Glucose and insulin induction of bile acid synthesis: mechanisms and implication in diabetes and obesity

Bile acids facilitate postprandial absorption of nutrients. Bile acids also activate the farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5 and play a major role in regulating lipid, glucose, and energy metabolism. Transgenic expression of cholesterol 7α-hydroxylase (CYP7A1) prevented high fat diet-induced diabetes and obesity in mice. In this study, we investigated the nutrient effects on bile acid synthesis. Refeeding of a chow diet to fasted mice increased CYP7A1 expression, bile acid pool size, and serum bile acids in wild type and humanized CYP7A1-transgenic mice. Chromatin immunoprecipitation assays showed that glucose increased histone acetylation and decreased histone methylation on the CYP7A1 gene promoter. Refeeding also induced CYP7A1 in fxr-deficient mice, indicating that FXR signaling did not play a role in postprandial regulation of bile acid synthesis. In streptozocin-induced type I diabetic mice and genetically obese type II diabetic ob/ob mice, hyperglycemia increased histone acetylation status on the CYP7A1 gene promoter, leading to elevated basal Cyp7a1 expression and an enlarged bile acid pool with altered bile acid composition. However, refeeding did not further increase CYP7A1 expression in diabetic mice. In summary, this study demonstrates that glucose and insulin are major postprandial factors that induce CYP7A1 gene expression and bile acid synthesis. Glucose induces CYP7A1 gene expression mainly by epigenetic mechanisms. In diabetic mice, CYP7A1 chromatin is hyperacetylated, and fasting to refeeding response is impaired and may exacerbate metabolic disorders in diabetes.     

Activation of farnesoid X receptor attenuates hepatic injury in a murine model of alcoholic liver disease

Alcoholic liver disease (ALD) is a common cause of advanced liver disease, and considered as a major risk factor of morbidity and mortality worldwide. Hepatic cholestasis is a pathophysiological feature observed in all stages of ALD. The farnesoid X receptor (FXR) is a member of the nuclear hormone receptor superfamily, and plays an essential role in the regulation of bile acid, lipid and glucose homeostasis. However, the role of FXR in the pathogenesis and progression of ALD remains largely unknown. Mice were fed Lieber-DeCarli ethanol diet or an isocaloric control diet. We used a specific agonist of FXR WAY-362450 to study the effect of pharmacological activation of FXR in alcoholic liver disease. In this study, we demonstrated that FXR activity was impaired by chronic ethanol ingestion in a murine model of ALD. Activation of FXR by specific agonist WAY-362450 protected mice from the development of ALD. We also found that WAY-362450 treatment rescued FXR activity, suppressed ethanol-induced Cyp2e1 up-regulation and attenuated oxidative stress in liver. Our results highlight a key role of FXR in the modulation of ALD development, and propose specific FXR agonists for the treatment of ALD patients.     

Loss of small heterodimer partner expression in the liver protects against dyslipidemia

Multiple studies suggest increased conversion of cholesterol to bile acids by cholesterol 7alpha-hydroxylase (CYP7A1) protects against dyslipidemia and atherosclerosis. CYP7A1 expression is repressed by the sequential activity of two nuclear hormone receptors, farnesoid X receptor (FXR) and small heterodimer partner (SHP). Here we demonstrate 129 strain SHP(-/-) mice are protected against hypercholesterolemia resulting from either a cholesterol/cholic acid (chol/CA) diet or from hypothyroidism. In a mixed 129-C57Bl/6 background, LDLR(-/-) and LDLR(-/-)SHP(-/-) mice had nearly identical elevations in hepatic cholesterol content and repression of cholesterol regulated genes when fed a Western diet. However, the LDLR(-/-)SHP(-/-) mice had greatly reduced elevations in serum VLDL and LDL cholesterol levels and triglyceride (TG) levels as compared with LDLR(-/-) mice. Additionally, the hepatic inflammation produced by the Western diet in the LDLR(-/-) mice was abolished in the LDLR(-/-)SHP(-/-) mice. CYP7A1 expression was induced 10-fold by the Western diet in the LDLR(-/-)SHP(-/-) mice but not in the LDLR(-/-) mice. Finally, hepatocyte-specific deletion of SHP expression was also protective against dyslipidemia induced by either a chol/CA diet or by hypothyroidism. While no antagonist ligands have yet been identified for SHP, these results suggest selective inhibition of hepatic SHP expression may provide protection against dyslipidemia.     

Differential regulation of hepatic cytochrome P450 monooxygenases in streptozotocin-induced diabetic rats

The present investigation was carried out to study the expression of major cytochrome P450 (CYP) isozymes in streptozotocin-induced diabetes with concomitant insulin therapy. Male Sprague-Dawley rats were randomly assigned to untreated control, streptozotocin-induced diabetic, insulin-treated groups and monitored for 4 weeks. Uncontrolled hyperglycemia in the early phase of diabetes resulted in differential regulation of cytochrome P450 isozymes. CYP1B1, CYP1A2, heme oxygenase (HO)-2 proteins and CYP1A2-dependent 7-ethoxyresorufin O-deethylase (EROD) activity were upregulated in the hepatic microsomes of diabetic rats. Insulin therapy ameliorated EROD activity and the expression of CYP1A2, CYP1B1 and HO-2 proteins. In addition, CYP2B1 and 2E1 proteins were markedly induced in the diabetic group. Insulin therapy resulted in complete amelioration of CYP2E1 whereas CYP2B1 protein was partially ameliorated. By contrast, CYP2C11 protein was decreased over 99% in the diabetic group and was partially ameliorated by insulin therapy. These results demonstrate widespread alterations in the expression of CYP isozymes in diabetic rats that are ameliorated by insulin therapy.     

Cerebrotendinous xanthomatosis: An inborn error in bile acid synthesis with defined mutations but still a challenge

Cerebrotendinous xanthomatosis [CTX] is a rare disease characterized by the accumulation of cholesterol and cholestanol in brain and tendons caused by a mutation in the sterol 27-hydroxylase gene [CYP27A1] involved in bile acid synthesis. Disruption of this gene in mice does not give rise to xanthomas. The gene defect leads to reduced bile acid synthesis with a compensatory increase in the activity of the rate-limiting enzyme in bile acid synthesis, cholesterol 7α-hydroxylase. This leads to a marked accumulation of 7α-hydroxylated bile acid precursors, in particular 7α-hydroxy-4-cholesten-3-one. The latter oxysterol passes the blood-brain barrier and is an efficient precursor to cholestanol. The activity of cholesterol 7α-hydroxylase is normalized by treatment with bile acids. Such treatment reduces the xanthomas in CTX patients in parallel with decreased cholestanol levels. The relationship between the accumulation of cholestanol and the development of cholesterol-rich xanthomas has however not been clarified and a suitable animal model is still lacking.     

Lowering bile acid pool size with a synthetic farnesoid X receptor (FXR) agonist induces obesity and diabetes through reduced energy expenditure

We evaluated the metabolic impact of farnesoid X receptor (FXR) activation by administering a synthetic FXR agonist (GW4064) to mice in which obesity was induced by a high fat diet. Administration of GW4064 accentuated body weight gain and glucose intolerance induced by the high fat diet and led to a pronounced worsening of the changes in liver and adipose tissue. Mechanistically, treatment with GW4064 decreased bile acid (BA) biosynthesis, BA pool size, and energy expenditure, whereas reconstitution of the BA pool in these GW4064-treated animals by BA administration dose-dependently reverted the metabolic abnormalities. Our data therefore suggest that activation of FXR with synthetic agonists is not useful for long term management of the metabolic syndrome, as it reduces the BA pool size and subsequently decreases energy expenditure, translating as weight gain and insulin resistance. In contrast, expansion of the BA pool size, which can be achieved by BA administration, could be an interesting strategy to manage the metabolic syndrome.     

Differential regulation of NMDA receptor function by DJ‐1 and PINK1

Dysfunction of PTEN‐induced kinase 1 (PINK1) or DJ‐1 promotes neuronal death and is implicated in the pathogenesis of Parkinson’s disease, but the underlying mechanisms remain unclear. Given the roles of N‐methyl‐d‐ aspartate receptor (NMDAr)‐mediated neurotoxicity in various brain disorders including cerebral ischemia and neurodegenerative diseases, we investigated the effects of PINK1 and DJ‐1 on NMDAr function. Using protein overexpression and knockdown approaches, we showed that PINK1 increased NMDAr‐mediated whole‐cell currents by enhancing the function of NR2A‐containing NMDAr subtype (NR2ACNR). However, DJ‐1 decreased NMDAr‐mediated currents, which was mediated through the inhibition of both NR2ACNR and NR2B‐containing NMDAr subtype (NR2BCNR). We revealed that the knockdown of DJ‐1 enhanced PTEN expression, which not only potentiated NR2BCNR function but also increased PINK1 expression that led to NR2ACNR potentiation. These results indicate that NMDAr function is differentially regulated by DJ‐1‐dependent signal pathways DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR. Our results further showed that the suppression of DJ‐1, while promoted NMDA‐induced neuronal death through the overactivation of PTEN/NR2BCNR‐dependent cell death pathway, induced a neuroprotective effect to counteract DJ‐1 dysfunction‐mediated neuronal death signaling through activating PTEN/PINK1/NR2ACNR cell survival–promoting pathway. Thus, PINK1 acts with DJ‐1 in a common pathway to regulate NMDAr‐mediated neuronal death. This study suggests that the DJ‐1/PTEN/NR2BCNR and DJ‐1/PTEN/PINK1/NR2ACNR pathways may represent potential therapeutic targets for the development of neuroprotection strategy in the treatment of brain injuries and neurodegenerative diseases such as Parkinson’s disease.     

Conformationally constrained farnesoid X receptor (FXR) agonists: alternative replacements of the stilbene

To further explore the optimum placement of the acid moiety in conformationally constrained analogs of GW 4064 1a, a series of stilbene replacements were prepared. The benzothiophene 1f and the indole 1g display the optimal orientation of the carboxylate for enhanced FXR agonist potency.     

Conformationally constrained farnesoid X receptor (FXR) agonists: heteroaryl replacements of the naphthalene

To improve on the drug properties of GSK8062 1b, a series of heteroaryl bicyclic naphthalene replacements were prepared. The quinoline 1c was an equipotent FXR agonist with improved drug developability parameters relative to 1b. In addition, analog 1c lowered body weight gain and serum glucose in a DIO mouse model of diabetes.     

Ethanol stimulates bile acid formation in primary human hepatocytes

The conversion of cholesterol to bile acids is a key pathway for elimination of cholesterol from the body, thereby reducing the risk of arteriosclerosis. Moderate consumption of ethanol has been shown to have preventive effects on cardiovascular disease and decrease the risk of gallstone formation. In the present study primary human hepatocytes were used to investigate if ethanol affected bile acid synthesis. Hepatocytes were prepared from donor liver (n = 11) and treated with ethanol, 7.7 or 50 mM, for 24 h. mRNA levels for enzymes in bile acid synthesis pathways were studied and bile acid synthesis was analyzed. Treatment with 7.7 mM ethanol increased cholic acid synthesis by 20% and treatment with 50 mM ethanol up-regulated cholic acid formation by 60%. The synthesis of cholic acid increased more than that of chenodeoxycholic acid, indicating that the classical pathway for bile acid synthesis was up-regulated. Increased bile acid levels in the cells treated with ethanol were seen after approximately 20 h. mRNA expression of CYP7A1, CYP27A1, and CYP8B1 in the hepatocytes was not affected by alcohol exposure.     

Molecular control of systemic bile acid homeostasis by the liver glucocorticoid receptor

Systemic bile acid (BA) homeostasis is a critical determinant of dietary fat digestion, enterohepatic function, and postprandial thermogenesis. However, major checkpoints for the dynamics and the molecular regulation of BA homeostasis remain unknown. Here we show that hypothalamic-pituitary-adrenal (HPA) axis impairment in humans and liver-specific deficiency of the glucocorticoid receptor (GR) in mice disrupts the normal changes in systemic BA distribution during the fasted-to-fed transition. Fasted mice with hepatocyte-specific GR knockdown had smaller gallbladder BA content and were more susceptible to developing cholesterol gallstones when fed a cholesterol-rich diet. Hepatic GR deficiency impaired liver BA uptake/transport via lower expression of the major hepatocyte basolateral BA transporter, Na(+)-taurocholate transport protein (Ntcp/Slc10a1), which affected dietary fat absorption and brown adipose tissue activation. Our results demonstrate a role of the HPA axis in the endocrine regulation of BA homeostasis through the liver GR control of enterohepatic BA recycling.     

Complementary roles of farnesoid X receptor,pregnane X receptor,and constitutive androstane receptor in protection against bile acid toxicity

The nuclear receptors, farnesoid X receptor (FXR) and pregnane X receptor (PXR), are important in maintaining bile acid homeostasis. Deletion of both FXR and PXR in vivo by cross-breeding B6;129-Fxrtm1Gonz (FXR-null) and B6;129-Pxrtm1Glaxo-Wellcome (PXR-null) mice revealed a more severe disruption of bile acid, cholesterol, and lipid homeostasis in B6;129-Fxrtm1Gonz Pxrtm1Glaxo-Wellcome (FXR-PXR double null or FPXR-null) mice fed a 1% cholic acid (CA) diet. Hepatic expression of the constitutive androstane receptor (CAR) and its target genes was induced in FXR- and FPXR-null mice fed the CA diet. To test whether up-regulation of CAR represents a means of protection against bile acid toxicity to compensate for the loss of FXR and PXR, animals were pretreated with CAR activators, phenobarbital or 1,4-bis[2-(3,5-dichlorpyridyloxy)]benzene (TCPOBOP), followed by the CA diet. A role for CAR in protection against bile acid toxicity was confirmed by a marked reduction of serum bile acid and bilirubin concentrations, with an elevation of the expression of the hepatic genes involved in bile acid and/or bilirubin metabolism and excretion (CYP2B, CYP3A, MRP2, MRP3, UGT1A, and glutathione S-transferase alpha), following pretreatment with phenobarbital or TCPOBOP. In summary, the current study demonstrates a critical and combined role of FXR and PXR in maintaining not only bile acid but also cholesterol and lipid homeostasis in vivo. Furthermore, FXR, PXR, and CAR protect against hepatic bile acid toxicity in a complementary manner, suggesting that they serve as redundant but distinct layers of defense to prevent overt hepatic damage by bile acids during cholestasis.     

Complex interaction between circadian rhythm and diet on bile acid homeostasis in male rats

Desynchronization between the master clock in the brain, which is entrained by (day) light, and peripheral organ clocks, which are mainly entrained by food intake, may have negative effects on energy metabolism. Bile acid metabolism follows a clear day/night rhythm. We investigated whether in rats on a normal chow diet the daily rhythm of plasma bile acids and hepatic expression of bile acid metabolic genes is controlled by the light/dark cycle or the feeding/fasting rhythm. In addition, we investigated the effects of high caloric diets and time-restricted feeding on daily rhythms of plasma bile acids and hepatic genes involved in bile acid synthesis. In experiment 1 male Wistar rats were fed according to three different feeding paradigms: food was available ad libitum for 24 h (ad lib) or time-restricted for 10 h during the dark period (dark fed) or 10 h during the light period (light fed). To allow further metabolic phenotyping, we manipulated dietary macronutrient intake by providing rats with a chow diet, a free choice high-fat-high-sugar diet or a free choice high-fat (HF) diet. In experiment 2 rats were fed a normal chow diet, but food was either available in a 6-meals-a-day (6M) scheme or ad lib. During both experiments, we measured plasma bile acid levels and hepatic mRNA expression of genes involved in bile acid metabolism at eight different time points during 24 h. Time-restricted feeding enhanced the daily rhythm in plasma bile acid concentrations. Plasma bile acid concentrations are highest during fasting and dropped during the period of food intake with all diets. An HF-containing diet changed bile acid pool composition, but not the daily rhythmicity of plasma bile acid levels. Daily rhythms of hepatic Cyp7a1 and Cyp8b1 mRNA expression followed the hepatic molecular clock, whereas for Shp expression food intake was leading. Combining an HF diet with feeding in the light/inactive period annulled CYp7a1 and Cyp8b1 gene expression rhythms, whilst keeping that of Shp intact. In conclusion, plasma bile acids and key genes in bile acid biosynthesis are entrained by food intake as well as the hepatic molecular clock. Eating during the inactivity period induced changes in the plasma bile acid pool composition similar to those induced by HF feeding.     

Identification of intermediates in the bile acid synthetic pathway as ligands for the farnesoid X receptor

Bile acid synthesis from cholesterol is tightly regulated via a feedback mechanism mediated by the farnesoid X receptor (FXR), a nuclear receptor activated by bile acids. Synthesis via the classic pathway is initiated by a series of cholesterol ring modifications and followed by the side chain cleavage. Several intermediates accumulate or are excreted as end products of the pathway in diseases involving defective bile acid biosynthesis. In this study, we investigated the ability of these intermediates to activate human FXR. In a cell-based reporter assay and coactivator recruitment assays in vitro, early intermediates possessing an intact cholesterol side chain were inactive, whereas 26- or 25-hydroxylated bile alcohols and C27 bile acids were highly efficacious ligands for FXR at a level comparable to that of the most potent physiological ligand, chenodeoxycholic acid. Treatment of HepG2 cells with these precursors repressed the rate-limiting cholesterol 7alpha-hydroxylase mRNA level and induced the small heterodimer partner and the bile salt export pump mRNA, indicating the ability to regulate bile acid synthesis and excretion. Because 26-hydroxylated bile alcohols and C27 bile acids are known to be evolutionary precursors of bile acids in mammals, our findings suggest that human FXR may have retained affinity to these precursors during evolution.