mRNA Expression Of Farnesoid X Receptor Research Articles

Peroxisome proliferator-activated receptors mediate phytochemical-induced upregulation of farnesoid X receptor expression and activity in colonic epithelial cells

Introduction: The nuclear bile acid receptor, farnesoid X receptor (FXR), is expressed on intestinal epithelial cells where its potential as a new drug target for treatment of intestinal and metabolic disorders, such as chronic diarrhoea, colitis, colon cancer, obesity and diabetes, has been well-established. We have previously shown that pentacyclic triterpenes (PCTs), a class of dietary phytochemical, enhance the expression and activity of colonic epithelial FXR. Here, we investigated if another common class of dietary phytochemical, polyunsaturated fatty acids (PUFAs), also modulate FXR signalling in these cells. Furthermore, since peroxisome proliferator-activated receptors (PPARs) have been shown to regulate FXR expression in other cell types, we investigated their potential in mediating the effects of PCTs and PUFAs on FXR expression in the colonic epithelium. Methods: All experiments were conducted on monolayers of T 84 colonic epithelial cells grown on permeable supports. Alpha-linolenic acid (ALA; 100 μM) and hederagenin (HG; 5 μM) were used as prototypical PUFAs or PCTs, respectively. Rosiglitazone (1 μM) and WY14643 (1 – 20 μM) were employed as PPARγ and PPARα agonists, whereas GW9662 (20 μM) and GW6471 (1 μM) were used as PPARγ and PPARα antagonists, respectively. Levels of FXR, FGF19, an index of FXR activation, and ANGPTL4, a PPAR target gene, were measured by qRT-PCR, western blotting, or ELISA. Results: Similar to our previous studies with HG, treatment of T 84 cells with ALA upregulated FXR mRNA and protein expression to 11.43 ± 2.1 fold (n = 7, p < 0.01) and 6.4 ± 1.2 fold (n = 6, p < 0.05) of that in control cells, respectively. ALA treatment also enhanced the effects of an FXR agonist, GW4064 (5 μM), on FGF19 mRNA and protein expression by 4.4 ± 1.6 (n = 7) and 6.5 ± 1.4 fold (n = 8, p < 0.05), respectively. Treatment with HG and ALA (3 hrs) also increased mRNA expression of the PPAR target gene, ANGPTL4, to 5.7 ± 1.0 (n = 5; p < 0.05) and 66.3 ± 9.5 fold (n = 7; p < 0.01) of that in untreated cells, respectively. Furthermore, the effects of the phytochemicals on FXR expression were mimicked by the PPARγ and PPARα agonists, rosiglitazone and WY14643, respectively. The PPARγ antagonist, GW9662, inhibited ALA-induced FXR mRNA expression by 0.5 ± 0.1 fold (n = 11; p < 0.001) but did not alter responses to HG. In contrast, the PPARα antagonist, GW6471, inhibited both ALA and HG-induced FXR mRNA expression by 0.71 ± 0.1 and 0.58 ± 0.1 fold (n = 4; p < 0.05) of that in control cells, respectively. Conclusion: The dietary phytochemicals, HG and ALA, upregulate colonic epithelial FXR through mechanisms that appear to involve differential activation of PPARs. By virtue of their ability to upregulate FXR expression and activity, foods or food supplements, rich in such PCTs and PUFAs have excellent potential for development as a new class of “FXR-targeted nutraceutical” for treatment and prevention of intestinal and metabolic disorders. Science Foundation Ireland This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Duodenal-jejunal bypass increases intraduodenal bile acids and upregulates duodenal SIRT1 expression in high-fat diet and streptozotocin-induced diabetic rats.

BACKGROUNDThe mechanisms underlying diabetes remission after duodenal-jejunal bypass (DJB) remain elusive. In DJB surgery, the duodenum is excluded. However, the duodenum has emerged as an important regulator of glucose homeostasis, and elevated duodenal SIRT1 leads to improved hepatic insulin sensitivity. After DJB, bile acids (BAs) in the duodenum are not mixed and diluted by the ingested food. And activation of BA receptors promotes SIRT1 expression in many tissues. We hypothesized that BA-mediated upregulation of SIRT1 may contribute to diabetic control after DJB.AIMTo investigate the surgical effects of DJB on duodenal SIRT1 expression and uncover the potential crosslinks between BAs and SIRT1.METHODSTwenty diabetic rats were randomly allocated to the sham (n = 10) and DJB (n = 10) groups. Body weight, food intake, fasting blood glucose (FBG), serum and intraduodenal total BA (TBA) levels were measured accordingly. Oral glucose tolerance test (OGTT) and intraperitoneal pyruvate tolerance test (ipPTT) were performed to evaluate the effects of surgeries on systemic glucose disposal and hepatic gluconeogenesis. The key genes of BA signaling pathway in the duodenal mucosa, including farnesoid X receptor (FXR), small heterodimer partner (SHP), and Takeda G-protein-coupled receptor 5 (TGR5) were evaluated by real-time quantitative polymerase chain reaction 8 wk postoperatively. The duodenal SIRT1, AMPK, and phosphorylated AMPK (p-AMPK) levels were evaluated by western blotting. Rat small intestine epithelial IEC-6 cells were treated with GW4064 and INT-777 to verify the effects of BAs on SIRT1 expression in enterocytes.RESULTSThe DJB group exhibited body weight and food intake comparable to those of the sham group at all postoperative time points. The FBG level and area under the curve for the OGTT and ipPTT were significantly lower in the DJB group. The DJB group exhibited higher fasting and postprandial serum TBA levels than the sham group at both 2 and 8 wk postoperatively. At 8 wk after surgery, the DJB group showed higher intraluminal TBA concentration, upregulated mRNA expression of FXR and SHP, and elevated protein expression of SIRT1 and p-AMPK in the descending and horizontal segments of the duodenum. Activation of FXR and TGR5 receptors by GW4064 and INT-777 increased the mRNA and protein expression of SIRT1 and promoted the phosphorylation of AMPK in IEC-6 cells.CONCLUSIONDJB elevates intraduodenal BA levels and activates the duodenal BA signaling pathway, which may upregulate duodenal SIRT1 and further contribute to improved glucose homeostasis after DJB.

Silymarin protects the liver from α-naphthylisothiocyanate-induced cholestasis by modulating the expression of genes involved in bile acid homeostasis

Cholestasis is characterized by impaired bile flow which results in inflammation, cirrhosis, and ultimately liver failure. The current study is aimed to evaluate the anti-cholestatic effect of silymarin against α-naphthylisothiocyanate (ANIT) induced cholestasis. Mice were gavaged with various doses of silymarin or ursodeoxycholic acid (UDCA) for 19 days. Then they were challenged with α-naphthylisothiocyanate (ANIT) and after 48 hours the animals were sacrificed to obtain blood and liver sections. Serum levels of bilirubin, aspartate transaminase (AST), alanine transaminase (ALP), and liver histology were analyzed. mRNA expression of selected transporters (Bile salt export pump (BSEP) and sodium taurocholate cotransporting polypeptide (NTCP)) and proteins (farnesoid x receptor (FXR) and Cytochrome P450 Family 7 Subfamily A Member 1 (Cyp7a1)) involved in bile acids biosynthesis, excretion and uptake were also evaluated by quantitative PCR. The results indicated that the serum levels of bilirubin, AST, and ALP were significantly higher in a cholestatic model group as compared to an untreated control group. However, in silymarin groups, the serum level of these parameters is significantly lower than in a cholestatic model group. Liver histology also showed that silymarin prevents ANIT-induced hepatic injury. mRNA expression of FXR, BSEP, and NTCP was downregulated and expression of Cyp7a1 was upregulated in a cholestatic model group as compared to an untreated control group. However, in silymarin treatment groups, the expression of FXR, BSEP and NTCP was upregulated and the expression of Cyp7a1 was downregulated as compared to the cholestatic model group. In conclusion, silymarin could alleviate hepatic injury by modulating the expression of genes involved in bile acid homeostasis.

Regulation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) by the Nuclear Bile Acid Receptor, Farnesoid X Receptor

Introduction and AimsCFTR, a Cl‐ channel important in regulating intestinal fluid and electrolyte secretion, is implicated in the pathogenesis of a number of intestinal disorders. We have previously shown that bile acids, acting via the nuclear receptor, farnesoid X receptor (FXR), inhibit colonic epithelial CFTR expression. Dietary phytochemicals have been reported to have the capacity to modulate FXR signalling. Here, we set out to investigate mechanisms underlying FXR regulation of CFTR expression, and the potential for therapeutically targeting the receptor with dietary phytochemicals.MethodsMonolayers of T84 colonic epithelial cells were treated with the FXR agonist, GW4064 (5 µM) in the absence or presence of a plant phytochemical, designated here as KFS1 (5 µM). mRNA and protein expression were measured by qPCR and Western blotting. FXR binding to the CFTR promoter was investigated by chromatin immunoprecipitation (ChIP). Electrophysiological studies of T84cells were conducted in Ussing chambers. Studies on human and murine enteroids were carried out with ethical approval from Johns Hopkins University School of Medicine Institutional Review Board and the Institutional Review Board of the University of California San Diego, respectively.ResultsTreatment of T84 monolayers with GW4064 downregulated CFTR mRNA to 0.51 ± 0.06 fold after 12 hrs (n = 12; p < 0.001) and protein levels to 0.28 ± 0.06 fold after 48 hrs, compared to controls (n = 8; p < 0.001). Studies in Ussing chambers showed that GW4064 treatment for 48 hrs inhibited Cl‐ secretory responses to the Ca2+‐ and cAMP‐dependent agonists, carbachol (CCh; 100mM) and forskolin (FSK; 10mM), by 79.9 ± 7.5 % and 74.2 ± 8.9 %, respectively. Transcriptomic analysis of human colonic enteroids revealed FXR to be robustly expressed in secretory (crypt‐like) cells and that its activation also induced CFTR downregulation. FXR activation did not alter expression or phosphorylation of the p65 subunit of NF‐κB, or inhibit its translocation to the nucleus. ChIP‐qPCR analysis appears to show enhanced binding of FXR to the CFTR promoter upon GW4064 treatment. The phytochemical, KFS1 (5 mM; 24hrs), upregulated FXR mRNA and protein expression in T84 cells and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.28 ± 0.05 fold (n = 8; p <0.01) and protein by 0.25 ± 0.11 fold (n = 4) after 24 hours. Similarly, KFS1 significantly upregulated FXR mRNA expression in murine colonic epithelial enteroids (2.3 ± 0.2; n = 4; p < 0.01) and enhanced GW4064‐induced downregulation of CFTR mRNA by 0.5 ± 0.1 fold (n = 4; p < 0.05) compared to GW4064 alone. Finally, KFS1 enhanced FXR inhibition of agonist‐induced Cl‐ secretory responses across T84 cells mounted in Ussing chambers.ConclusionFXR regulates colonic epithelial CFTR expression and function by a mechanism which appears to involve direct binding of FXR to the CFTR promoter. By virtue of their ability to upregulate FXR expression, and thereby enhance its antisecretory actions, plant extracts containing KFS1 have excellent potential to be developed as FXR‐targeted nutraceuticals for the treatment and prevention of intestinal disease.

Regulation of the Nuclear Bile Acid Receptor, Farnesoid X Receptor, by the Omega‐3 Polyunsaturated Fatty Acid, Alpha‐Linolenic Acid, in Colonic Epithelial Cells

Introduction and AimsThe intestinal epithelium forms the interface between the body and the luminal contents, facilitating fluid and electrolyte transport, while also acting as a barrier to harmful pathogens. Dysregulation of barrier and transport function is associated with the pathogenesis of a number of conditions, including chronic diarrhoeal and inflammatory bowel diseases (IBD). Previous studies from ours, and other laboratories, have identified the nuclear bile acid receptor, farnesoid x receptor (FXR), as an excellent target for the development of new anti‐diarrhoeal and anti‐inflammatory therapeutics. Polyunsaturated fatty acids (PUFAs), such as alpha‐linolenic acid (ALA), are a group of bioactive phytochemicals, which have been shown in other systems to modulate FXR activity. PUFAs are also thought to be ligands for another group of nuclear receptors, known as the peroxisome proliferator‐activated receptors (PPARs), which have been recently shown to modulate FXR expression. Here, we set out to investigate a possible role for ALA in regulating farnesoid x receptor signalling in colonic epithelial cells.MethodsT84 human colonic epithelial cells were cultured as polarised monolayers on permeable supports and treated bilaterally with alpha‐linolenic acid (ALA) (100 µM) for 1 – 24 hours, in the presence or absence of the FXR agonist, GW4064 (5 µM), or the PPARg antagonist, GW9662 (20 µM). Levels of FXR, FGF19, an index of FXR activation, and ANGPTL4, a PPAR target gene, were measured by qRT‐PCR, western blotting, or ELISA.ResultsTreatment of T84 cells with ALA increased FXR expression in a time dependent fashion. Maximal effects of ALA occurred after 6 hrs when increases in FXR mRNA and protein were 8.65 ± 0.80 fold (n = 4; p < 0.01) and 4.44 ± 0.85 fold (n = 4) of untreated controls, respectively. Moreover, treatment of the cells with ALA potentiated GW4064‐induced expression of the FXR target gene, FGF‐19, at both mRNA and protein levels by 2.66 ± 0.56 (n = 4) and 5.52 ± 1.52 (n = 6), respectively. The effects of ALA on FXR expression and activity were mimicked by the PPARg agonist, rosiglitazone (1 mM). Treatment with ALA also significantly increased mRNA expression of the PPAR target gene, ANGPTL4, by 56.69 ± 5.7 (n = 4; p < 0.01), while the PPARg antagonist, GW9662 (20 µM; 1 hr), inhibited ALA‐induced FXR mRNA expression by 0.44 ± 0.12 fold (n =4) and protein by 0.23 ± 0.06 fold (n = 3).ConclusionThe dietary PUFA, ALA, regulates colonic epithelial FXR expression through mechanisms that involve activation of PPARγ. By virtue of their ability to upregulate FXR expression and activity, PUFAs, such as ALA, have excellent potential for development as FXR‐targeted nutraceuticals for the treatment and prevention of intestinal disease.

Intestinal farnesoid X receptor mediates the effect of dietary berberine on lipid accumulation in grass carp (Ctenopharyngodon idella)

Berberine (BBR) suppresses lipid accumulation in fish, but mechanisms underlying this effect remain poorly understood. The aim of this study was to test the hypothesis that intestinal farnesoid X receptor (FXR) signaling is involved in this physiological process. Thus, juvenile grass carp (9.90 ± 0.07 g) were fed experimental diets (control, 0.1% BBR, and 0.1% BBR + Gly-β-MCA [an intestine-specific FXR inhibitor]) for 8 weeks. Dietary BBR significantly reduced intraperitoneal fat, hepatic triglyceride (TG), total cholesterol (TC), and whole-body lipid levels, whereas additional supplementation with Gly-β-MCA restored these levels. Dietary BBR also significantly reduced serum TG and high-density lipoprotein cholesterol (HDL-c), and additional supplementation with Gly-β-MCA significantly increased serum alanine aminotransferase activity and levels of TG, TC, HDL-c, and low-density lipoprotein cholesterol. Dietary BBR significantly increased FXR mRNA and protein expression, as well as fibroblast growth factor 19 mRNA expression in the intestine, whereas additional supplementation with Gly-β-MCA reversed these trends. Gly-β-MCA also restored BBR-reduced hepatopancreatic cholesterol 7 alpha-hydroxylase, sterol 12α-hydroxylase, and sterol 27-hydroxylase expression, sterol-regulatory element binding proteins-1c and fatty acid synthase expression, and attenuated BBR-increased peroxisome proliferator activated receptor α, adipose triglyceride lipase, and carnitine palmitoyltransferase 1 expression. Both BBR and BBR + Gly-β-MCA diets significantly reduced the richness and diversity of the fish gut microbiota. The abundance of Firmicutes showed a significant negative correlation with the mRNA expression of FXR in the intestine. Overall, these findings indicate that FXR signaling is involved in lipid suppression induced by dietary BBR in grass carp.

Bile Salt and FGF19 Signaling in the Early Phase of Human Liver Regeneration.

The involvement of bile salt–fibroblast growth factor 19 (FGF19) signaling in human liver regeneration (LR) is not well studied. Therefore, we studied aspects of bile salt–FGF19 signaling shortly after liver resection in patients. We compared plasma bile salt and FGF19 levels in arterial, portal and hepatic venous blood, calculated venous‐arterial differences (ΔVA), and determined hepatic transcript levels on two intra‐operative time points: before (< 1 hour) and immediately after (> 2‐3 hours) liver resection (i.e., following surgery). Postoperative bile salt and FGF19 levels were assessed on days 1, 2, and 3. LR was studied by computed tomography (CT)–liver volumetry. Following surgery, the liver, arterial, and portal bile salt levels were elevated (P < 0.05). Furthermore, an increased amount of bile salts was released in portal blood and extracted by the remnant liver (P < 0.05). Postoperatively, bile salt levels were elevated from day 1 onward (P < 0.001). For FGF19, intra‐operative or postoperative changes of ΔVA or plasma levels were not observed. The bile salt–homeostatic regulator farnesoid X receptor (FXR) was markedly up‐regulated following surgery (P < 0.001). Cell‐cycle re‐entry priming factors (interleukin 6 [IL‐6], signal transducer and activator of transcription 3 [STAT3], and cJUN) were up‐regulated following surgery and were positively correlated with FXR expression (P < 0.05). Postoperative hyperbilirubinemia was preceded by postsurgery low FXR and high Na+/Taurocholate cotransporting polypeptide (NTCP) expression in the remnant liver coupled with higher liver bile salt content (P < 0.05). Finally, bile salt levels on postoperative day 1 were an independent predictor of LR (P < 0.05). Conclusion: Systemic, portal, and liver bile salt levels are rapidly elevated after liver resection. Postoperative bile salts were positively associated with liver volume gain. In the studied time frame, FGF19 levels remained unaltered, suggesting that FGF19 plays a minor role in human LR. These findings indicate a more relevant role of bile salts in human LR.

Decreased expression of farnesoid X receptor may indicate poor prognosis in patients with colorectal cancer.

BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide. Many studies showed that increased secretion of bile acid may contribute to the pathogenesis of CRC. Farnesoid X receptor (FXR) plays an important role in several physiological functions of bile acid metabolism and circulation, while the diagnostic and prognostic values of FXR remain unknown.MethodsWe used immunohistochemistry (IHC) to examine FXR expression in 132 formalin-fixed paraffin-embedded (FFPE) CRC samples, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was also conducted to compare the mRNA expression of FXR between 50 fresh CRC tissues and 50 adjacent normal tissues. Immunohistochemical method has been used to compare the protein expression of FXR in FFPE samples, and the associate between FXR expression and clinical characteristics of the patients were also analyzed. Moreover, RT-qPCR has been applied to quantify the mRNA expression of FXR, and the diagnostic value and prognostic value of FXR were analyzed by receiver operating characteristics (ROC) analysis and Kaplan-Meier survival analysis, respectively.ResultsThe expression of FXR was significantly decreased in CRC tumor samples compared with normal tissues on both protein and mRNA levels, and the expression of FXR was positively correlated with the tumor size, lymph node metathesis and distant metathesis of the patients. Moreover, the area under curve (AUC) of FXR for the diagnosis of CRC was 0.8088, and low expression of FXR was associated with low overall survival (OS) and disease-free survival (DFS) rate.ConclusionsFXR was down-regulated in CRC and may serve as potential diagnostic and prognostic biomarkers.

Chenodeoxycholic Acid Releases Proinflammatory Cytokines from Small Intestinal Epithelial Cells Through the Farnesoid X Receptor

Background/Aims: Bile acids have recently been associated with the pathogenesis of irritable bowel syndrome (IBS). We therefore evaluated the expression of bile acid receptors in the intestinal mucosa of IBS patients as well as the effects of bile acids on small intestinal epithelial cells. Methods: Intestinal biopsy specimens were obtained from 15 IBS patients and 15 healthy controls. The effects of bile acid stimulation on trans-epithelial electrical resistance (TEER) and permeability in differentiated Caco-2 cells were measured. Proinflammatory cytokines were measured by enzyme-linked immunosorbent assay. mRNA levels of bile acid receptors, including farnesoid X receptor (FXR), and cytokines were determined by real-time reverse transcription-PCR. Caco-2 cells were pre-incubated with the FXR antagonist guggulsterone. Results: FXR mRNA expression at the terminal ileum was increased in IBS patients. Chenodeoxycholic acid (CDCA) significantly decreased TEER, increased permeability, and increased interleukin-8 (IL-8) release from Caco-2 cells. Pre-incubation with guggulsterone blocked CDCA-mediated IL-8 release; however, the decrease in TEER was not reversed. CDCA-induced IL-6 and IL-8 mRNA levels were blocked by guggulsterone. CDCA increased IL-6, tumor necrosis factor-α (TNF-α), and vascular endothelial growth factor release, whereas guggulsterone significantly blocked IL-6 and TNF-α release. Conclusions: FXR expression was elevated at the terminal ileum in IBS patients. CDCA increased proinflammatory cytokines, while guggulsterone blocked these increases.

IPEG 2018The 27th Annual Congress for Endosurgery in ChildrenApril 11–14, 2018, Seattle, Washington

IPEG 2018The 27^th Annual Congress for Endosurgery in ChildrenApril 11–14, 2018, Seattle, Washington

Adiponectin determines farnesoid X receptor agonism-mediated cardioprotection against post-infarction remodelling and dysfunction.

The farnesoid X receptor (FXR) is a member of the metabolic nuclear receptor superfamily that plays a critical regulatory role in cardiovascular physiology/pathology. However, the role of systemic FXR activation in the chronic phase in myocardial infarction (MI)-induced cardiac remodelling and dysfunction remains unclear. In this study, we aimed to elucidate the role of long-term FXR activation on post-MI cardiac remodelling and dysfunction. Mice underwent either MI surgery or sham operation. At 1 week after MI, both sham and MI mice were gavaged with 25 mg/kg/d of a synthetic FXR agonist (GW4064) or a vehicle control for 7 weeks, and cardiac performance was assessed by consecutive echocardiography studies. Administration of GW4064 significantly increased left ventricular ejection fraction at 4 weeks and 8 weeks after MI (both P < 0.01). Moreover, GW4064 treatment increased angiogenesis and mitochondrial biogenesis, reduced cardiomyocyte loss and inflammation, and ameliorated cardiac remodelling as evidenced by heart weight, lung weight, atrial natriuretic peptide/brain natriuretic peptide levels, and myocardial fibrosis at 8 weeks post-MI. At the molecular level, GW4064 significantly increased FXR mRNA expression and transcriptional activity in heart tissue. Moreover, over-expression of myocardial FXR failed to exert significant cardioprotection in vivo, indicating that GW4064 improved post-MI heart remodelling and function independent of myocardial FXR expression/activity. Among the four down-stream soluble molecules of FXR, plasma adiponectin was most significantly increased by GW4064. In cultured adipocytes, GW4064 increased mRNA levels and protein expression of adiponectin. Conditioned medium of GW4064-treated adipocytes activated AMPK-PGC-1α signalling and reduced hypoxia-induced cardiomyocyte apoptosis, all of which were attenuated by an adiponectin neutralizing anti-body. More importantly, when knocking-out adiponectin in mice, the cardioprotective effects of GW4064 were attenuated. We are the first to show that FXR agonism ameliorated post-MI cardiac dysfunction and remodelling by stimulating adiponectin secretion. Thus, we demonstrated that FXR agonism is a potential therapeutic strategy in post-MI heart failure.

Effects of Sleeve Gastrectomy on Nonalcoholic Fatty Liver Disease in an Obese Rat Model.

Laparoscopic sleeve gastrectomy (SG) is an increasingly used bariatric surgery, which is reported to be effective for nonalcoholic fatty liver disease (NAFLD). Recently, activation of farnesoid X receptor (FXR), which is a nuclear receptor of bile acid (BA), was reported to contribute to the resolution of NAFLD. However, it is unclear whether SG has an effect on expression of FXR in the liver. We aimed to investigate the expression of FXR and its related factors in the liver after SG and to clarify the relationship between changes in FXR expression and NAFLD in an obese rat model. Thirty male Zucker fatty rats were divided into three groups: sham-operated (SO) control, pair-fed (PF) control, and SG. Eight weeks after the surgery, metabolic parameters, plasma levels of total BA and liver enzymes, liver triglyceride (TG) content, and mRNA expression of FXR and its related factors, such as small heterodimer partner (SHP) and peroxisome proliferator-activated receptor α (PPARα), were measured. Metabolic parameters in the SG group were significantly improved compared with the SO group. Liver enzymes and TG were significantly lower in the SG group than in the SO group. Plasma levels of BA were significantly higher in the SG group than in the SO and PF groups. mRNA expression of FXR, SHP, and PPARα in the liver was significantly higher in the SG group than in the SO group. These results suggest that the effects of SG on NAFLD should be associated with the expression of the FXR pathway in the liver in a Zucker fatty rat model.

The relationship between bile acid concentration, glucagon-like-peptide 1, fibroblast growth factor 15 and bile acid receptors in rats during progression of glucose intolerance

BackgroundRecent studies show that bile acids are involved in glucose and energy homeostasis through activation of G protein coupled membrane receptor (TGR5) and farnesoid X receptor (FXR). A few researches have explored changes of TGR5 and FXR in animals with impaired glucose regulation. This study aimed to observe changes of plasma total bile acids (TBA), glucagon-like-peptide 1 (GLP-1), fibroblast growth factor 15 (FGF15), intestinal expressions of TGR5 and FXR, and correlations between them in rats with glucose intolerance.MethodsBesides plasma fasting glucose, lipid, TBAs, alanine transaminase (ALT), active GLP-1(GLP-1A) and FGF15, a postprandial meal test was used to compare responses in glucose, insulin and GLP-1A among groups. The expressions of TGR5 and FXR in distal ileum and ascending colon were quantified by real-time PCR and western blot.ResultsTGR5 expression was significantly decreased in distal ileum in DM group compared to other groups, and TGR5 and FXR expressions in ascending colon were also decreased in DM group compared to other groups. Correlation analysis showed correlations between TBA and GLP-1A or FGF15. GLP-1A was correlated with TGR5 mRNA expression in colon, and FGF15 was correlated with FXR mRNA expression in colon.ConclusionsThese results indicates that bile acid-TGR5/FXR axis contributes to glucose homeostasis.

Glycosides Based Standardized Fenugreek Seed Extract Ameliorates Bleomycin-induced Liver Fibrosis in Rats Via Modulation of Endogenous Enzymes.

Background:Liver fibrosis a complex process of excess collagen deposition resulted in disturbance of hepatic cellar function. Glycosides based standardized fenugreek seed extract (SFSE-G) has potent anti-inflammatory, antioxidant, and anti-fibrotic properties.Objective:The aim of this study is to evaluate the hepatoprotective potential of SFSE-G against bleomycin (BLM)-induced liver fibrosis in laboratory animals.Materials and Methods:Sprague-Dawley rats (180–220 g) were assigned to various groups, namely, normal, sham, BLM control, SFSE-G (5, 10, 20, and 40 mg/kg, p.o.), methylprednisolone (10 mg/kg, p.o.), and sildenafil (25 mg/kg, p.o.). Liver fibrosis was induced in various groups (except normal and sham) by single intratracheal BLM (6 IU/kg) injection. Various biochemical, molecular (reverse transcription polymerase chain reaction) and histological parameters were evaluated.Results:Intratracheal BLM administration caused significant induction (P < 0.001) of hepatotoxicity and liver fibrosis reflected by elevated levels of serum aspartate transaminase (AST), alanine transaminase (ALT), total as well as direct bilirubin, and gamma-glutamyl transferase (GGT). Administration of SFSE-G (20 and 40 mg/kg, p.o.) significantly reduced (P < 0.001) levels of AST, ALT, and GGT and significantly increased (P < 0.001) the level of serum albumin. BLM-induced elevated liver oxidative stress and decreased total antioxidant capacity was significantly restored (P < 0.001) by SFSE-G (20 and 40 mg/kg) treatment. It also significantly inhibited BLM-induced alteration in liver Farnesoid X receptor (FXR) mRNA expression. SFSE-G treatment reduced histopathological alteration induced by BLM in liver.Conclusion:SFSE-G exerts its hepatoprotective potential via inhibition of oxido-nitrosative stress and modulation of FXR mRNA expression thus ameliorates BLM-induced liver fibrosis.

Role of farnesoid X receptor in rats with acute cholestatic hepatitis

To investigate the role of farnesoid X receptor (FXR) and its downstream molecules small heterodimer partner (SHP), UDP-glucuronosyltransferase 2B4 (UGT2B4), and bile salt export pump (BSEP) in rats with acute cholestatic hepatitis. A total of 20 Sprague-Dawley rats were randomly divided into normal control group and model group, with 10 rats in each group. The rats in the model group were given a single dose (50 mg/kg) ofα-naphthyl isothiocyanate by gavage to establish the animal model of acute cholestatic hepatitis. Quantitative real-time PCR was used to measure the mRNA expression of FXR, UGT2B4, and BSEP in liver tissue at 48 hours after gavage. An automatic biochemical analyzer was used to measure the serum levels of total bilirubin, direct bilirubin, alanine aminotransferase, total bile acid, aspartate aminotransferase, alkaline phosphatase, andγ-glutamyl transferase. The independent samples t-test was used for comparison of means between groups. The model group had significantly lower mRNA expression of FXR, SHP, UGT2B4, and BSEP in liver tissue than the normal control group (0.152±0.088/0.559±0.194/0.177±0.039/0.561±0.123 vs 1.137±0.215/1.512±0.309/2.394±0.462/1.631±0.376, t = 13.408, 8.260, 15.121, and 8.553, all P < 0.05). The model group had significantly higher liver function parameters than the normal control group (all P < 0.01). FXR, SHP, UGT2B4, and BSEP are involved in the development of acute cholestatic hepatitis. Reduced expression of FXR may cause reduced expression of downstream SHP, UGT2B4, and BSEP, increase the synthesis of bile acid, weaken detoxicating and transporting functions, and thus mediate the development of cholestatic hepatitis.

Abstract 905: Influence of high fat diet and APC status on epigenetic regulation of FXR in colon cells

Abstract Bile acids (BAs) not recycled through the enterohepatic system can be modified in the colon into cancer-promoting secondary BAs such as deoxycholic acid (DCA). The farnesoid-X-receptor (FXR) regulates hepatic and intestinal BA homeostasis. However, in intestinal cells carrying defective adenomatous polyposis coli (Apc) gene the expression of FXR is reduced through yet unknown mechanisms. To investigate if Western high-fat diet (HFD) and Apc status influence epigenetic regulation of the Fxr in colonic mucosa. Weaned C57BL/6J male mice were fed a HFD (22% safflower oil = SO) or control low fat diet (LFD; 5% SO) for 6 wks. We examined the effects on CpG pmethylation of Fxr, and expression of FXR, peroxisome-proliferator activated receptor-gamma (PPARγ), and cyclooxygenase-2 (COX-2) mRNA. Also, we studied the influence of APC status on CpG methylation of the Fxr gene, and expression of FXR, ileal bile acid-binding protein (IBABP), small heterodimer partner (SHP), and COX-2 mRNA in normal colonic mucosa and colon tumors from APCMin/+ mice. Mice fed the HFD had reduced (60%) Fxr promoter methylation and increased (2∼3-fold) FXR, COX-2, and PPARγ mRNA levels. Conversely, APC-deficiency was associated with constitutive hypermethylation of the Fxr gene, elevation of COX-2, and reduced (60-90%) baseline FXR, IBABP, and SHP mRNA. In human HCT-116 colon cells, siRNA knock-down of APC reduced (50%) basal FXR mRNA expression, which was further reduced to 80% by DCA. We conclude that APC deficiency leads to constitutive epigenetic silencing of Fxr increasing the risk of inflammation and colon cancer associated with a Western HFD. Note: This abstract was not presented at the meeting. Citation Format: Ornella I. Selmin, Adam M. Lyon, Changming Fang, Tom C. Doetschman, Patricia A. Thompson, Jesse D. Martinez, Jeffrey Smith, Peter M. Lance, Donato F. Romagnolo. Influence of high fat diet and APC status on epigenetic regulation of FXR in colon cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 905. doi:10.1158/1538-7445.AM2015-905

Effects of alfalfa saponin extract on mRNA expression of Ldlr, LXRα, and FXR in BRL cells.

We studied the effects of alfalfa saponin extract (ASE) on low density lipoprotein receptor (Ldlr), liver X receptor α (LXRα), and farnesoid X receptor (FXR) in normal and hyperlipidemic Buffalo rat liver (BRL) cells. Normal and hyperlipidemic BRL cells were divided into eight groups: normal, or normal cells treated with 50, 100, and 150 mg/L ASE, hyperlipidemic, or hyperlipidemic cells treated with 50, 100, and 150 mg/L ASE. After treatment for 24 h, Ldlr, LXRα, and FXR mRNA expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Data showed that mRNA expression of Ldlr in normal BRL cells was significantly up-regulated by ASE treatment and mRNA expressions of LXRα and FXR were significantly down-regulated both in normal and hyperlipidemic BRL cells after ASE treatment. Thus, ASE might ameliorate hepatic steatosis by regulating genes involved in cholesterol metabolism, including up-regulation of Ldlr as well as down-regulation of LXRα and FXR.

Pravastatin activates the expression of farnesoid X receptor and liver X receptor alpha in Hep3B cells

Statins are suggested to preserve gallbladder function by suppressing pro-inflammatory cytokines and preventing cholesterol accumulation in gallbladder epithelial cells. They also affect cross-talk among the nuclear hormone receptors that regulate cholesterol-bile acid metabolism in the nuclei of hepatocytes. However, there is controversy over whether or how statins change the expression of peroxisome proliferator-activated receptor (PPAR)alpha, PPARgamma, liver X receptor alpha (LXRalpha), farnesoid X receptor (FXR), ABCG5, ABCG8, and 7alpha-hydroxylase (CYP7A1) which are directly involved in the cholesterol saturation index in bile. Human Hep3B cells were cultured on dishes. MTT assays were performed to determine the appropriate concentrations of reagents to be used. The protein expression of PPARalpha and PPARgamma was measured by Western blotting analysis, and the mRNA expression of LXRalpha, FXR, ABCG5, ABCG8 and CYP7A1 was estimated by RT-PCR. In cultured Hep3B cells, pravastatin activated PPARalpha and PPARgamma protein expression, induced stronger expression of PPARgamma than that of PPARalpha, increased LXRalpha mRNA expression, activated ABCG5 and ABCG8 mRNA expression mediated by FXR as well as LXRalpha, enhanced FXR mRNA expression, and increased CYP7A1 mRNA expression mediated by the PPARgamma and LXRalpha pathways, together or independently. Our data suggested that pravastatin prevents cholesterol gallstone diseases via the increase of FXR, LXRalpha and CYP7A1 in human hepatocytes.

GW4064, a farnesoid X receptor agonist, upregulates adipokine expression in preadipocytes and HepG2 cells.

To investigate the effect of GW4064 on the expression of adipokines and their receptors during differentiation of 3T3-L1 preadipocytes and in HepG2 cells. The mRNA expression of farnesoid X receptor (FXR), peroxisome proliferator-activated receptor-gamma 2 (PPAR-γ2), adiponectin, leptin, resistin, adiponectin receptor 1 (AdipoR1), adiponectin receptor 2 (AdipoR2), and the long isoform of leptin receptor (OB-Rb) and protein levels of adiponectin, leptin, and resistin were determined using fluorescent real-time PCR and enzyme linked immunosorbent assay, respectively, on days 0, 2, 4, 6, and 8 during the differentiation of 3T3-L1 preadipocytes exposed to GW4064. Moreover, mRNA expression of AdipoR2 and OB-Rb was also examined using fluorescent real-time PCR at 0, 12, 24, and 48 h in HepG2 cells treated with GW4064. The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, resistin, AdipoR1, AdipoR2, and OB-Rb and protein levels of adiponectin, leptin, and resistin increased along with differentiation of 3T3-L1 preadipocytes (P < 0.05 for all). The mRNA expression of FXR, PPAR-γ2, adiponectin, leptin, and AdipoR2 in 3T3-L1 preadipocytes, and AdipoR2 and OB-Rb in HepG2 cells was significantly increased after treatment with GW4064, when compared with the control group (P < 0.05 for all). A similar trend was observed for protein levels of adipokines (including adiponectin, leptin and resistin). However, the expression of resistin, AdipoR1, and OB-Rb in 3T3-L1 cells did not change after treatment with GW4064. The FXR agonist through regulating, at least partially, the expression of adipokines and their receptors could offer an innovative way for counteracting the progress of metabolic diseases such as nonalcoholic fatty liver disease.

Hypocholesterolemia of Rhizoma Coptidis alkaloids is related to the bile acid by up-regulated CYP7A1 in hyperlipidemic rats

This study is to investigate the cholesterol-lowering effect and the new mode of action of coptis alkaloids on high lipid diet-induced hyperlipidemic rats. Coptis alkaloids extract (CAE) was prepared by alcohol extraction from Rhizoma Coptidis that have been quality-controlled according to the protocol. The cholesterol-lowering effect of CAE was evaluated on SD rats fed with high-lipid diet. Serum level of lipid, Bile acid and cholesterol in the liver and feces of the rats were measured using colorimetric assay kit. RT-PCR and Western blot were used to analyze the mRNA and protein expression of cholesterol metabolism-related genes including cholesterol 7α-hydroxylase (CYP7A1), peroxisome proliferator-activated receptor-alpha (PPARα) and farnesoid X receptor (FXR) in the livers of the rats. A HPLC analysis was used to assess the activity of CYP7A1. The results showed that CAE reduced the levels of serum total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C). CYP7A1 gene expression and its activity was up-regulated dose-dependently acompanying with the increased level of bile acid and the reduced cholesterol level in the livers of the CAE treated hyperlipidemic rats. Meanwhile, the mRNA expression of PPARα was also up-regulated in dose-dependent way acompanying the down-modulation of the FXR mRNA expression in the livers of the CAE treated hyperlipidemic rats.The results indicate that the cholesterol-lowering effect of coptis alkaloid extract is at least partly attributed to its promoting the cholesterol conversion into bile acids by up-regulating the gene expression of CYP7A1 and thus increasing its activity in the liver of the hyperlipidemic rats, which might related to the positive regulation of PPARα and the negative modulation of FXR.