Cholate Diet Research Articles

Hepatoprotective Effect of Leaves of Morinda tinctoria Roxb. Against Non-alcoholic Fatty Liver Disease in Rats

Aim: To study the hepatoprotective and antioxidant activity of 50% aqueous-alcoholic leaves extract of Morinda tinctoria (Rubiaceae) against non-alcoholic fatty liver disease.
 Study Design: All experiments involving animals complies with the ethical standards of animal handling and approved by institutional animal ethical and welfare committee of the Institute of Pharmacy, PSIT (1273/AC/09/ CPCSEA) and plant were collected from Ranan Nagar, Madurai, Tamil Nadu, India and was authenticated by Dr. Navin K. Ambasht, Head and Associate Professor, Botany Department, Christ Church College, Kanpur, Uttar Pradesh, India.
 Place and Duration of Study: The study was carried out at Institute of Pharmacy, PSIT, Kanpur, Uttar Pradesh, India, during 2018-21.
 Methodology: The hepatoprotective potential of Morinda tinctoria leaves extract (MTLE) 150 and 300 mg/kg body weight was studied on Methionine and Choline deficient diet, High Fat Diet, Cholesterol and Cholate diet, and Streptozotocin + HFD induced non-alcoholic fatty liver disease. At the end of the treatment blood sample was collected from direct cardiac puncture and analysed for various parameter like alanine aminotransferase, aspartate transaminase, low density lipoprotein, high density lipoprotein, triglycerides, total cholesterol, free fatty acid and malondialdehyde.
 Results: The phytochemical investigation of extract showed presence of alkaloids, flavonoids, amino acid, saponin, tannins, phenols, carbohydrate and for the first time the present study showed that Morinda tinctoria leaves extract reduced level of alanine aminotransferase, aspartate transaminase, triglycerides, total cholesterol, low density lipoprotein, free fatty acid, malondialdehyde and enhance the level of Superoxide dismutase, High Density Lipoprotein and it also returned hepatic damage towards normal which further supports hepatoprotective and antioxidant activity of M. tinctoria. leaves extracts.
 Conclusions: M. tinctoria leaves extract showed maximum curation in the dose 300 mg/kg body weight against non-alcoholic fatty liver disease.

Tocopheryl quinone improves non-alcoholic steatohepatitis (NASH) associated dysmetabolism of glucose and lipids by upregulating the expression of glucagon-like peptide 1 (GLP-1) via restoring the balance of intestinal flora in rats

Context Glucagon-like peptide 1 (GLP-1) and α-tocopheryl quinone can promote the growth of intestinal flora and affect the pathogenesis of non-alcoholic steatohepatitis (NASH). Objective This study determines the molecular mechanism of the effect of tocopheryl quinone in the treatment of high cholesterol and cholate diet (HFCC)-induced NASH. Materials and methods Thirty-two male Sprague Dawley (SD) rats grouped as lean control (LC), LC + tocopheryl quinone (1 mL of 3 × 106 dpm tocopheryl quinone via i.p. injection), HFCC (5.1 kcal/g of fat diet), and HFCC + tocopheryl quinone. Profiles of intestinal flora were assessed by 16S ribosomal ribonucleic acid–based analysis. Levels and activity of GLP-1, interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-α) in intestinal tissues were detected by immunohistochemistry (IHC), Western blot and enzyme-linked immunosorbent assay (ELISA). Results HFCC rats presented higher levels of cholesterol, low-density lipoprotein (LDL) and high-density lipoprotein (HDL), while tocopheryl quinone reversed the effects of HFCC. HFCC dysregulated malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD), Vitamin E, 12-hydroxyeicosatetraenoic acid (12-HETE), 13-hydroxyoctadecadienoic acid (13-HODE) and nuclear factor kappa B (NF-κB), and the effects of HFCC were reversed by the treatment of tocopheryl quinone. Also, GLP-1 in the HFCC group was down-regulated while the IL-6 and TNF-α activity and endotoxins were all up-regulated. HFCC significantly decreased the number and diversity of bacteria, whereas tocopheryl quinone substantially restored the balance of intestinal flora and promoted the growth of both Bacteroides and Lactobacilli in vitro. Discussion and conclusions α-Tocopheryl quinone relieves HFCC-induced NASH via regulating oxidative stress, GLP-1 expression, intestinal flora imbalance, and the metabolism of glucose and lipids.

Non-Alcoholic Fatty Liver Disease, and the Underlying Altered Fatty Acid Metabolism, Reveals Brain Hypoperfusion and Contributes to the Cognitive Decline in APP/PS1 Mice.

Non-alcoholic fatty liver disease (NAFLD), the leading cause of chronic liver disease, is associated with cognitive decline in middle-aged adults, but the mechanisms underlying this association are not clear. We hypothesized that NAFLD would unveil the appearance of brain hypoperfusion in association with altered plasma and brain lipid metabolism. To test our hypothesis, amyloid precursor protein/presenilin-1 (APP/PS1) transgenic mice were fed a standard diet or a high-fat, cholesterol and cholate diet, inducing NAFLD without obesity and hyperglycemia. The diet-induced NAFLD disturbed monounsaturated and polyunsaturated fatty acid (MUFAs, PUFAs) metabolism in the plasma, liver, and brain, and particularly reduced n-3 PUFAs levels. These alterations in lipid homeostasis were associated in the brain with an increased expression of Tnfα, Cox2, p21, and Nox2, reminiscent of brain inflammation, senescence, and oxidative stress. In addition, compared to wild-type (WT) mice, while brain perfusion was similar in APP/PS1 mice fed with a chow diet, NAFLD in APP/PS1 mice reveals cerebral hypoperfusion and furthered cognitive decline. NAFLD reduced plasma β40- and β42-amyloid levels and altered hepatic but not brain expression of genes involved in β-amyloid peptide production and clearance. Altogether, our results suggest that in a mouse model of Alzheimer disease (AD) diet-induced NAFLD contributes to the development and progression of brain abnormalities through unbalanced brain MUFAs and PUFAs metabolism and cerebral hypoperfusion, irrespective of brain amyloid pathology that may ultimately contribute to the pathogenesis of AD.

Xanthine Oxidase Inhibition Attenuates Insulin Resistance and Diet-Induced Steatohepatitis in Mice

Background: Hyperuricemia drives the development of nonalcoholic fatty liver disease (NAFLD). Pharmacological inhibition of xanthine oxidase (XO), a rate-limiting enzyme for uric acid (UA) production, has been demonstrated to improve hepatic steatosis in diet-induced obese mice. However, it remains unclear whether inhibition of XO could improve nonalcoholic steatohepatitis (NASH), a more advanced form of NAFLD, in terms of both liver inflammation and fibrosis. Methods: To investigate the effects of febuxostat and allopurinol, XO inhibitors clinically used for gout, on diet-induced NASH, C57BL/6J mice were fed a high-fat, high-cholesterol, and cholate diet (CL diet) with or without either 0.001% febuxostat or 0.003% allopurinol for 18 weeks. Systemic glucose tolerance and insulin sensitivity, liver histology, inflammation, and oxidative stress were tested. Furthermore, we conducted a single-arm, open-label intervention study with febuxostat for NAFLD patients with hyperuricemia. Findings: Despite a similar hypouricemic effect of the XO inhibitors on blood UA level, febuxostat, but not allopurinol, significantly decreased hepatic UA levels in CL diet-fed mice. This reduction in hepatic UA levels was accompanied by alleviated insulin resistance, lipid peroxidation, and classically activated M1-like macrophage accumulation in the liver. Furthermore, in NAFLD patients with hyperuricemia, treatment with febuxostat for 24 weeks decreased the serum UA level, accompanied by a reduction in serum levels of liver enzymes, alanine aminotransferase and aspartate aminotransferase. Interpretation: XO may represent a promising therapeutic target in NAFLD/NASH, especially in patients with hyperuricemia. Trial Registration: The study has been registered with UMIN-CTR (#UMI08686). Funding Statement: This work was supported by Grants-in-aid for Scientific Research (B) (25282017) and Challenging Exploratory Research (15K12698) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and the Japan Diabetes Foundation (to T.O.). This study was also supported by Teijin Pharma Limited. Declaration of Interests: The authors declare no conflict of interest associated with this manuscript. Ethics Approval Statement: The study was approved by the Ethics Committee of Kanazawa University Hospital. All animal procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals of Kanazawa University, Japan.

Liver-directed gene therapy results in long-term correction of progressive familial intrahepatic cholestasis type 3 in mice

Progressive familial intrahepatic cholestasis type 3 (PFIC3), for which there are limited therapeutic options, often leads to end-stage liver disease before adulthood due to impaired ABCB4-dependent phospholipid transport to bile. Using adeno-associated virus serotype 8 (AAV8)-mediated gene therapy, we aimed to restore the phospholipid content in bile to levels that prevent liver damage, thereby enabling stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Ten-week-old Abcb4-/- mice received a single dose of AAV8-hABCB4 (n = 10) or AAV8-GFP (n = 7) under control of a liver specific promoter via tail vein injection. Animals were sacrificed either 10 or 26 weeks after vector administration to assess transgene persistence, after being challenged with a 0.1% cholate diet for 2 weeks. Periodic evaluation of plasma cholestatic markers was performed and bile duct cannulation enabled analysis of biliary phospholipids. Liver fibrosis and the Ki67 proliferation index were assessed by immunohistochemistry. Stable transgene expression was achieved in all animals that received AAV8-hABCB4 up to 26 weeks after administration. AAV8-hABCB4 expression restored biliary phospholipid excretion, increasing the phospholipid and cholesterol content in bile to levels that ameliorate liver damage. This resulted in normalization of the plasma cholestatic markers, alkaline phosphatase and bilirubin. In addition, AAV8-hABCB4 prevented progressive liver fibrosis and reduced hepatocyte proliferation for the duration of the study. Liver-directed gene therapy provides stable hepatic ABCB4 expression and long-term correction of the phenotype in a murine model of PFIC3. Translational studies that verify the clinical feasibility of this approach are warranted. Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a severe genetic liver disease that results from impaired transport of lipids to bile, which makes the bile toxic to liver cells. Because therapeutic options are currently limited, this study aims to evaluate gene therapy to correct the underlying genetic defect in a mouse model of this disease. By introducing a functional copy of the missing gene in liver cells of mice, we were able to restore lipid transport to bile and strongly reduce damage to the liver. The proliferation of liver cells was also reduced, which contributes to long-term correction of the phenotype. Further studies are required to evaluate whether this approach can be applied to patients with PFIC3.

Biochemical and histological characterisation of an experimental rodent model of non-alcoholic steatohepatitis – Effects of a peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist and a glucagon-like peptide-1 analogue

BackgroundNon-alcoholic steatohepatitis (NASH) is a prevalent disease that is highly associated with the metabolic syndrome and type II diabetes. The development of in vivo models that reflect all nuances of the human NASH pathology is essential for drug discovery and development. We aimed to further characterise a dietary induced model of NASH both biochemically and histologically. In addition, we also investigated whether pioglitazone and liraglutide, drugs that have both been investigated as potential NASH treatments, could modulate the pathological changes induced by the NASH diet. Furthermore, to aid the translation of data from pre-clinical in vivo models, we aimed to adapt the NASH Clinical Research Network (CRN) histological score system for use in rodent studies. MethodsSprague Dawley rats were fed a high-fat diet (HFD) for 9 weeks, after which they were switched to a high fat, high cholesterol and cholate diet (HFCC) for 12 weeks. The rats were divided into treatment groups, receiving either 30 mg/kg pioglitazone p.o. SID or liraglutide s.c. 200 μg/kg BID or the respective vehicles. Serum levels of triglycerides (TG), cholesterol (Chol), LDL, HDL, AST and ALT, as well as body weight were assessed in all subjects. Upon termination, the liver was weighed and evaluated histologically using modified NASH-CRN criteria. ResultsHFCC feeding induced severe hepatic injury and hepatomegaly as indicated by significant increases in AST, ALT and an increased liver weight. Additionally, HFCC feeding induced dyslipidaemia, significant increases in circulating cholesterol and LDL were observed. No obesogenic effect of the HFCC diet was observed, though the diet did induce insulin resistance. Histological analysis showed that the HFCC diet induced several NASH like features, though it did not induce the development of severe fibrosis. However, microgranulomas were often prevalent in addition to lobular inflammatory foci. Pioglitazone showed little efficacy upon both biochemical and histological features. However, liraglutide induced weight loss, improved glycaemic control, reduced ALT and AST and showed some beneficial effects upon steatosis and lobular inflammation. ConclusionSimilar to previous reports we have shown that the atherogenic diet, HFCC, induces a phenotype akin to that seen in human NASH patients. Despite inducing all histological features of NASH, HFCC feeding does not promote the development of significant fibrosis within rodents. Pioglitazone and liraglutide have been investigated as potential NASH treatments. Within this model of NASH we have shown that pioglitazone has little efficacy, whereas liraglutide reduced the levels of circulating aminotransferases and had some beneficial effects upon NASH histological parameters.

Mango phenolics increase the serum apolipoprotein A1/B ratio in rats fed high cholesterol and sodium cholate diets.

Serum lipoproteins are in dynamic equilibrium, partially controlled by the apolipoprotein A1 to apolipoprotein B ratio (APOA1/APOB). Freeze-dried mango pulp (FDM) is a rich source of phenolic compounds (MP) and dietary fiber (MF), although their effects on lipoprotein metabolism have not yet been studied. Thirty male Wistar rats were fed with four different isocaloric diets (3.4 kcal g-1 ) for 12 weeks: control diet, high cholesterol (8 g kg-1 ) + sodium cholate (2 g kg-1 ) diet either alone or supplemented with MF (60 g kg-1 ), MP (1 g kg-1 ) or FDM (50 g kg-1 ). MP and FDM reduced food intake, whereas MF and MP tended to increase serum APOA1/APOB ratio, independently of their hepatic gene expression. This suggests that lipoprotein metabolism was favorably altered by mango bioactives, MP also mitigated the non-alcoholic steatohepatitis that resulted from the intake of this diet. We propose that phenolics are the most bioactive components of mango pulp, acting as anti-atherogenic and hepatoprotective agents, with a mechanism of action tentatively based on changes to the main protein components of lipoproteins. © 2018 Society of Chemical Industry.

Cholesterol induces lipoprotein lipase expression in a tree shrew (Tupaia belangeri chinensis) model of non-alcoholic fatty liver disease.

Animal models are indispensible to investigate the pathogenesis and treatments of non-alcoholic fatty liver diseases (NAFLD). Altered cholesterol metabolism has been implicated into the pathogenesis of NAFLD. Here, using high fat, cholesterol and cholate diet (HFHC), we generated a novel tree shrew (Tupaia belangeri chinensis) model of NAFLD, which displayed dyslipidemia with increased levels of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-c) and high density lipoprotein-cholesterol (HDL-c), but decreased level of triglycerides (TG). Liver histopathology and genes expression indicated that HFHC diet successfully induced liver steatosis to inflammation and fibrosis progressively within 10 weeks. Moreover, HFHC induced the transcriptional expression of lipoprotein lipase (lpl) in the liver, but repressed the expression of LDL receptor, and the endogenous synthesis pathway and excretion of cholesterol. Notably, Poloxamer 407 (P-407) inhibition of LPL improved the severity of steatosis and reduced inflammation. These results illustrated that LPL plays an important role in cholesterol metabolism in NAFLD, and the tree shrew may be a valuable animal model for further research into NAFLD.

Cysteine sulfinic acid decarboxylase regulation: A role for farnesoid X receptor and small heterodimer partner in murine hepatic taurine metabolism

AimBile acid synthesis is regulated by nuclear receptors including farnesoid X receptor (FXR) and small heterodimer partner (SHP), and by fibroblast growth factor 15/19 (FGF15/19). We hypothesized that hepatic cysteine sulfinic acid decarboxylase (CSAD) (a key enzyme in taurine synthesis) is regulated by bile acids (BA). The aim of this study was to investigate CSAD regulation by BA dependent regulatory mechanisms.MethodsMice were fed a control diet or a diet supplemented with either 0.5% cholate or 2% cholestyramine. To study BA dependent pathways, we utilized GW4064 (FXR agonist), FGF19 or T‐0901317 (liver X receptor [LXR] agonist) and Shp−/− mice. Tissue mRNA was determined by quantitative reverse transcription polymerase chain reaction. Amino acids were measured by high‐performance liquid chromatography.ResultsMice supplemented with dietary cholate exhibited reduced hepatic CSAD mRNA while those receiving cholestyramine exhibited increased mRNA. Activation of FXR suppressed CSAD mRNA expression whereas CSAD expression was increased in Shp−/− mice. Hepatic hypotaurine concentration (the product of CSAD) was higher in Shp−/− mice with a corresponding increase in serum taurine conjugated BA. FGF19 administration suppressed hepatic cholesterol 7‐α‐hydroxylase (CYP7A1) mRNA but did not change CSAD mRNA expression. LXR activation induced CYP7A1 mRNA yet failed to induce CSAD mRNA expression.ConclusionBA regulate CSAD mRNA expression in a feedback fashion via mechanisms involving SHP and FXR but not FGF15/19 or LXR. These findings implicate BA as regulators of CSAD mRNA via mechanisms shared with CYP7A1.

Dietary Folic Acid Activates AMPK and Improves Insulin Resistance and Hepatic Inflammation in Dietary Rodent Models of the Metabolic Syndrome

The AMP activated kinase plays an important role in metabolic control, and pharmacologic enhancement of AMPK activity is used to improve insulin resistance. We hypothesized that high dose of folic acid supplementation might improve insulin sensitivity and hepatic inflammation and examined this by a dietary intervention in (a) the high fat fed rat model of the metabolic syndrome, which shows sole hepatic steatosis as well as (b) in rats fed with a high cholesterol, high cholate diet inducing nonalcoholic steatohepatitis (NASH). Male Wistar rats were fed with folic acid supplemented (40 mg/kg) high fat diet [based on lard, fat content 25% (wt/wt)] or NASH inducing diet (containing 15% fat, 1.25% cholesterol, 0.5% sodium cholate). Metabolic profiling was performed by measuring the animals' visceral fat pads, fasting plasma glucose, insulin, and adipokines as well as in vivo insulin tolerance tests. Hepatic steatosis and inflammation were analyzed semiquantitatively by histological analysis. Folic acid supplementation reduced visceral obesity and improved plasma adiponectin levels. In vivo insulin sensitivity was improved, and in HF-FA rats folic acid increased activation of hepatic AMPK. Further, folic acid supplementation improved hepatic inflammation in animals fed with NASH-inducing diet. Dietary folic acid improved parameters of insulin resistance and hepatic inflammation in rodent models. This might be due to an increased AMK activation.

Hypocholesterolemic Mechanism ofChlorella:Chlorellaand Its Indigestible Fraction Enhance Hepatic Cholesterol Catabolism through Up-Regulation of Cholesterol 7α-Hydroxylase in Rats

Chlorella powder (CP) has a hypocholesterolemic effect and high bile acid-binding capacity; however, its effects on hepatic cholesterol metabolism are still unclear. In the present study, male Wistar rats were divided into four groups and fed a high sucrose + 10% lard diet (H), an H + 10% CP diet (H+CP), an H + 0.5% cholesterol + 0.25% sodium cholate diet (C), or a C + 10% CP diet (C+CP) for 2 weeks. CP decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but did not affect these parameters in rats fed H-based diets. CP increased the hepatic mRNA level and activity of cholesterol 7alpha-hydroxylase (CYP7A1). CP increased hepatic HMG-CoA reductase (HMGR) activity in the rats fed H-based diets, but not in rats fed C-based diets. CP did not affect hepatic mRNA levels of sterol 27-hydroxylase, HMGR, low-density lipoprotein (LDL) receptor, scavenger receptor class B1, ATP-binding cassette (ABC) A1, ABCG5, or ABCB11. Furthermore, the effect of a 3.08% Chlorella indigestible fraction (CIF, corresponding to 10% CP) on hepatic cholesterol metabolism was determined using the same animal models. CIF also decreased serum and liver cholesterol levels significantly in rats fed C-based diets. CIF increased hepatic CYP7A1 mRNA levels. These results suggest that the hypocholesterolemic effect of CP involves enhancement of cholesterol catabolism through up-regulation of hepatic CYP7A1 expression and that CIF contributes to the hypocholesterolemic effect.

Effects of taurine on plasma and liver lipids, erythrocyte ouabain sensitive Na efflux and platelet aggregation in Sprague Dawley rats

The effects of taurine on plasma and liver cholesterol, erythrocyte ouabain sensitive Na efflux and platelet aggregation were examined in Sprague Dawley rats fed control or 0.5% cholesterol with 0.2% cholate diet. Plasma and liver levels of total cholesterol were increased significantly (p<0.05) in rats fed cholesterol diet compared to the control, and taurine significantly decreased the elevated plasma level of cholesterol in rats fed cholesterol diet (p<0.05). HDL-cholesterol was decreased in groups fed the cholesterol diet regardless of taurine supplementation and the difference between groups with and without cholesterol was significant (p<0.01). Plasma triglyceride was decreased and liver triglyceride was increased both significantly (p<0.05) in rats fed cholesterol compared to the control. Plasma and liver triglyceride in rats fed taurine was decreased significantly compared to the control (p<0.05). Intracellular Na tended to be lower in rats fed cholesterol or taurine and higher in rats fed cholesterol plus taurine compared to the control. Na efflux through Na-K ATPase and the passive leak of Na was somewhat reduced in rats fed cholesterol or taurine and was augmented in rats fed cholesterol plus taurine compared to the control, which showed a similar trend to the intracellular Na. Taurine supplementation caused a suppression of Na efflux in groups fed control diet and restored the suppressed Na efflux in groups fed cholesterol. Platelet aggregation was significantly decreased in the group fed taurine compared to the control (p<0.05) and the group fed cholesterol plus taurine was also a little lower in aggregation than the group fed cholesterol. Microscopic examination showed that taurine prevented fatty liver in rats fed cholesterol diet. Taurine known for stimulating Na-K ATPase in some cell types rather decreased erythrocyte ouabain sensitive Na-K ATPase in the present study. Taurine had hypolipidemic and hypocholesterolemic effects and inhibited platelet aggregation which may be favorable for prevention of cardiovascular diseases.

Roles of nuclear receptors in the up-regulation of hepatic cholesterol 7α-hydroxylase by cholestyramine in rats

Nuclear receptors are involved in regulating the expression of cholesterol 7α-hydroxylase (CYP7A1), however, their roles in the up-regulation of CYP7A1 by cholestyramine (CSR) are still unclear. In the present study, male Wistar rats were divided into four groups and fed [high sucrose + 10% lard diet] (H), [H + 3% CSR diet] (H + CSR), [H + 0.5% cholesterol + 0.25% sodium cholate diet] (C), or [C + 3% CSR diet] (C + CSR) for 2 weeks. Cholestyramine decreased serum and liver cholesterol levels significantly in rats fed C-based diets, but had no effect on these parameters in rats fed H-based diets. Cholestyramine raised hepatic levels of CYP7A1 mRNA and activity in both groups. The gene expression of hepatic ATP-binding cassettes A1 and G5, regulated by liver X receptor (LXR), were unchanged and down-regulated by cholestyramine, respectively. The mRNA levels of the hepatic ATP-binding cassette B11 and short heterodimer partner (SHP), regulated by farnesoid X receptor (FXR), were not changed by cholestyramine. C-based diets, which contained cholesterol and cholic acid, increased SHP mRNA levels compared to H-based diets. Consequently, in rats fed the C + CSR diet, hepatic FXR was activated by dietary bile acids, but the hepatic CYP7A1 mRNA level was increased 16-fold compared to that in rats fed an H diet. These results suggest that cholestyramine up-regulates the expression of CYP7A1 independently via LXR- or FXR-mediated pathways in rats.

Effect of diets on lipoprotein concentrations in heterozygous apolipoprotein E-deficient mice.

Loss of apolipoprotein E synthesis causes increased serum cholesterol concentrations and the sensitivity to high-fat diet in mice. We analyzed the changes in lipoprotein and hepatic structures in apolipoprotein E-deficient mice kept on control diet and cholesterol diets. Basal cholesterolemia of heterozygous (+/-) mice (2.2+/-0.28 mmol/l) was the same compared to wild-type (+/+) mice (2.3+/-0.15 mmol/l), but was lower compared to homozygous (-/-) mice (10.3+/-1.40 mmol/l). In +/- mice, cholesterolemia rose to 3.2 mmol/l on cholesterol diet and to 9 mmol/l on cholate diet, to 3 mmol/l and 3.6 mmol/l in +/+ mice, and to 23.4 mmol/l and 70.5 mmol/l in -/- mice, respectively. While the ratio of cholesterol/triglyceride concentrations in VLDL, IDL and LDL fractions was not increased in +/- mice and +/+ mice, it was increased in -/- mice on control diet. On the cholesterol diet, this ratio rose and was dramatically increased by cholate diet in all groups of mice. Even though cholate supplementation increased cholesterol concentration, it led to substantial toxic changes in hepatic morphology of all animals. In conclusion, one functional apo E allele in +/- mice is effective in keeping serum cholesterol concentrations in normal range on a control diet, but not on the cholesterol and cholate diets.

Comparative regulation of major enzymes in the bile acid biosynthesis pathway by cholesterol, cholate and taurine in mice and rats

These enzymes play important roles in the biosynthesis of bile acids. They are cholesterol 7α-hydroxylase (CYP7A1), the rate limiting enzyme in the classic pathway, sterol 12α-hydroxylase (CYP8B1), the key enzyme for synthesis of cholic acid (CA), and sterol 27-hydroxylase (CYP27), the initial enzyme in the alternative pathway. In the present study, the susceptibility of these three enzymes to dietary cholesterol and cholate, and the cholesterol lowering effect of taurine were determined in male C57BL/6 mice and Wistar rats. Both mice and rats were divided into 6 groups: control group (N), high cholesterol diet group (C), high cholesterol and cholate diet group (CB), and their 1% taurine-supplemented groups (NT, CT, CBT, respectively). After animals were fed with the respective diets for one week, the mRNA levels of CYP7A1 increased in the C-group compared with those of the N-group, and decreased in the CB-group compared with those of the C-group in both mice and rats. But the extent of decrease is different between the two species. CYP8B1 was also markedly repressed by cholate in mice, but not in rats. These results are consistent with the changes in serum and liver cholesterol concentrations. Taurine significantly increased CYP7A1 mRNA levels in the CBT-group compared with the CB-group in both animal models, with a subsequent decrease in serum and liver cholesterol levels and increase in fecal bile acid excretion. Up-regulated CYP8B1 was also observed after taurine supplementation in the CBT-group in mice. No increase in CYP7A1 was produced by taurine in the CT-group compared with that of the C-group in mice, although the changes of serum and liver cholesterol and fecal bile acids indicated taurine showed an efficient cholesterol lowering effect. In addition, CYP27 was induced in both C- and CB-groups of rats but not of mice, and no changes were produced by taurine. The overall results suggest that there are differences between mice and rats in susceptibility of the three enzymes to dietary cholesterol and cholate, and taurine induced CYP7A1 to produce its cholesterol-lowering effect only in the presence of cholate in the cholesterol diet.

Dietary cholate is required for antiatherogenic effects of ethanol in mouse models.

Human consumption of moderate amounts of ethanol is associated with reduced cardiovascular events. Studies examining the effect of ethanol on atherosclerosis in mouse models have yielded conflicting results that may be due to differences in dietary fat and cholate content. To determine if dietary cholate influences ethanol's effect on atherosclerosis, we fed apolipoprotein E-/- and low-density lipoprotein receptor (LDLR)-/- mice different liquid diets with or without ethanol. Apolipoprotein E-/- mice were fed a low-fat or high saturated fat, cholate-containing diet with or without ethanol for 3 to 10 weeks, and LDLR-/- mice were fed a low-fat, high saturated fat, or high saturated fat diet with cholate with or without ethanol for 7 weeks. At the end of the feeding study, aortic root lesion size was determined and compared with serum cholesterol, triglycerides, and high-density lipoprotein cholesterol. Because dietary cholate increases hepatic nuclear factor (NF)-kappaB and ethanol inhibits NF-kappaB, we also examined the effect of ethanol on aortic NF-kappaB binding activity. Adding ethanol to a low-fat diet had no effect on lesion size. Similarly, ethanol had no effect on lesion size in LDLR-/- mice consuming a high saturated fat diet. Adding ethanol to a high-fat, cholate-containing diet for either strain resulted in a 25% to 50% reduction in lesion size. Dietary cholate increased and ethanol reduced NF-kappaB binding activity in the aorta. These results suggest that ethanol inhibits atherosclerosis in the presence of dietary cholate, which may occur via an anti-inflammatory mechanism.

Dietary cholate increases plasma levels of apolipoprotein B in mice by posttranscriptional mechanisms

To induce atherogenesis in mice, a high fat (HF) diet is supplemented with cholic acid (CA), which increases apoB-containing particles and lower apoA-I-containing particles. HF diet without CA increases levels of both HDL and LDL, suggesting that CA may be responsible for the elevation of LDL and lowering of HDL. The mechanism of dietary CA-induced lowering of apoA-I-containing particles has recently been reported. In this study, we examined the mechanism of CA- and HF-induced elevation of apoB-containing lipoproteins in mice. Mice were fed the following four diets: control chow (C), high fat high cholesterol, (HF), control and 0.5% cholate (CA), and HF+CA. Dietary CA increased the plasma levels of apoB-containing particles by ∼2-fold when compared to control; VLDL levels increased 2-fold, and LDL levels increased 1.3-fold. On HF diet, VLDL increased by 1.4-fold, and LDL by 2-fold, suggesting that CA and HF-induced increases of apoB-containing particles occurred by different mechanisms. We investigated the potential mechanisms regulating plasma levels of apoB in CA- and HF-fed mice. Although hepatic apoB mRNA levels did not change on CA diet, apoB-100 mRNA increased relative to B-48 as a result of decreased editing of apoB mRNA. Measurements of hepatic LDL receptor mRNA suggested that CA diet down-regulated LDL receptor mRNA, possibly by increasing the levels of hepatic cholesterol. Since plasma and hepatic vitamin E levels did not show significant changes on CA-containing diets, it suggests that dietary CA did not act by increasing the absorption of dietary fat. Hepatic lipase, known to modulate plasma levels of apoB-containing particles, did not show changes in CA- or HF-fed mice. Taken together, these results suggest that dietary CA increased apoB-containing particles both in chow-fed and fat-fed mice by enhancing the relative production of apoB-100, and also by reducing LDL receptor-mediated clearance of apoB-containing particles. Thus, dietary cholate modulates plasma levels of apoB primarily by posttranscriptional mechanisms.

Dietary cholic acid lowers plasma levels of mouse and human apolipoprotein A-I primarily via a transcriptional mechanism

To induce dietary atherosclerosis in mice, high-fat/high-cholesterol (HF) diets are frequently supplemented with cholic acid (CA). This diet produces low plasma levels of high-density lipoprotein (HDL) and high levels of low-density lipoprotein (LDL). However, HF diets without any added CA, which more closely resemble human diets, increase levels of both HDL and LDL, suggesting that CA may be responsible for the lowering of HDL. Our aim was to examine the potential mechanism responsible for the lowering of HDL. Nontransgenic (NTg) C57BL mice and apoA-I-transgenic (apoAI-Tg) mice, with greatly increased basal apoA-I and HDL levels, were used. Mice were fed the following four diets: control (C), high-fat/high-cholesterol (HF), control and 1% cholate (CA) and HF + CA. Dietary CA reduced plasma HDL levels by 35% in NTg and 250% in apoAI-Tg mice, independent of the fat or cholesterol content of the diet. Hepatic apoA-I mRNA decreased 30% in NTg and 180% in apoAI-Tg mice. Hepatic apoA-I synthesis and apoA-I mRNA transcription rates also decreased in parallel with apoA-I mRNA levels, suggesting that the CA-induced decreases in plasma apoA-I levels occurred primarily via decreasing apoA-I mRNA transcription rates. An HF diet increased HDL levels 1.8-fold in NTg and 1.5-fold in apoAI-Tg mice. Addition of CA to the HF diet lowered HDL levels by 1.6-fold in NTg and 2.5-fold in apoAI-Tg mice. Transfection studies with the apoA-I promoter suggested the presence of a putative cis-acting element responsible for the CA-mediated down-regulation of the apoA-I promoter activity. Measurements of apoA-I regulatory protein-1 (ARP-1) mRNA, a negative regulator of the apoA-I gene in the mouse liver showed that CA increased the ARP-1 mRNA levels. Because apoA-I gene transcription alone was not sufficient to account for the lowering of plasma HDL levels, scavenger receptor-B1 (SR-B1) and hepatic lipase (HL) mRNAs levels were quantitated. The levels of SR-B1 and HL mRNA were not changed by dietary CA. These studies suggest that dietary cholate regulates plasma levels of apoA-I primarily by a transcriptional mechanism via a putative bile acid response element involving a negative regulator of apoA-I, and partly by an unidentified post-transcriptional mechanism.

Dietary cholic acid lowers plasma levels of mouse and human apolipoprotein A-I primarily via a transcriptional mechanism.

To induce dietary atherosclerosis in mice, high-fat/high-cholesterol (HF) diets are frequently supplemented with cholic acid (CA). This diet produces low plasma levels of high-density lipoprotein (HDL) and high levels of low-density lipoprotein (LDL). However, HF diets without any added CA, which more closely resemble human diets, increase levels of both HDL and LDL, suggesting that CA may be responsible for the lowering of HDL. Our aim was to examine the potential mechanism responsible for the lowering of HDL. Nontransgenic (NTg) C57BL mice and apoA-I-transgenic (apoAI-Tg) mice, with greatly increased basal apoA-I and HDL levels, were used. Mice were fed the following four diets: control (C), high-fat/high-cholesterol (HF), control and 1% cholate (CA) and HF + CA. Dietary CA reduced plasma HDL levels by 35% in NTg and 250% in apoAI-Tg mice, independent of the fat or cholesterol content of the diet. Hepatic apoA-I mRNA decreased 30% in NTg and 180% in apoAI-Tg mice. Hepatic apoA-I synthesis and apoA-I mRNA transcription rates also decreased in parallel with apoA-I mRNA levels, suggesting that the CA-induced decreases in plasma apoA-I levels occurred primarily via decreasing apoA-I mRNA transcription rates. An HF diet increased HDL levels 1.8-fold in NTg and 1.5-fold in apoAI-Tg mice. Addition of CA to the HF diet lowered HDL levels by 1.6-fold in NTg and 2. 5-fold in apoAI-Tg mice. Transfection studies with the apoA-I promoter suggested the presence of a putative cis-acting element responsible for the CA-mediated down-regulation of the apoA-I promoter activity. Measurements of apoA-I regulatory protein-1 (ARP-1) mRNA, a negative regulator of the apoA-I gene in the mouse liver showed that CA increased the ARP-1 mRNA levels. Because apoA-I gene transcription alone was not sufficient to account for the lowering of plasma HDL levels, scavenger receptor-B1 (SR-B1) and hepatic lipase (HL) mRNAs levels were quantitated. The levels of SR-B1 and HL mRNA were not changed by dietary CA. These studies suggest that dietary cholate regulates plasma levels of apoA-I primarily by a transcriptional mechanism via a putative bile acid response element involving a negative regulator of apoA-I, and partly by an unidentified post-transcriptional mechanism.

Elevated Cholesterol Metabolism and Bile Acid Synthesis in Mice Lacking Membrane Tyrosine Kinase Receptor FGFR4

Heparan sulfate-regulated transmembrane tyrosine kinase receptor FGFR4 is the major FGFR isotype in mature hepatocytes. Fibroblast growth factor has been implicated in the definition of liver from foregut endoderm where FGFR4 is expressed and stimulation of hepatocyte DNA synthesis in vitro. Here we show that livers of mice lacking FGFR4 exhibited normal morphology and regenerated normally in response to partial hepatectomy. However, the FGFR4 (-/-) mice exhibited depleted gallbladders, an elevated bile acid pool and elevated excretion of bile acids. Cholesterol- and bile acid-controlled liver cholesterol 7alpha-hydroxylase, the limiting enzyme for bile acid synthesis, was elevated, unresponsive to dietary cholesterol, but repressed normally by dietary cholate. Expression pattern and cholate-dependent, cholesterol-induced hepatomegaly in the FGFR4 (-/-) mice suggested that activation of receptor interacting protein 140, a co-repressor of feed-forward activator liver X receptor alpha, may mediate the negative regulation of cholesterol- and bile acid-controlled liver cholesterol 7alpha-hydroxylase transcription by FGFR4 and cholate. The results demonstrate that transmembrane sensors interface with metabolite-controlled transcription networks and suggest that pericellular matrix-controlled liver FGFR4 in particular may ensure adequate cholesterol for cell structures and signal transduction.