FXR Activation Research Articles

PS-038-A humanized bile acid pool induced by somatic CRISPR/Cas9-mediated deletion of Cyp2c70 modulates effects of pharmacological FXR activation in mice

PS-038-A humanized bile acid pool induced by somatic CRISPR/Cas9-mediated deletion of Cyp2c70 modulates effects of pharmacological FXR activation in mice

Autophagy Deficiency in the Liver Altered Pathogenesis of Alcoholic Liver Disease and Profile of Gut Microbiota

Autophagy is an evolutionarily conserved cellular degradation process that is critical to maintain metabolism homeostasis in the liver. To investigate the role of autophagy in liver diseases, we examined the impact of autophagy deficiency on ethanol‐induced liver injury and the gut‐liver relationship. First, hepatic autophagy deficient mice were challenged with ethanol by either acute or acute‐on‐chronic scheme. Our results demonstrated a protective role of autophagy in ethanol‐induced liver injury in a genetic and an inducible hepatic Atg7 knockout mice. Surprisingly, hepatic Atg5 knockout mice, which had a minor presentation of liver injury, were resistant to acute ethanol treatment, and were actually improved in the liver presentation following the treatment of in the chronic‐plus‐binge regime. A series of ethanol metabolism‐related genes were altered in livers of ethanol‐fed Atg5Δhep mice, which was accompanied with a faster clearance of plasma ethanol. This may partially explain the seemingly paradoxical observations. Second, gut microbiota was analyzed in fecal samples from hepatic autophagy deficient mice by 16S sequencing. The profile of gut microbiota was altered by hepatic autophagy deficiency with a notable increase of bile acids metabolism related bacteria. In addition to this, significant increase of total bile acids was observed in serum, liver, and feces. Moreover, ileal FXR was activated as increased gene expression of its target genes, including Shp, Fgf15 and Ibabp. Finally, modulation of gut microbiota by antibiotics treatment suppressed ileal FXR activation, increased enterohepatic bile acids pool, and aggravated liver injury in hepatic autophagy‐deficient mice. Collectively, our results indicate that hepatic autophagy has significant impacts on liver homeostasis and gut microbiota. The alteration of gut microbiota in hepatic autophagy deficiency can have in turn notable effects on liver injury.Support or Funding InformationThis research was partially supported by NIH R01AA021751 (X.‐M.Y.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Hepatic Autophagy Deficiency Compromises Farnesoid X Receptor Functionality and Causes Cholestatic Injury

Autophagy is important for hepatic homeostasis, nutrient regeneration, and organelle quality control. We investigated the mechanisms by which liver injury occurred in the absence of autophagy function. We found that mice deficient in autophagy because of the lack of autophagy-related gene 7 or autophagy-related gene 5, key autophagy-related genes, manifested intracellular cholestasis with increased levels of serum bile acids, a higher ratio of tauromuricholic acid/taurocholic acid in the bile, increased hepatic bile acid load, abnormal bile canaliculi, and altered expression of hepatic transporters. In determining the underlying mechanism, we found that autophagy sustained and promoted the basal and up-regulated expression of farnesoid X receptor (Fxr) in the fed and starved conditions, respectively. Consequently, expression of Fxr and its downstream genes, particularly bile salt export pump, and the binding of FXR to the promoter regions of these genes, were suppressed in autophagy-deficient livers. In addition, codeletion of nuclear factor erythroid 2-related factor 2 (Nrf2) in autophagy deficiency status reversed the FXR suppression. Furthermore, the cholestatic injury of autophagy-deficient livers was reversed by enhancement of FXR activity or expression, or by Nrf2 deletion. Conclusion: Together with earlier reports that FXR can suppress autophagy, our findings indicate that autophagy and FXR form a regulatory loop and deficiency of autophagy causes abnormal FXR functionality, leading to the development of intracellular cholestasis and liver injury.

In-silico Evaluation of Rare Codons and their Positions in the Structure of ATP8b1 Gene

Progressive familial intrahepatic cholestases (PFIC) are a spectrum of autosomal progressive liver diseases developing to end-stage liver disease. ATP8B1 deficiency caused by mutations in ATP8B1 gene encoding a P-type ATPase leads to PFIC1. The gene for PFIC1 has been mapped on a 19-cM region of 18q21-q22, and a gene defect in ATP8B1 can cause deregulations in bile salt transporters through decreased expression and/or activity of FXR. Point mutations are the most common, with the majority being missense or nonsense mutations. In addition, approximately 15% of disease-causing ATP8B1 mutations are annotated as splicing disrupting alteration given that they are located at exon-intron borders. Here, we describe the hidden layer of computational biology information of rare codons in ATP8B1, which can help us for drug design. Some rare codons in different locations of ATP8b1 gene were identified using several web servers and by in-silico modelling of ATP8b1 in Phyre2 and I-TASSER server, some rare codons were evaluated. Some of these rare codons were located at special positions which seem to have a critical role in proper folding of ATP8b1 protein. Structural analysis showed that some of rare codons are related to mutations in ATP8B1 that are responsible for PFIC1 disease, which may have a critical role in ensuring the correct folding. Investigation of such hidden information can enhance our understanding of ATP8b1 folding. Moreover, studies of these rare codons help us to clarify their role in rational design of new and effective drugs.

Targeting FXR in Cholestasis.

The farnesoid X receptor (FXR, NR1H4) is a bile acid (BA)-activated transcription factor, which is essential for BA homeostasis. FXR and its hepatic and intestinal target genes, small heterodimer partner (SHP, NR0B2) and fibroblast growth factor 15/19 (Fgf15 in mice, FGF19 in humans), transcriptionally regulate BA synthesis, detoxification, secretion, and absorption in the enterohepatic circulation. Furthermore, FXR modulates a large variety of physiological processes, such as lipid and glucose homeostasis as well as the inflammatory response. Targeted deletion of FXR renders mice highly susceptible to cholic acid feeding resulting in cholestatic liver injury, weight loss, and increased mortality. Combined deletion of FXR and SHP spontaneously triggers early-onset intrahepatic cholestasis in mice resembling human progressive familial intrahepatic cholestasis (PFIC). Reduced expression levels and activity of FXR have been reported in human cholestatic conditions, such as PFIC type 1 and intrahepatic cholestasis of pregnancy. Recently, two pairs of siblings with homozygous FXR truncation or deletion variants were identified. All four children suffered from severe, early-onset PFIC and liver failure leading to death or need for liver transplantation before the age of 2. These findings underscore the central role of FXR as regulator of systemic and hepatic BA levels. Therefore, targeting FXR has been exploited in different animal models of both intrahepatic and obstructive cholestasis, and the first FXR agonist obeticholic acid (OCA) has been approved for the treatment of primary biliary cholangitis (PBC). Further FXR agonists as well as a FGF19 analogue are currently tested in clinical trials for different cholestatic liver diseases. This chapter will summarize the current knowledge on the role of FXR in cholestasis both in rodent models and in human diseases.

Activation of the Nuclear Receptor Fxr Improves Intestinal Cell Tolerance to Ischemia-Reperfusion Injury.

The farnesoid X receptor (FXR) plays an important role in bile acid metabolism, intestinal homeostasis, and intestinal ischemia-reperfusion (I/R) injury. We aimed to clarify the potential effects of FXR on intestinal epithelial cell tolerance to intestinal I/R injury and reveal the underlying mechanisms. An intestinal I/R injury model was established by the occlusion of the superior mesenteric artery for ischemia for 1 h, followed by reperfusion for 4 h in C57BL/6 (wild type [WT]) and FXR mice. The small intestine injury was assessed by histological analysis. Diamine oxidase and TNF-α levels in the serum were measured. Expressions of Bcl-2, Bax, caspase-3, and cystathionine-γ-lyase (CSE) were determined by immunohostochemical staining. Oxygen-glucose deprivation/reperfusion (OGD/R) was used to make injury in cultured Caco-2 cells pretreated with FXR agonist (INT-747) or DL-propargylglycine (PAG) for 24 h. Cell viability and the expressions of NF-κB, TNF-α, and IL-6 were assessed. Compared with WT I/R mice, FXR knockout mice exacerbated intestinal I/R injury, intestinal epithelial apoptosis, and inflammatory response. The I/R injury in WT mice was alleviated with INT-747 pretreatment. CSE expression increased after intestinal I/R injury in WT but not in FXR mice. INT-747 enhanced Caco-2 cell viability and inhibited inflammatory response by blocking the NF-κB pathway after OGD/R injury, which was diminished by a CSE-specific inhibitor (PAG). Thus, we demonstrated that FXR activation enhances intestinal epithelial cell tolerance to I/R by suppressing the inflammatory response and NF-κB pathway via CSE mediation.

The nuclear bile acid receptor FXR is a PKA- and FOXA2-sensitive activator of fasting hepatic gluconeogenesis

Embedded into a complex signaling network that coordinates glucose uptake, usage and production, the nuclear bile acid receptor FXR is expressed in several glucose-processing organs including the liver. Hepatic gluconeogenesis is controlled through allosteric regulation of gluconeogenic enzymes and by glucagon/cAMP-dependent transcriptional regulatory pathways. We aimed to elucidate the role of FXR in the regulation of fasting hepatic gluconeogenesis. The role of FXR in hepatic gluconeogenesis was assessed in vivo and in mouse primary hepatocytes. Gene expression patterns in response to glucagon and FXR agonists were characterized by quantitative reverse transcription PCR and microarray analysis. FXR phosphorylation by protein kinase A was determined by mass spectrometry. The interaction of FOXA2 with FXR was identified by cistromic approaches and in vitro protein-protein interaction assays. The functional impact of the crosstalk between FXR, the PKA and FOXA2 signaling pathways was assessed by site-directed mutagenesis, transactivation assays and restoration of FXR expression in FXR-deficient hepatocytes in which gene expression and glucose production were assessed. FXR positively regulates hepatic glucose production through two regulatory arms, the first one involving protein kinase A-mediated phosphorylation of FXR, which allowed for the synergistic activation of gluconeogenic genes by glucagon, agonist-activated FXR and CREB. The second arm involves the inhibition of FXR's ability to induce the anti-gluconeogenic nuclear receptor SHP by the glucagon-activated FOXA2 transcription factor, which physically interacts with FXR. Additionally, knockdown of Foxa2 did not alter glucagon-induced and FXR agonist enhanced expression of gluconeogenic genes, suggesting that the PKA and FOXA2 pathways regulate distinct subsets of FXR responsive genes. Thus, hepatic glucose production is regulated during physiological fasting by FXR, which integrates the glucagon/cAMP signal and the FOXA2 signal, by being post-translationally modified, and by engaging in protein-protein interactions, respectively. Activation of the nuclear bile acid receptor FXR regulates gene expression networks, controlling lipid, cholesterol and glucose metabolism, which are mostly effective after eating. Whether FXR exerts critical functions during fasting is unknown. The results of this study show that FXR transcriptional activity is regulated by the glucagon/protein kinase A and the FOXA2 signaling pathways, which act on FXR through phosphorylation and protein-protein interactions, respectively, to increase hepatic glucose synthesis.

Intestine farnesoid X receptor agonist and the gut microbiota activate G-protein bile acid receptor-1 signaling to improve metabolism.

This study uncovered a mechanism in which activation of intestinal FXR shaped the gut microbiota to activate TGR5/GLP-1 signaling to improve hepatic glucose and insulin sensitivity and increase adipose tissue browning; the gut microbiota plays a critical role in bile acid metabolism and signaling to regulate metabolic homeostasis in health and disease. (Hepatology 2018).

Effects of Farnesoid X Receptor Activation on Arachidonic Acid Metabolism, NF-kB Signaling, and Hepatic Inflammation.

Inflammation has a recognized role in nonalcoholic fatty liver disease (NAFLD) progression. In the present work, we studied the effect of high-fat diet (HFD) on arachidonic acid metabolism in the liver and investigated the role of the farnesoid X receptor (FXR, NR1H4) in eicosanoid biosynthetic pathways and nuclear factor κ light-chain enhancer of activated B cells (NF-kB) signaling, major modulators of the inflammatory cascade. Mice were fed an HFD to induce NAFLD and then treated with the FXR ligand obeticholic acid (OCA). Histology and gene expression analyses were performed on liver tissue. Eicosanoid levels were measured from serum and urine samples. The molecular mechanism underlying the effect of FXR activation on arachidonic acid metabolism and NF-kB signaling was studied in human liver Huh7 cells and primary cultured hepatocytes. NAFLD was characterized by higher (∼25%) proinflammatory [leukotrienes (LTB4)] and lower (∼3-fold) anti-inflammatory [epoxyeicosatrienoic acids (EETs)] eicosanoid levels than in chow mice. OCA induced the expression of several hepatic cytochrome P450 (P450) epoxygenases, the enzymes responsible for EET synthesis, and mitigated HFD-induced hepatic injury. In vitro, induction of CYP450 epoxygenases was sufficient to inhibit NF-kB signaling and cell migration. The CYP450 epoxygenase pan-inhibitor gemfibrozil fully abolished the protective effect of OCA, indicating that OCA-mediated inhibition of NF-kB signaling was EET-dependent. In summary, NAFLD was characterized by an imbalance in arachidonate metabolism. FXR activation reprogramed arachidonate metabolism by inducing P450 epoxygenase expression and EET production. In vitro, FXR-mediated NF-kB inhibition required active P450 epoxygenases.

Abstract 202: Zfp36l1 is a Post transcriptional Regulator of Hepatic Bile Acid and Lipid Metabolism

Bile acids are detergents and important signaling molecules that activate the nuclear receptor FXR to control key metabolic processes, including feedback mechanisms to maintain bile acid homeostasis. FXR is the central rheostat of bile acid metabolism, and activation of FXR decreases the mRNA levels of bile acid synthetic genes, including Cyp 7 a 1 , the gene encoding the rate-limiting enzyme of bile acid synthesis. We show that Cyp 7 a 1 mRNA levels were rapidly reduced after pharmacologic FXR activation in wild-type, but not Fxr – / – or liver-specific Fxr knockout mice ( Fxr L - KO ). The rapid decrease in Cyp 7 a 1 mRNA suggested a previously unidentified post-transcriptional mechanism. To identify the mechanism, we used synthetic and endogenous FXR agonists and found the RNA binding protein ZFP36L1 as a novel FXR target gene. ZFP36L1 mRNA and protein levels were increased as early as 30 minutes after FXR activation. ZFP36L1 is a bona - fide RNA binding protein that promotes degradation of mRNA targets by binding to AU-rich elements (AREs) in the 3’ UTR. We generated in vivo and in vitro gain-of-function models and we used reporter assays to show that ZFP36L1 targets the Cyp7a1 UTR . In mice, hepatic overexpression of ZFP36L1 decreased Cyp 7 a 1 mRNA and protein and decreased bile acid levels. To complement our gain-of-function studies, we generated liver-specific Zfp 36 l 1 knockout mice ( Zfp 36 l 1 L - KO ) and we show that loss of Zfp 36 l 1 resulted in elevated Cyp 7 a 1 mRNA and protein, and increased bile acid levels. Given that bile acids are important metabolites that control lipid absorption and signaling, we investigated whether loss of hepatic Zfp 36 l 1 resulted in more broad metabolic dysfunction. Western diet fed Zfp 36 l 1 L - KO mice had reduced body weight gain, specifically in adipose tissue depots compared to littermate Zfp 36 l 1 flox-flox mice. The differences in adiposity and steatosis were attributed to reduced lipid absorption, as Zfp 36 l 1 L - KO mice have increased fecal caloric content and reduced triglyceride absorption as determined by an intragastric fat tolerance test. The decreased lipid absorption is consistent with an altered bile acid metabolism. Thus, we have identified a novel pathway that controls Cyp 7 a 1 and bile acid metabolism but may also have wider implications in diseases such as obesity and hepatosteatosis.

550 - Concurrent PPARα and FXR Activation Mediates Nash Improvement after Gastric Bypass Surgery: A Geo Datasets Analysis

550 - Concurrent PPARα and FXR Activation Mediates Nash Improvement after Gastric Bypass Surgery: A Geo Datasets Analysis

FXR deletion in hepatocytes does not affect the severity of alcoholic liver disease in mice

Emerging evidence has shown that FXR activation ameliorates the development of alcoholic liver diseases (ALD) while whole-body deficiency of FXR in mice leads to more severe ALD. However, it’s unknown whether the enhanced susceptibility to ALD development in FXR−/− mice is due to deficiency of hepatic FXR or increased toxicity secondary to increased bile acid (BA) levels. Hepatocyte-specific FXR knockout mice (FXRhep−/−) present similar BA levels compared to wild-type mice, and are therefore a useful model to study a direct role of hepatic FXR in ALD development. FXRhep−/− mice were subject to an ALD model with chronic plus binge drinking of alcohol to determine the effects of hepatic FXR deficiency on ALD development. The FXRhep−/− mice showed an altered expression of genes involved in BA and lipid homeostasis with alcohol treatment. Despite a slightly increased trend in hepatic lipid deposition and collagen accumulation in FXRhep−/− mice, there were no significant differences in the severity of steatosis, inflammation, or fibrosis between WT and FXRhep−/− mice. Therefore, these findings indicate that FXR deficiency in hepatocytes might only play a minor role in ALD development. Deficiency of FXR in other non-hepatic tissues and/or increased BA levels resultant from whole-body FXR deficiency might be responsible for more severe ALD development.

ZFP36L1 is a post‐transcriptional regulator of lipid metabolism.

Bile acids are detergents and important signaling molecules that activate the nuclear receptor FXR to control key metabolic processes, including feedback mechanisms to maintain bile acid homeostasis. FXR agonists are currently promising therapeutic agents to treat non‐alcoholic steatohepatitis (NASH), a significant risk factor for cardiovascular disease. FXR is the central rheostat of bile acid metabolism, and activation of FXR decreases the mRNA levels of several bile acid synthetic genes, including the expression of the gene encoding the rate‐limiting enzyme of bile acid synthesis, Cyp7a1. We show that Cyp7a1 mRNA levels were rapidly reduced after pharmacologic FXR activation in wild‐type, but not Fxr−/− or liver‐specific Fxr knockout mice (FxrL‐KO). The rapid decrease in Cyp7a1 mRNA suggested a previously unidentified post‐transcriptional mechanism. To identify the molecular mechanism, we used FXR ChIP‐Seq and RNA‐Seq to identify the RNA binding protein Zfp36l1 as a novel FXR target gene. We showed that FXR activation using synthetic and endogenous agonists increases ZFP36L1 mRNA and protein levels in wild‐type, but not Fxr−/− mice. Further, the increased ZFP36L1 mRNA and protein was observed as early as 30 minutes after FXR activation. ZFP36L1 is a bona‐fide RNA binding protein that promotes degradation of mRNA targets by binding to AU‐rich elements (AREs) in the 3′ UTR. We generated in vivo and in vitro gain‐of‐function models and we used reporter assays to show that ZFP36L1 targets the Cyp7a1 UTR. In mice, hepatic overexpression of ZFP36L1 decreased Cyp7a1 mRNA and protein and decreased bile acid levels. To complement our gain‐of‐function studies, we generated liver‐specific Zfp36l1 knockout mice (Zfp36l1L‐KO) and we show that loss of Zfp36l1 resulted in elevated Cyp7a1 mRNA and protein, and increased bile acid levels. Given that bile acids are important metabolites that control lipid absorption and signaling, we investigated whether loss of hepatic Zfp36l1 resulted in more broad metabolic dysfunction. Western diet fed Zfp36l1L‐KO mice had reduced body weight gain, specifically in adipose tissue depots compared to littermate Zfp36l1flox‐flox mice. In addition, Zfp36l1L‐KO mice also had reduced hepatic steatosis, without any differences in energy expenditure compared to littermate Zfp36l1flox‐flox mice. The differences in adiposity and steatosis were attributed to reduced lipid absorption, as Zfp36l1L‐KO mice have increased fecal caloric content and reduced triglyceride absorption as determined by an intragastric fat tolerance test. The decreased lipid absorption is consistent with an altered bile acid metabolism. Thus, we have identified a novel pathway that controls Cyp7a1 and bile acid metabolism but may also have wider implications in diseases such as obesity and hepatosteatosis, which are both significant risk factors for cardiovascular disease.Support or Funding InformationNIH grants HL118161 and HL136543 to E.J.T., and AHA grant SDG18440015 and NIH grants HL122677 and DK102559 to T.Q.de A.V.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Cenicriviroc, a cytokine receptor antagonist, potentiates all-trans retinoic acid in reducing liver injury in cholestatic rodents.

Cholestatic liver injury is mediated by bile acid-induced inflammatory responses. We hypothesized that superior therapeutic effects might be achieved by combining treatments that reduce the bile acid pool size with one that blocks inflammation. Bile duct-ligated (BDL) rats and Mdr2(Abcb4)-/- mice were treated with all-trans retinoic acid (atRA), a potent inhibitor of bile acid synthesis, 5mg/kg/d by gavage, or Cenicriviroc (CVC), a known antagonist of CCR2 and CCR5, 50mg/kg/d alone or in combination for 14 days and 1month respectively. All-trans retinoic acid alone reduced bile acid pool size and liver necrosis in BDL rats. However, the combination with CVC further reduced liver to body weight ratio, bile acid pool size, plasma liver enzyme, bilirubin, liver necrosis and fibrosis when compared to the atRA treatment. The assessment of hepatic hydroxyproline content further confirmed the reduced liver injury concurrent with reduction of pro-inflammatory cytokines emphasizing the synergistic effects of these two agents. Profiling of hepatic inflammatory cells revealed that combination therapy reduced neutrophils and T cells but not macrophages. The superior therapeutic effects of combination treatment were also confirmed in Mdr2-/- mice where a significant reduction in plasma liver enzymes, bilirubin, liver fibrosis, bile duct proliferation and hepatic infiltration of neutrophils and T cells and expression of cytokines were found. Multitargeted therapy is an important paradigm for treating cholestatic liver injury. The combination of CVC with atRA or other FXR activators may warrant a clinical trial in patients with cholestatic liver disease.

A gut–brain axis regulating glucose metabolism mediated by bile acids and competitive fibroblast growth factor actions at the hypothalamus

ObjectiveBile acids have been implicated as important regulators of glucose metabolism via activation of FXR and GPBAR1. We have previously shown that FGF19 can modulate glucose handling by suppressing the activity of hypothalamic AGRP/NPY neurons. As bile acids stimulate the release of FGF19/FGF15 into the circulation, we pursued the potential of bile acids to improve glucose tolerance via a gut–brain axis involving FXR and FGF15/FGF19 within enterocytes and FGF receptors on hypothalamic AGRP/NPY neurons. MethodsA 5-day gavage of taurocholic acid, mirroring our previous protocol of a 5-day FGF19 treatment, was performed. Oral glucose tolerance tests in mice with genetic manipulations of FGF signaling and melanocortin signaling were used to define a gut–brain axis responsive to bile acids. ResultsThe taurocholic acid gavage led to increased serum concentrations of taurocholic acid as well as increases of FGF15 mRNA in the ileum and improved oral glucose tolerance in obese (ob/ob) mice. In contrast, lithocholic acid, an FXR antagonist but a potent agonist for GPBAR1, did not improve glucose tolerance. The positive response to taurocholic acid is dependent upon an intact melanocortinergic system as obese MC4R-null mice or ob/ob mice without AGRP did not show improvements in glucose tolerance after taurocholate gavage. We also tested the FGF receptor isoform necessary for the bile acid response, using AGRP:Fgfr1−/− and AGRP:Fgfr2−/− mice. While the absence of FGFR1 in AGRP/NPY neurons did not alter glucose tolerance after taurocholate gavage, manipulations of Fgfr2 caused bidirectional changes depending upon the experimental model. We hypothesized the existence of an endogenous hypothalamic FGF, most likely FGF17, that acted as a chronic activator of AGRP/NPY neurons. We developed two short peptides based on FGF8 and FGF17 that should antagonize FGF17 action. Both of these peptides improved glucose homeostasis after a 4-day course of central and peripheral injections. Significantly, daily average blood glucose from continuous glucose monitoring was reduced in all tested animals but glucose concentrations remained in the euglycemia range. ConclusionsWe have defined a gut–brain axis that regulates glucose metabolism mediated by antagonistic fibroblast growth factors. From the intestine, bile acids stimulate FGF15 secretion, leading to activation of the FGF receptors in hypothalamic AGRP/NPY neurons. FGF receptor intracellular signaling subsequently silences AGRP/NPY neurons, leading to improvements of glucose tolerance that are likely mediated by the autonomic nervous system. Finally, short peptides that antagonize homodimeric FGF receptor signaling within the hypothalamus have beneficial effects on glucose homeostasis without inducing hypoglycemia. These peptides could provide a new mode of regulating glucose metabolism.

Glycyrrhetic Acid Derivative TY501 Protects Against Lithocholic Acid-Induced Cholestasis.

The aim of the study is to investigate the protective effects of TY501 against LCA-induced cholestasis in mice and to explore the potential mechanisms. It was demonstrated that TY501(5, 15 or 45 mg/kg, i.g.) can markedly reduced the level of ALT, AST and ALP which increased by LCA treatment. Meanwhile, TY501 also lowered total bile acids, total bilirubin and total cholesterol levels in serum. Furthermore, TY501 can protect HepG2 cell cultures from LCA-induced cytotoxicity. RT-PCR and Western Blot analysis showed that TY501 recovered the expression of BSEP, MRP2 and NTCP which were down-regulated by LCA. Moreover, mRNA and protein of FXR was also observed in TY501 treated mice significantly accumulation in nucleus. Taken together, It can be concluded that TY501 exerted beneficial effects on LCA-induced cholestasis, possibly via activation of FXR mediated upregulation of BSEP, MRP2 and NTCP.

Impaired vagus function in rats suppresses bile acid synthesis in the liver by disrupting tight junctions and activating Fxr-Fgf15 signaling in the intestine

Bile acids (BAs) circulate between the liver and intestine, and regulate the homeostasis of glucose, lipid, and energy. Recent studies demonstrated an essential role of BAs in neurological diseases, suggesting an interaction between BAs and the nervous system. In the present study, we showed that impaired vagus function in rats induced by vagotomy resulted in an increase in bile flow without causing liver injury. The concentrations of unconjugated and glycine-conjugated BAs were increased in both serum and bile of rats after vagotomy, which was due to impaired tight junctions and thus increased passive absorption of BAs in the intestine. Vagotomy markedly suppressed the expression of the rate-limiting BA synthetic enzyme Cyp7a1, which was not due to activation of Fxr-Shp signaling in the liver, but due to activation of Fxr-Fgf15 signaling in the intestine. Furthermore, vagotomy produced a BA profile in the bile favorable for Fxr activation by decreasing tauro-β-muricholic acid, a natural Fxr antagonist, and increasing glyco-chenodeoxycholic acid, a natural Fxr agonist. In summary, the present study provides the first comprehensive analysis of the critical role of the vagus nerve in regulating BA metabolism and signaling pathway.

Organophosphate Flame-Retardants Alter Adult Mouse Homeostasis and Gene Expression in a Sex-Dependent Manner Potentially Through Interactions With ERα.

Flame retardants (FRs) such as polybrominated diphenyl ethers and organophosphate FR (OPFR) persist in the environment and interact with multiple nuclear receptors involved in homeostasis, including estrogen receptors (ERs). However, little is known about the effects of FR, especially OPFR, on mammalian neuroendocrine functions. Therefore, we investigated if exposure to FR alters hypothalamic gene expression and whole-animal physiology in adult wild-type (WT) and ERα KO mice. Intact WT and KO males and ovariectomized WT and KO females were orally dosed daily with vehicle (oil), 17α-ethynylestradiol (2.5 μg/kg), 2,2', 4,4-tetrabromodiphenyl ether (BDE-47, 1 or 10 mg/kg), or an OPFR mixture {1 or 10 mg/kg of tris(1, 3-dichloro-2-propyl)phosphate, triphenyl phosphate, and tricresyl phosphate each} for 28 days. Body weight, food intake, body composition, glucose and insulin tolerance, plasma hormone levels, and hypothalamic and liver gene expression were measured. Expression of neuropeptides, receptors, and cation channels was differentially altered between WT males and females. OPFR suppressed body weight and energy intake in males. FR increased fasting glucose levels in males, and BDE-47 augmented glucose clearance in females. Liver gene expression indicated FXR activation by BDE-47 and PXR and CAR activation by OPFR. In males, OPFR increased ghrelin but decreased leptin and insulin independent of body weight. The loss of ERα reduced the effects of both FR on hypothalamic and liver gene expression and plasma hormone levels. The physiological implications are that males are more sensitive than ovariectomized females to OPFR exposure and that these effects are mediated, in part, by ERα.

Differential effects of FXR or TGR5 activation in cholangiocarcinoma progression

Background and aimsCholangiocarcinoma (CCA) is an aggressive tumor type affecting cholangiocytes. CCAs frequently arise under certain cholestatic liver conditions. Intrahepatic accumulation of bile acids may facilitate cocarcinogenic effects by triggering an inflammatory response and cholangiocyte proliferation. Here, the role of bile acid receptors FXR and TGR5 in CCA progression was evaluated. MethodsFXR and TGR5 expression was determined in human CCA tissues and cell lines. An orthotopic model of CCA was established in immunodeficient mice and tumor volume was monitored by magnetic resonance imaging under chronic administration of the specific FXR or TGR5 agonists, obeticholic acid (OCA) or INT-777 (0,03% in chow; Intercept Pharmaceuticals), respectively. Functional effects of FXR or TGR5 activation were evaluated on CCA cells in vitro. ResultsFXR was downregulated whereas TGR5 was upregulated in human CCA tissues compared to surrounding normal liver tissue. FXR expression correlated with tumor differentiation and TGR5 correlated with perineural invasion. TGR5 expression was higher in perihilar than in intrahepatic CCAs. In vitro, FXR was downregulated and TGR5 was upregulated in human CCA cells compared to normal human cholangiocytes. OCA halted CCA growth in vivo, whereas INT-777 showed no effect. In vitro, OCA inhibited CCA cell proliferation and migration which was associated with decreased mitochondrial energy metabolism. INT-777, by contrast, stimulated CCA cell proliferation and migration, linked to increased mitochondrial energy metabolism. ConclusionActivation of FXR inhibits, whereas TGR5 activation may promote, CCA progression by regulating proliferation, migration and mitochondrial energy metabolism. Modulation of FXR or TGR5 activities may represent potential therapeutic strategies for CCA.

In vivo therapeutic effect of combination treatment with metformin and Scutellaria baicalensis on maintaining bile acid homeostasis.

The radix of Scutellaria baicalensis (SB) is a herb widely used in traditional Chinese medicine to treat metabolic diseases. Several main components, including baicalin and wogonoside, possess anti-dyslipidemia, anti-obesity and anti-diabetic effects. We hypothesized that co-administration of SB extract and metformin exerts a better effect on obesity-induced insulin resistance and lipid metabolism than treatment with metformin alone. We compared the effect of metformin (100 mg/10 mL/kg/day) alone with co-administration of metformin (100 mg/5 mL/kg/day) and SB extract (200 mg/5 mL/kg/day) on Otsuka Long Evans Tokushima Fatty rats, a useful model of type II diabetes with obesity, and used Long-Evans Tokushima Otsuka rats as a control. Weight, fasting glucose, oral glucose tolerance test, intraperitoneal insulin tolerance test, and serum total cholesterol were measured after 12 weeks of drug administration. We observed a synergetic effect of metformin and SB on lowering cholesterol level by excretion of bile acid through feces. We found that this accompanied activation of FXR, CYP7A1 and LDLR genes and repression of HMGCR in the liver. Although there were no significant changes in BSH-active gut microbiota due to high variability, functional prediction with 16S sequences showed increased primary and secondary bile acid biosynthesis in the combination treatment group. Further study is needed to find the specific strains of bacteria which contribute to FXR-related cholesterol and bile acid regulations.