Increased Bile Acid Synthesis Research Articles

Antibody-Mediated Inhibition of Fibroblast Growth Factor 19 Results in Increased Bile Acids Synthesis and Ileal Malabsorption of Bile Acids in Cynomolgus Monkeys

Fibroblast growth factor 19 (FGF19) represses cholesterol 7α-hydroxylase (Cyp7α1) and inhibits bile acid synthesis in vitro and in vivo. Previous studies have shown that anti-FGF19 antibody treatment reduces growth of colon tumor xenografts and prevents hepatocellular carcinomas in FGF19 transgenic mice and thus may be a useful cancer target. In a repeat dose safety study in cynomolgus monkeys, anti-FGF19 treatment (3-100 mg/kg) demonstrated dose-related liver toxicity accompanied by severe diarrhea and low food consumption. The mechanism of anti-FGF19 toxicity was investigated using in vitro and in vivo approaches. Our results show that anti-FGF19 antibody had no direct cytotoxic effect on monkey hepatocytes. Anti-FGF19 increased Cyp7α1, as expected, but also increased bile acid efflux transporter gene (bile salt export pump, multidrug resistant protein 2 [MRP2], and MRP3) expression and reduced sodium taurocholate cotransporting polypeptide and organic anion transporter 2 expression in liver tissues from treated monkeys and in primary hepatocytes. In addition, anti-FGF19 treatment increased solute transporter gene (ileal bile acid-binding protein, organic solute transporter α [OST-α], and OST-β) expression in ileal tissues from treated monkeys but not in Caco-2 cells. However, deoxycholic acid (a secondary bile acid) increased expression of FGF19 and these solute transporter genes in Caco-2 cells. Gas chromatography-mass spectrometry analysis of monkey feces showed an increase in total bile acids and cholic acid derivatives. These findings suggest that high doses of anti-FGF19 increase Cyp7α1 expression and bile acid synthesis and alter the expression of bile transporters in the liver resulting in enhanced bile acid efflux and reduced uptake. Increased bile acids alter expression of solute transporters in the ileum causing diarrhea and the enhanced enterohepatic recirculation of bile acids leading to liver toxicity.

Subcellular localization and regulation of StarD4 protein in macrophages and fibroblasts

StarD4 is a member of the StarD4 subfamily of START domain proteins with a characteristic lipid binding pocket specific for cholesterol. The objective of this study was to define StarD4 subcellular localization, regulation, and function. Immunobloting showed that StarD4 is highly expressed in the mouse fibroblast cell line 3T3-L1, in human THP-1 macrophages, Kupffer cells (liver macrophages), and hepatocytes. In 3T3-L1 cells and THP-1 macrophages, StarD4 protein appeared localized to the cytoplasm and the endoplasmic reticulum (ER). More specifically, in THP-1 macrophages StarD4 co-localized to areas of the ER enriched in Acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), and was closely associated with budding lipid droplets. The addition of purified StarD4 recombinant protein to an in vitro assay increased ACAT activity 2-fold, indicating that StarD4 serves as a rate-limiting step in cholesteryl ester formation by delivering cholesterol to ACAT-1-enriched ER. In addition, StarD4 protein was found to be highly regulated and to redistribute in response to sterol levels. In summary, these observations, together with our previous findings demonstrating the ability of increased StarD4 expression to increase bile acid synthesis and cholesteryl ester formation, provide strong evidence for StarD4 as a highly regulated, non-vesicular, directional, intracellular transporter of cholesterol which plays a key role in the maintenance of intracellular cholesterol homeostasis.

A Randomized Placebo-Controlled Phase IIb Trial of A3309, A Bile Acid Transporter Inhibitor, for Chronic Idiopathic Constipation

OBJECTIVES:A3309 is a minimally absorbed ileal bile acid (BA) transporter (IBAT) inhibitor. We conducted an 8-week, multicenter, randomized, double-blind, placebo-controlled, parallel group, phase IIb study, which evaluated A3309 in patients with chronic idiopathic constipation (CIC).METHODS:Patients with CIC (modified Rome III criteria and <3 complete (CSBM) spontaneous bowel movements (SBMs)/week during the 2-week baseline) were randomized to 5, 10, or 15 mg A3309 or placebo once daily. The primary end point was change in SBM number during week 1 compared with baseline. Other bowel and abdominal symptoms were assessed as secondary end points. Serum 7αC4 and lipids were evaluated as biomarkers of BA synthesis/loss.RESULTS:In all, 190 patients (mean 48 years, 90% female) were randomized. Mean increase (95% confidence interval) in SBM for week 1 were 1.7 (0.7–2.8) for placebo vs. 2.5 (1.5–3.5), 4.0 (2.9–5.0), and 5.4 (4.4–6.4) for 5 mg, 10 mg (P<0.002), and 15 mg (P<0.001) A3309, respectively. Increased stool frequency was maintained over 8 weeks. Time to first SBM and CSBM were significantly reduced in the 10- and 15-mg A3309 groups compared with placebo. Straining and bloating decreased with A3309 compared with placebo (P<0.05). Increased 7αC4 and reduced low-density lipoprotein cholesterol with A3309 suggested increased BA synthesis and BA loss. The most common adverse events (AEs) were abdominal pain and diarrhea, which occurred most commonly in the 15-mg A3309 group. No drug-related serious AEs were observed.CONCLUSIONS:A3309 increased stool frequency and improved constipation-related symptoms in CIC; effects were maintained over 8 weeks of treatment.

Randomised clinical trial: the ileal bile acid transporter inhibitor A3309 vs. placebo in patients with chronic idiopathic constipation - a double-blind study

One half of patients with constipation are not satisfied with available therapies, hence there is a need for more effective and well-tolerated drugs. To evaluate the effects of a specific inhibitor of the Ileal Bile Acid Transporter (IBAT; syn apical sodium-dependent bile acid transporter; ASBT) in patients with chronic idiopathic constipation (CIC) with focus on safety, colonic transit and efficacy signals. This was a single-centre, prospective, randomised, double-blind, placebo-controlled study with a dose-escalating design in patients with CIC. In addition to evaluation of conventional safety and tolerability parameters, (i) colonic transit time (CTT) was measured using radio-opaque markers, (ii) metabolic parameters [lipid profile, C4 (7α-hydroxy-4-cholesten-3-one) and FGF19 (Fibroblast Growth Factor 19)] were evaluated, and (iii) constipation parameters, such as changes in stool frequency and consistency, were analysed. Thirty patients were randomised into five dose-levels (range: 0.1-10 mg/day) or to placebo. All patients completed a 14-day treatment period, and the safety/tolerability analysis was favourable. A3309, present in picomolar concentrations in plasma, induced up to a three-fold increase in bile acid synthesis (C4) and a reduction of plasma FGF19, as well as reduction in total and LDL cholesterol. CTT was reduced in the highest dose groups; the main acceleration was identified in the left colon. Efficacy parameters showed trends for increased number of spontaneous bowel movements and improved stool consistency. Ileal Bile Acid Transporter inhibition is a novel mechanism for treatment of patients with chronic idiopathic constipation and has additional benefits of improving metabolic parameters (EudraCT 2008-003255-72).

Flaxseed but Not Flaxseed Oil Prevented the Rise in Serum Cholesterol Due to Ovariectomy in the Golden Syrian Hamsters

This study was designed to investigate whether flaxseed oil (FO) exerts hypocholesterolemic effects similar to ground whole flaxseed (WF) and to gain insight into its hypocholesterolemic mechanism. Forty-eight 6-month-old female Golden Syrian hamsters were either sham-operated (Sham) or ovariectomized (Ovx) and randomly assigned to one of four treatment groups (n = 12/group) for 90 days: Sham, Ovx, Ovx+WF, or Ovx+FO. Hamsters in the Sham and Ovx groups were fed a semipurified diet (control), whereas Ovx+WF and Ovx+FO received the same basic diet supplemented with either WF (15% wt/wt) or FO (amount equivalent to the oil contribution of WF). Ovariectomy significantly (P < .05) increased serum total concentrations by approximately 15%. WF, but not FO, prevented (P < .05) the ovariectomy-induced increase in serum total cholesterol concentration (12% and 4% reduction by WF and FO, respectively). Hamsters fed FO or WF had high-density lipoprotein (HDL)-cholesterol concentrations similar to those of the Ovx hamsters receiving the control diet. Non-HDL-cholesterol concentrations were lowest in the WF group, albeit not statistically different from the other treatment groups. There were no significant differences among groups in serum triglyceride concentration and liver lipids. Both WF and FO more than doubled the hepatic protein levels of 7α-hydroxylase in comparison to the Ovx hamsters receiving the control diet (P < .05). Our findings suggest that increased bile acid synthesis is one of the major cholesterol-lowering mechanisms of flaxseed and that other flaxseed components, aside from its oil, contribute to its hypocholesterolemic property. The cholesterol-lowering effects of other components of flaxseed and their mechanisms of action need to be further explored.

Abolished synthesis of cholic acid reduces atherosclerotic development in apolipoprotein E knockout mice

To investigate the effects of abolished cholic acid (CA) synthesis in the ApoE knockout model [apolipoprotein E (apoE) KO],a double-knockout (DKO) mouse model was created by crossbreeding Cyp8b1 knockout mice (Cyp8b1 KO), unable to synthesize the primary bile acid CA, with apoE KO mice. After 5 months of cholesterol feeding, the development of atherosclerotic plaques in the proximal aorta was 50% less in the DKO mice compared with the apoE KO mice. This effect was associated with reduced intestinal cholesterol absorption, decreased levels of apoB-containing lipoproteins in the plasma, enhanced bile acid synthesis, reduced hepatic cholesteryl esters, and decreased hepatic activity of ACAT2. The upregulation of Cyp7a1 in DKO mice seemed primarily caused by reduced expression of the intestinal peptide FGF15. Treatment of DKO mice with the farnesoid X receptor (FXR) agonist GW4064 did not alter the intestinal cholesterol absorption, suggesting that the action of CA in this process is confined mainly to formation of intraluminal micelles and less to its ability to activate the nuclear receptor FXR. Inhibition of CA synthesis may offer a therapeutic strategy for the treatment of hyperlipidemic conditions that lead to atherosclerosis.

Defining primary bile acid diarrhea: making the diagnosis and recognizing the disorder

Chronic diarrhea due to bile acid malabsorption may be considered as contributing to the diagnosis when it results from secondary causes, such as ileal resection affecting the enterohepatic circulation. However, the primary form (also known as idiopathic bile acid malabsorption) is not well recognized as a common condition and patients are left undiagnosed. Primary bile acid diarrhea can be diagnosed by the nuclear medicine 75Se-homocholyltaurine (SeHCAT) test, although this is unavailable or underutilized in many settings. A systematic review suggests that approximately 30% of patients who would otherwise be diagnosed with diarrhea-predominant irritable bowel syndrome or functional diarrhea have abnormal SeHCAT retention. Serum 7α-hydroxy-4-cholesten-3-one can also be measured to show increased bile acid synthesis. The reasons for the lack of recognition of primary bile acid diarrhea are discussed, and these are compared with the other common cause of malabsorption, celiac disease. The lack of a clear pathophysiological mechanism has been a problem, but recent evidence suggests that impaired feedback control of hepatic bile acid synthesis by the ileal hormone FGF19 results in overproduction of bile acids. The identification of FGF19 as the central mechanism opens up new areas for development in the diagnosis and treatment of primary bile acid diarrhea.

Improved glycemic control with colesevelam treatment in patients with type 2 diabetes is not directly associated with changes in bile acid metabolism

Bile acids (BAs) are essential for fat absorption and appear to modulate glucose and energy metabolism. Colesevelam, a BA sequestrant, improves glycemic control in type 2 diabetes mellitus (T2DM). We aimed to characterize the alterations in BA metabolism associated with T2DM and colesevelam treatment and to establish whether metabolic consequences of T2DM and colesevelam are related to changes in BA metabolism. Male subjects with T2DM (n = 16) and controls (n = 12) were matched for age and body mass index. BA pool sizes and synthesis/input rates were determined before and after 2 and 8 weeks of colesevelam treatment. T2DM subjects had higher cholic acid (CA) synthesis rate, higher deoxycholic acid (DCA) input rate, and enlarged DCA pool size. Colesevelam resulted in a preferential increase in CA synthesis in both groups. CA pool size was increased whereas chenodeoxycholic acid and DCA pool sizes were decreased upon treatment. Fasting and postprandial fibroblast growth factor 19 (FGF19) levels did not differ between controls and diabetics, but were decreased by treatment in both groups. Colesevelam treatment reduced hemoglobin A1C by 0.7% (P < 0.01) in diabetics. Yet, no relationships between BA kinetic parameters and changes in glucose metabolism were found in T2DM or with colesevelam treatment. Our results reveal significant changes in BA metabolism in T2DM, particularly affecting CA and DCA. Colesevelam treatment reduced FGF19 signaling associated with increased BA synthesis, particularly of CA, and resulted in a more hydrophilic BA pool without altering total BA pool size. However, these changes could not be related to the improved glycemic control in T2DM.

S1302 Dose-Related Effects of Chenodeoxycholate on Gastrointestinal and Colonic Transit and Bowel Function in Female Patients With Constipation-Predominant Irritable Bowel Syndrome

Background: A3309 modulates the enterohepatic circulation of bile acids (BA) by inhibiting the intestinal bile acid transporter (IBAT). This will result in an increased concentration of BA in the colon. The synthesis of bile acids in the liver can be measured indirectly by plasma concentrations of the intermediate 7-α-hydroxy-4-cholesten-3-one (C4). Methods: In Vitro part: A3309 potency was tested in transfected HEK293 cells. The cells expressing the various BA transporters were grown in Cytostar 96 w Scintillation Proximity Assay plates and the effect of increasing concentrations of A3309 on accumulation of 30 μM of radiolabelled glycocholic acid was monitored. In Vivo part: In dogs, constipation was induced by shifting the standard diet to ground beef dosed once daily until the faeces weight was <30 g/day. Vehicle or test compounds (A3309 or the active comparator tegaserod) were administered once daily for 3 days, and faeces was collected and weighed on a daily basis. The pharmacodynamic effect of A3309 was investigated in a 1-month study in 24 dogs by measuring the plasma concentration of C4. Results: A3309 was found to be highly potent and selective for IBAT with IC50 =0.53±0.17, 0.13±0.03 and 5.8±1.6 nM for the human, mouse and canine transporters, respectively, whereas the corresponding value for the human liver (basolateral) sodium/bile acid co-transporter was found to be 0.24±0.02 μM (400-fold higher than for IBAT inhibition). Furthermore, inhibition of amino acid uptake via the neutral amino acid transporter(s) in the HEK293 cells was at least 1000 times lower than that of BA transport via the human ileal bile acid transporter. In the constipated dog, A3309 increased faeces weight from 9.4+12.4 g/day (mean±SEM n=36) after vehicle to 25.8 g/day (1.5 μmol/kg, n=2), 34.4 g/day (5 μmol/kg, n=2), 31.3±4,5 g/day (15 μmol/kg, n=6) and for tegaserod 14 g/day (3.3 μmol/kg, n=1), 18.1±10,2g/day (3.3 μmol /kgx2, n=3), 23.9±7,1 g/day (10 μmol /kg, n=3) and 68.3 g/day (33 μmol /kgx2, n=2). In the dog, a dose dependant increase of C4 in plasma was observed already after single doses. After 26 days treatment with A3309 the plasma concentration of C4 increased 3 to 7 fold (43.0±8.43,150±48.8 ,190±47.0 and 350±49.0 nmol/L, pre-values, 5, 25, 200 μmol/kg, respectivly). Conclusion: A3309 was found to be highly potent and selective for IBAT and ameliorate meat-induced constipation in dogs. The increased BA synthesis effect last for at least 1 month. IBAT inhibitors have a potential clinical utility in the treatment of constipation.

Glucose stimulates cholesterol 7α-hydroxylase gene transcription in human hepatocytes

Bile acids play important roles in the regulation of lipid, glucose, and energy homeostasis. Recent studies suggest that glucose regulates gene transcription in the liver. The aim of this study was to investigate the potential role of glucose in regulation of bile acid synthesis in human hepatocytes. High glucose stimulated bile acid synthesis and induced mRNA expression of cholesterol 7alpha-hydroxylase (CYP7A1), the key regulatory gene in bile acid synthesis. Activation of an AMP-activated protein kinase (AMPK) decreased CYP7A1 mRNA, hepatocyte nuclear factor 4alpha (HNF4alpha) protein, and binding to CYP7A1 chromatin. Glucose increased ATP levels to inhibit AMPK and induce HNF4alpha to stimulate CYP7A1 gene transcription. Furthermore, glucose increased histone acetylation and decreased H3K9 di- and tri-methylation in the CYP7A1 chromatin. Knockdown of ATP-citrate lyase, which converts citrate to acetyl-CoA, decreased histone acetylation and attenuated glucose induction of CYP7A1 mRNA expression. These results suggest that glucose signaling also induces CYP7A1 gene transcription by epigenetic regulation of the histone acetylation status. This study uncovers a novel link between hepatic glucose metabolism and bile acid synthesis. Glucose induction of bile acid synthesis may have an important implication in metabolic control of glucose, lipid, and energy homeostasis under normal and diabetic conditions.

Hepatic bile acid metabolism in the neonatal hamster: expansion of the bile acid pool parallels increased Cyp7a1 expression levels

Intraluminal concentrations of bile acids are low in newborn infants and increase rapidly after birth, at least partly owing to increased bile acid synthesis rates. The expansion of the bile acid pool is critical since bile acids are required to stimulate bile flow and absorb lipids, a major component of newborn diets. The purpose of the present studies was to determine the mechanism responsible for the increase in bile acid synthesis rates and the subsequent enlargement of bile acid pool sizes (BAPS) during the neonatal period, and how changes in circulating hormone levels might affect BAPS. In the hamster, pool size was low just after birth and increased modestly until 10.5 days postpartum (dpp). BAPS increased more significantly ( approximately 3-fold) between 10.5 and 15.5 dpp. An increase in mRNA and protein levels of cholesterol 7alpha-hydroxylase (Cyp7a1), the rate-limiting step in classical bile acid synthesis, immediately preceded an increase in BAPS. In contrast, levels of oxysterol 7alpha-hydroxylase (Cyp7b1), a key enzyme in bile acid synthesis by the alternative pathway, were relatively elevated by 1.5 dpp. farnesyl X receptor (FXR) and short heterodimeric partner (SHP) mRNA levels remained relatively constant at a time when Cyp7a1 levels increased. Finally, although simultaneous increases in circulating cortisol and Cyp7a1 levels occurred, precocious expression of Cyp7a1 could not be induced in neonatal hamsters with dexamethasone. Thus the significant increase in Cyp7a1 levels in neonatal hamsters is due to mechanisms independent of the FXR and SHP pathway and cortisol.

Sequential effects of a high-fiber diet with psyllium husks on the expression levels of hepatic genes and plasma lipids

Objective We studied the sequential effects of a high-fiber diet using psyllium husks on hepatic gene expression and plasma lipid levels. Methods C57BL/6J mice were randomly assigned to a control diet or a high-fiber diet containing 10% psyllium husks for 3 weeks (PE-3wk) and 10 weeks (PE-10wk). Oligonucleotide microarrays were used to screen the transcriptional response at both time points. Genes encoding enzymes regulating key steps of lipid metabolism were then selected for further validation by quantitative real-time reverse transcription polymerase chain reaction and their protein expression by western blot assays. Results Plasma cholesterol and triacylglycerol levels were reduced in both high-fiber groups. Three weeks of high-fiber feeding downregulated genes involved in lipogenesis, whereas those involved in cholesterol and bile acid synthesis were upregulated. With prolonged high-fiber feeding, genes involved in lipogenesis such as fatty acid synthase (Fasn) were then upregulated. Additional genes in cholesterol synthesis such as 3-hydroxy-3-methylglutaryl coenzyme A reductase (Hmgcr) were also upregulated. At week 3, protein expression levels of Fasn were significantly lower in the high-fiber group but increased at week 10. Protein levels of Hmgcr were significantly increased in PE-10wk mice. Conclusion The high-fiber diet containing psyllium husks reduced plasma total cholesterol and triacylglycerol levels. Cholesterol lowering was most likely mediated by increased bile acid synthesis. The increased transcript levels of genes related to cholesterol synthesis throughout the entire feeding period and the subsequent increased lipogenic gene transcript levels could likely suggest a regulatory mechanism to restore the lowered plasma cholesterol and triacylglycerol levels.

Simultaneous Intake of β-Glucan and Plant Stanol Esters Affects Lipid Metabolism in Slightly Hypercholesterolemic Subjects

Intake of food products rich in water-soluble fiber β-glucan and products enriched with plant stanol esters lower serum cholesterol. Combining 2 functional food ingredients into one food product may achieve additional reductions of serum cholesterol. Our objective was to investigate the effects of a simultaneous intake of β-glucan plus plant stanol esters on lipid metabolism in mildly hypercholesterolemic volunteers. In a randomized, controlled, 3-period crossover study, 40 mildly hypercholesterolemic men and women received muesli in random order twice a day for 4 wk, which provided, in total, 5 g control fiber from wheat (control muesli), 5 g oat β-glucan (β-glucan muesli), or 5 g oat β-glucan plus 1.5 g plant stanols (combination muesli). β-Glucan muesli decreased serum LDL cholesterol by 5.0% compared with control muesli (P = 0.013). Combination muesli reduced LDL cholesterol by 9.6% compared with control muesli (P < 0.001), and by 4.4% compared with β-glucan muesli (P = 0.036). Serum HDL cholesterol and triacylglycerol concentrations did not differ after the 3 treatments. Compared with control muesli, β-glucan muesli increased bile acid synthesis (P = 0.043) and decreased cholesterol absorption (P = 0.011). Addition of plant stanols did not influence bile acid synthesis but decreased cholesterol absorption (P < 0.001) and raised cholesterol synthesis (P = 0.016) compared with control muesli, and the plant stanols decreased cholesterol absorption compared with β-glucan muesli (P = 0.004). The combination muesli decreased serum concentrations of sitostanol compared with control muesli (P = 0.010). Plasma concentrations of lipid-soluble antioxidants did not differ after the 3 treatments. β-Glucan muesli effectively lowered serum LDL cholesterol concentrations. The addition of plant stanol esters to β-glucan-enriched muesli further lowered serum LDL cholesterol, although effects were slightly less than predicted.

Fish Oil Increases Bile Acid Synthesis in Male Patients with Hypertriglyceridemia

Fibrates are drugs of choice in patients with hypertriglyceridemia (HTG), but may increase the risk for gallstones by decreasing bile acid synthesis. Fish oil might be a therapeutic alternative, but its effect on bile acid metabolism in humans is unknown. We compared the effects of triglyceride-lowering therapy by fish oil or bezafibrate on cholesterol synthesis and bile acid metabolism in HTG. Cholesterol synthesis, bile acid pool sizes, and synthesis rates were compared between 9 male HTG patients and 10 normolipidemic controls matched for age, sex, and BMI. Effects of bezafibrate or fish oil were studied only in HTG patients in a randomized crossover trial. Patients had 14-fold higher serum triglyceride concentrations and greater cholesterol synthesis, as indicated by a 107% higher ratio of serum lathosterol to cholesterol (P < 0.01) than controls. The groups did not differ in bile acid metabolism. Both bezafibrate and fish oil reduced serum TG concentration (-68 and -51% vs. baseline, respectively). Compared with baseline, bezafibrate therapy was associated with reduced cholesterol synthesis (-25%, P = 0.009) without changes in bile acid synthesis rate and pool size. In contrast, fish oil increased bile acid synthesis (+31% vs. baseline, P = 0.07 and +53% vs. bezafibrate, P = 0.02) and altered bile acid distribution, as reflected by an increased ratio of the cholic acid (CA) synthesis rate to the chenodeoxycholic acid (CDCA) synthesis rate (+35% vs baseline, P = 0.05 and + 32% vs bezafibrate, P = 0.07) without effects on bile acid pool size or cholesterol synthesis. In conclusion, cholesterol synthesis is greater in HTG patients than in controls, whereas bile acid synthesis does not differ. Bezafibrate and fish oil have similar triglyceride-lowering capacities, but distinct effects on cholesterol synthesis. Bile acid synthesis is increased by fish oil, but not by bezafibrate therapy.

Biliary lipids, cholesterol and bile synthesis: different adaptive mechanisms to dietary cholesterol in lean and obese subjects

Increased biliary cholesterol secretion together with elevated cholesterol synthesis may predispose obese subjects to cholesterol gallstone formation. To investigate whether processing of dietary cholesterol is altered in obesity, we enrolled eight lean and seven obese subjects in a double-blind crossover study. Cholesterol consumption was 300 mg/day on low and 1300 mg/day on high cholesterol diet. After 3 weeks on either diet, hepatic bile was collected to determine biliary lipid secretion, and bile salt composition by high-performance liquid chromatography and cholesterol saturation index was calculated. Cholesterol synthesis was measured employing mass isotopomer distribution analysis. Bile acid synthesis via neutral and acidic pathway was assessed by serum levels of 7alpha-hydroxy-4-cholesten-3-one and 27-hydroxycholesterol. Cholesterol synthesis was increased in obese compared with lean and feedback inhibited only in obese. On low cholesterol diet, cholesterol secretion was doubled in obese but bile acid composition and synthesis was similar between the two groups. After high cholesterol diet, cholesterol saturation index and bile secretion were unchanged. In contrast to obese, lean increased bile acid synthesis only via the acidic pathway. Dietary cholesterol appears to preferentially induce bile acid synthesis via the acidic pathway in lean, whereas cholesterol synthesis was inhibited in obese. Thus, stable cholesterol saturation index may be achieved by different mechanisms.

Clinical Utility of Bile Acid Sequestrants in the Treatment of Dyslipidemia: A Scientific Review

Bile acid sequestrants (BAS) continue to command a position in the treatment of dyslipidemias 25 years after their introduction. Partial diversion of the enterohepatic circulation using BAS depletes the endogenous bile acid pool by approximately 40%, thus stimulating an increase in bile acid synthesis from cholesterol, which lowers low-density lipoprotein cholesterol (LDL-C) by 15 to 26%. Three BAS are currently used for treating hypercholesterolemia in the United States: the conventional sequestrants, cholestyramine and colestipol, and the specifically engineered BAS, colesevelam hydrochloride (HCl). Compared with conventional BAS, colesevelam HCl has enhanced specificity, greater affinity, and higher capacity for binding bile acids, due to its polymer structure engineered for bile acid sequestration. BAS are not absorbed by the intestine and thus have no systemic drug-drug interactions, but may interfere with the absorption of some drugs. Although BAS monotherapy effectively lowers LDL-C, combination therapy, especially with BAS and statins, is becoming increasingly common due to complementary mechanisms of action. Low-dose statin plus BAS combinations lead to greater or similar LDL-C reductions compared with high-dose statin monotherapy and may have a better safety profile. Combinations of BAS with nonstatin lipid-lowering agents, including niacin, fibrates, and cholesterol absorption inhibitors, may be useful in those patients who require intensive lipid-lowering, but are statin intolerant. BAS treatment can significantly reduce coronary artery disease (CAD) progression and the risk of CAD-associated outcomes. It is also becoming clear that BAS and other therapies that manipulate the bile acid synthetic pathway may have clinically useful therapeutic effects on other metabolic disorders including type 2 diabetes.

Human liver cholesteryl ester hydrolase: cloning, molecular characterization, and role in cellular cholesterol homeostasis

The liver regulates cholesterol homeostasis and eliminates excess cholesterol as bile acids or biliary cholesterol. Free cholesterol for bile acid synthesis or biliary secretion is obtained by the hydrolysis of stored cholesteryl esters or from cholesteryl esters taken up by the liver from high-density lipoproteins via a selective uptake pathway. The present study was undertaken to characterize the enzyme catalyzing this reaction, namely, cholesterol ester hydrolase (CEH) from the human liver, and demonstrate its role in regulating bile acid synthesis. Two cDNAs were isolated from the human liver that differed only in the presence of an additional alanine at position 18 in one of the clones. Transient transfection of COS-7 cells with a eukaryotic expression vector containing either of these two cDNAs resulted in significant increase in the hydrolysis of cholesteryl esters, authenticating these clones as human liver CEH. CEH mRNA and protein expression in human hepatocytes were demonstrated by real-time PCR and Western blot analyses, respectively, confirming the location of this enzyme in the cell type involved in hepatic cholesterol homeostasis. Overexpression of these CEH clones in human hepatocytes resulted in significant increase in bile acid synthesis, demonstrating a role for liver CEH in modulating bile acid synthesis. This CEH gene mapped on human chromosome 16, and the two clones represent two different transcript variants resulting from splice shifts at exon 1. In conclusion, these data identify that human liver CEH was expressed in hepatocytes, where it potentially regulates the synthesis of bile acids and thus the removal of cholesterol from the body.

Effect of increasing the expression of cholesterol transporters (StAR, MLN64, and SCP-2) on bile acid synthesis

There are two major pathways of bile acid synthesis: the "neutral" pathway, initiated by highly regulated microsomal cholesterol 7alpha-hydroxylase (CYP7A1), and an "alternative" pathway, initiated by mitochondrial sterol 27-hydroxylase (CYP27A1). In hepatocyte cultures, overexpression of CYP7A1 increases bile acid synthesis by >8-fold. However, overexpression of CYP27A1 in hepatocytes only increases it by 1.5-fold, suggesting that additional rate-limiting steps must be involved in the regulation of this pathway. The effects of intracellular cholesterol transport proteins on bile acid synthesis have been investigated in the current study. Under culture conditions in which the neutral pathway was inactive, selective overexpression of the gene encoding steroidogenic acute regulatory protein (StAR), MLN64 (StAR homolog protein), and sterol carrier protein-2 (SCP-2) led to 5.7-, 1.2-, and 1.7-fold increases, respectively, in the rates of bile acid synthesis in primary rat hepatocytes. Surprisingly, co-overexpression of MLN64 with StAR, SCP-2, or CYP7A1 blunted the upregulated bile acid synthesis by 48, 47, and 45%, respectively. These results suggest that MLN64, in its full-length form, is not responsible for the transport of cholesterol to the mitochondria or the endoplasmic reticulum, where CYP27A1 or CYP7A1 is located, respectively.

Overexpression of cholesterol transporter StAR increasesIn Vivo rates of bile acid synthesis in the rat and mouse

Bile acid synthesis (BAS) occurs mainly via two pathways: the "neutral" pathway, which is initiated by highly regulated microsomal CYP7A1, and an "acidic" pathway, which is initiated by mitochondrial CYP27A1. Previously, we have shown that overexpression of the steroidogenic acute regulatory protein (StAR), a mitochondrial cholesterol transport protein, increases bile acid biosynthesis more than 5-fold via the acidic pathway in primary rat hepatocytes. This observation suggests that mitochondrial cholesterol transport is the rate-limiting step of BAS via this pathway. The objective of this study was to determine the effect of increased StAR on rates of BAS in vivo. Overexpression of StAR and CYP7A1 were mediated via infection with recombinant adenoviruses. BAS rates were determined in chronic biliary-diverted rats and mice, and in mice with an intact enterohepatic circulation. The protein/messenger RNA levels of StAR and CYP7A1 increased dramatically following overexpression. Overexpression of StAR or CYP7A1 led to a similar 2-fold (P <.01) increase in BAS over up-regulated (approximately 2-fold) 3-day chronic biliary-diverted control rats. Additionally, overexpression of StAR led to more than 3- and 6-fold increases over controls in the rates of BAS in biliary-diverted and intact mice, respectively (P <.01). In conclusion, in both rats and mice in vivo, overexpression of StAR led to a marked increase in the rates of BAS initiated by delivery of cholesterol to mitochondria containing CYP27A1.

Inhibition of ileal bile acid transport lowers plasma cholesterol levels by inactivating hepatic farnesoid X receptor and stimulating cholesterol 7α-hydroxylase

We investigated the effect of SC-435, a competitive inhibitor of ileal apical sodium-dependent bile acid cotransporter (ASBT) on ileal bile acid absorption and the hepatic nuclear receptor FXR (farnesoid X receptor), which regulates cholesterol 7α-hydroxylase (CYP7A1) activity and mRNA levels. Eighteen New Zealand White (NZW) rabbits were divided into 2 groups: controls (n = 10) and fed SC-435 125 mg/kg/d for 1 week (n = 8). In rabbits treated with SC-435, fecal bile acid outputs increased by more than 8 times, reflecting substantial bile acid malabsorption. Plasma cholesterol levels decreased 26%, while bile acid pool sizes and biliary bile acid outputs did not change after treatment. CYP7A1 activity increased 64% and mRNA rose by 4 times after treatment. The expression of FXR target genes in the liver, short heterodimer partner (SHP) and bile salt export pump (BSEP), decreased 11.6 and 2.6 times, respectively, after treatment, which indicates inactivation of hepatic FXR. However, the mRNA levels of ileal bile acid binding protein (IBABP) did not change significantly, while ileal ASBT mRNA expression increased by 2.4 times after treatment. Rabbits treated with SC-435 developed ileal bile acid malabsorption, which decreased the return of bile acids (FXR ligands) to the liver to inactivate hepatic FXR, which upregulated CYP7A1 and lowered plasma cholesterol levels. Although fecal bile acid malabsorption was substantial, increased bile acid production from hepatic cholesterol kept biliary bile acid outputs intact. Thus, a new balance was reached in the liver, where increased bile acid synthesis compensated for diminished ileal bile acid absorption to maintain the circulating enterohepatic bile acid pool.