-/-) mice. Gallstone formation in the mutant mice was accompanied by hyposecretion of bile acids with no alteration in fecal bile acid excretion, increased biliary cholesterol saturation and hydrophobicity indices, as well as hepatic p42/44(MAPK) activation, all of which enhance susceptibility to gallstone formation. Lithogenic diet-fed PKCβ(-/-) mice also displayed decreased expression of hepatic cholesterol-7α-hydroxylase (CYP7A1) and sterol 12α-hydroxylase (CYP8b1). Finally, feeding a modified lithogenic diet supplemented with milk fat, instead of cocoa butter, both increased the severity of and shortened the interval for gallstone formation in PKCβ(-/-) mice and was associated with dramatic increases in cholesterol saturation and hydrophobicity indices. Taken together, the findings reveal a hitherto unrecognized role of PKCβ in fine tuning diet-induced cholesterol and bile acid homeostasis, thus identifying PKCβ as a major physiological regulator of both triglyceride and cholesterol homeostasis.     

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3β-hydroxy-5-cholesten-(25R)26-oic acid (3βHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3βHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3βHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1^−/− mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3βHCA levels were maintained at basal levels in ND-fed Cyp7b1^−/− mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1^−/− mice developed insulin resistance (IR) with subsequent 26HC/3βHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1^−/− mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3βHCA accumulation. The results suggest 26HC/3βHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3βHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.     

Inhibition of ileal bile acid transport and reduced atherosclerosis in apoE-/- mice by SC-435

Blocking intestinal bile acid absorption by inhibiting the apical sodium codependent bile acid transporter (ASBT) is a target for increasing hepatic bile acid synthesis and reducing plasma LDL cholesterol. SC-435 was identified as a potent inhibitor of ASBT (IC50 = 1.5 nM) in cells transfected with the human ASBT gene. Dietary administration of 3 mg/kg to 30 mg/kg SC-435 to apolipoprotein E-/- (apoE-/-) mice increased fecal bile acid excretion by >2.5-fold. In vivo inhibition of ASBT also resulted in significant increases of hepatic mRNA levels for cholesterol 7alpha-hydroxylase and HMG-CoA reductase. Administration of 10 mg/kg SC-435 for 12 weeks to apoE-/- mice lowered serum total cholesterol by 35% and reduced aortic root lesion area by 65%. Treatment of apoE-/- mice also resulted in decreased expression of ileal bile acid binding protein and hepatic nuclear hormone receptor small heterodimer partner, direct target genes of the farnesoid X receptor (FXR), suggesting a possible role of FXR in SC-435 modulation of cholesterol homeostasis. In dogs, SC-435 treatment reduced serum total cholesterol levels by 相似文献   

Increased Hepatic Lipogenesis Elevates Liver Cholesterol Content

Cardiovascular diseases (CVDs) are the most common cause of death in patients with nonalcoholic fatty liver disease (NAFLD) and dyslipidemia is considered at least partially responsible for the increased CVD risk in NAFLD patients. The aim of the present study is to understand how hepatic de novo lipogenesis influences hepatic cholesterol content as well as its effects on the plasma lipid levels. Hepatic lipogenesis was induced in mice by feeding a fat-free/high-sucrose (FF/HS) diet and the metabolic pathways associated with cholesterol were then analyzed. Both liver triglyceride and cholesterol contents were significantly increased in mice fed an FF/HS diet. Activation of fatty acid synthesis driven by the activation of sterol regulatory element binding protein (SREBP)-1c resulted in the increased liver triglycerides. The augmented cholesterol content in the liver could not be explained by an increased cholesterol synthesis, which was decreased by the FF/HS diet. HMG-CoA reductase protein level was decreased in mice fed an FF/HS diet. We found that the liver retained more cholesterol through a reduced excretion of bile acids, a reduced fecal cholesterol excretion, and an increased cholesterol uptake from plasma lipoproteins. Very low-density lipoprotein-triglyceride and -cholesterol secretion were increased in mice fed an FF/HS diet, which led to hypertriglyceridemia and hypercholesterolemia in Ldlr^-/- mice, a model that exhibits a more human like lipoprotein profile. These findings suggest that dietary cholesterol intake and cholesterol synthesis rates cannot only explain the hypercholesterolemia associated with NAFLD, and that the control of fatty acid synthesis should be considered for the management of dyslipidemia.     

Medium-Chain Fatty Acids Enhanced the Excretion of Fecal Cholesterol and Cholic Acid in C57BL/6J Mice Fed a Cholesterol-Rich Diet

The objective of the present study was to investigate the cholesterol-reducing effect of medium-chain fatty acids (MCFAs) completed by elevated excretion of fecal neutral steroids and/or bile acids. Blood and liver lipid profiles, fecal neutral steroids, bile acids, and mRNA and protein expression of the genes relevant to cholesterol homeostasis were measured and analyzed in C57BL/6J mice fed a cholesterol-rich diet with 2% caprylic acid or capric acid for 12 weeks. Blood total cholesterol and low-density lipoprotein cholesterol (LDL-c) levels were reduced significantly as compared to diet with palmitic acid or stearic acid. Caprylic acid promoted the excretion of fecal neutral steroids, especially cholesterol. The excretion of fecal bile acids, mainly in the form of cholic acid was enhanced and accompanied by elevated expression of mRNA and the protein of hepatic cholesterol 7α-hydroxylase (CYP7A1). These results indicate that MCFAs can reduce blood cholesterol by promoting the excretion of fecal cholesterol and cholic acid.     

Deletion of PARP-2 induces hepatic cholesterol accumulation and decrease in HDL levels

Poly(ADP-ribose) polymerase-2 (PARP-2) is acknowledged as a DNA repair enzyme. However, recent investigations have attributed unique roles to PARP-2 in metabolic regulation in the liver. We assessed changes in hepatic lipid homeostasis upon the deletion of PARP-2 and found that cholesterol levels were higher in PARP-2^−/− mice as compared to wild-type littermates. To uncover the molecular background, we analyzed changes in steady-state mRNA levels upon the knockdown of PARP-2 in HepG2 cells and in murine liver that revealed higher expression of sterol-regulatory element binding protein (SREBP)-1 dependent genes. We demonstrated that PARP-2 is a suppressor of the SREBP1 promoter, and the suppression of the SREBP1 gene depends on the enzymatic activation of PARP-2. Consequently, the knockdown of PARP-2 enhances SREBP1 expression that in turn induces the genes driven by SREBP1 culminating in higher hepatic cholesterol content. We did not detect hypercholesterolemia, higher fecal cholesterol content or increase in serum LDL, although serum HDL levels decreased in the PARP-2^−/− mice. In cells and mice where PARP-2 was deleted we observed decreased ABCA1 mRNA and protein expression that is probably linked to lower HDL levels. In our current study we show that PARP-2 impacts on hepatic and systemic cholesterol homeostasis. Furthermore, the depletion of PARP-2 leads to lower HDL levels which represent a risk factor to cardiovascular diseases.     

microRNA-30c reduces plasma cholesterol in homozygous familial hypercholesterolemic and type 2 diabetic mouse models

High plasma cholesterol levels are found in several metabolic disorders and their reductions are advocated to reduce the risk of atherosclerosis. A way to lower plasma lipids is to curtail lipoprotein production; however, this is associated with steatosis. We previously showed that microRNA (miR)-30c lowers diet-induced hypercholesterolemia and atherosclerosis in C57BL/6J and Apoe^−/− mice. Here, we tested the effect of miR-30c on plasma lipids, transaminases, and hepatic lipids in different mouse models. Hepatic delivery of miR-30c to chow-fed leptin-deficient (ob/ob) and leptin receptor-deficient (db/db) hypercholesterolemic and hyperglycemic mice reduced cholesterol in total plasma and VLDL/LDL by ∼28% and ∼25%, respectively, without affecting triglyceride and glucose levels. And these mice had lower plasma transaminases and creatine kinase activities than controls. Moreover, miR-30c significantly lowered plasma cholesterol and atherosclerosis in Western diet-fed Ldlr^−/− mice with no effect on plasma triglyceride, glucose, and transaminases. In these studies, hepatic lipids were similar in control and miR-30c-injected mice. Mechanistic studies showed that miR-30c reduced hepatic microsomal triglyceride transfer protein activity and lipid synthesis. Thus miR-30c reduced plasma cholesterol in several diet-induced and diabetic hypercholesterolemic mice. We speculate that miR-30c may be beneficial in lowering plasma cholesterol in different metabolic disorders independent of the origin of hypercholesterolemia.     

A Grape Seed Procyanidin Extract Ameliorates Fructose-Induced Hypertriglyceridemia in Rats via Enhanced Fecal Bile Acid and Cholesterol Excretion and Inhibition of Hepatic Lipogenesis

The objective of this study was to determine whether a grape seed procyanidin extract (GSPE) exerts a triglyceride-lowering effect in a hyperlipidemic state using the fructose-fed rat model and to elucidate the underlying molecular mechanisms. Rats were fed either a starch control diet or a diet containing 65% fructose for 8 weeks to induce hypertriglyceridemia. During the 9^th week of the study, rats were maintained on their respective diet and administered vehicle or GSPE via oral gavage for 7 days. Fructose increased serum triglyceride levels by 171% after 9 weeks, compared to control, while GSPE administration attenuated this effect, resulting in a 41% decrease. GSPE inhibited hepatic lipogenesis via down-regulation of sterol regulatory element binding protein 1c and stearoyl-CoA desaturase 1 in the fructose-fed animals. GSPE increased fecal bile acid and total lipid excretion, decreased serum bile acid levels and increased the expression of genes involved in cholesterol synthesis. However, bile acid biosynthetic gene expression was not increased in the presence of GSPE and fructose. Serum cholesterol levels remained constant, while hepatic cholesterol levels decreased. GSPE did not modulate expression of genes responsible for esterification or biliary export of the newly synthesized cholesterol, but did increase fecal cholesterol excretion, suggesting that in the presence of GSPE and fructose, the liver may secrete more free cholesterol into the plasma which may then be shunted to the proximal small intestine for direct basolateral to apical secretion and subsequent fecal excretion. Our results demonstrate that GSPE effectively lowers serum triglyceride levels in fructose-fed rats after one week administration. This study provides novel insight into the mechanistic actions of GSPE in treating hypertriglyceridemia and demonstrates that it targets hepatic de novo lipogenesis, bile acid homeostasis and non-biliary cholesterol excretion as important mechanisms for reducing hypertriglyceridemia and hepatic lipid accumulation in the presence of fructose.     

l-Alanine-α-Ketobutyrate Aminotransferase of Pseudomonas sp.

Modified fungal product 4-O-methylascochlorin (MAC) is an experimental agent affecting lipid and carbohydrate metabolism in mammals. The hypocholesterolemic properties of MAC were studied using rats fed on a standard laboratory diet. Because of the insolubility in water, reproducibility of the hypocholesterolemic activity had usually been poor for rats fed ad libitum. The difficulty was overcome by controlled reverse-phase feeding; MAC significantly lowered serum total cholesterol (s-TC) in rats only when given by gastric intubation soon after diet intake.

MAC increased fecal excretion of neutral and acidic sterols and also increased biliary flow accompanying increments in biliary cholesterol, bile acids and phospholipids. A much larger increase in neutral sterols was characteristic for MAC. However, intestinal absorption of cholesterol and cholic acid was unaffected by MAC. Three mechanisms therefore seemed to be working in hypocholesterolemic activity: (a) withdrawal of hepatic cholesterol into bile, (b) a larger fecal loss of sterols following increment of biliary sterols and (c) enhanced bile acid synthesis compensating the larger fecal loss. A negative sterol balance often leads to an increase in hepatic cholesterogenesis. However, cholesterogenesis, as judged from incorporation of the precursors, was unchanged by MAC.     

Identification of Novel Pathways That Control Farnesoid X Receptor-mediated Hypocholesterolemia

Farnesoid X receptor (FXR) plays important regulatory roles in bile acid, lipoprotein, and glucose homeostasis. Here, we have utilized Fxr^−/− mice and mice deficient in scavenger receptor class B type I (SR-BI), together with an FXR-specific agonist and adenovirus expressing hepatocyte nuclear factor 4α or constitutively active FXR, to identify the mechanisms by which activation of FXR results in hypocholesterolemia. We identify a novel pathway linking FXR to changes in hepatic p-JNK, hepatocyte nuclear factor 4α, and finally SR-BI. Importantly, we demonstrate that the FXR-dependent increase in SR-BI results in both hypocholesterolemia and an increase in reverse cholesterol transport, a process involving the transport of cholesterol from peripheral macrophages to the liver for excretion into the feces. In addition, we demonstrate that FXR activation also induces an SR-BI-independent increase in reverse cholesterol transport and reduces intestinal cholesterol absorption. Together, these data indicate that FXR is a promising therapeutic target for treatment of hypercholesterolemia and coronary heart disease.     

Hypercholesterolemia and changes in lipid and bile acid metabolism in male and female cyp7A1-deficient mice

Cholesterol 7alpha-hydroxylase, a rate-limiting enzyme for bile acid synthesis, has been implicated in genetic susceptibility to atherosclerosis. The gene, CYP7A1, encoding a protein with this activity, is expressed normally only in hepatocytes and is highly regulated. Our cyp7A1 gene knockout mouse colony, as young adults on a chow diet, is hypercholesterolemic. These mice were characterized extensively to understand how cyp7A1 affects lipid and bile acid homeostasis in different tissue compartments and whether gender plays a modifying role. Both male and female cyp7A1-deficient mice had decreased hepatic LDL receptors, unchanged hepatic cholesterol synthesis, increased intestinal cholesterol synthesis and bile acid transporters, and decreased fecal bile acids but increased fecal sterols. In females, cyp7A1 deficiency also caused changes in hepatic fatty acid metabolism, decreased hepatic canalicular bile acid transporter, Bsep, and gallbladder bile composition altered to a lithogenic profile. Taken together, the data suggest that cyp7A1 deficiency results in a proatherogenic phenotype in both genders and leads to a prolithogenic phenotype in females.