In the recent years, bile acid receptors FXR and GPBAR1 have attracted the interest of scientific community and companies, as they proved promising targets for the treatment of several diseases, ranging from liver cholestatic disorders to metabolic syndrome, inflammatory states, nonalcoholic steatohepatitis (NASH), and diabetes.Consequently, the development of dual FXR/GPBAR1 agonists, as well as selective targeting of one of these receptors, is considered a hopeful possibility in the treatment of these disorders. Because endogenous bile acids and steroidal ligands, which cover the same chemical space of bile acids, often target both receptor families, speculation on nonsteroidal ligands represents a promising and innovative strategy to selectively target GPBAR1 or FXR.In this review, we summarize the most recent acquisition on natural, semisynthetic, and synthetic steroidal and nonsteroidal ligands, able to interact with FXR and GPBAR1.

OBJECTIVE

NGM282 is an analog of fibroblast growth factor 19 (FGF19), a potent inhibitor of bile acid (BA) synthesis in animals and humans. In phase 2 trials in type 2 diabetes and primary biliary cholangitis, NGM282 was associated with dose-related abdominal cramping and diarrhea. We aimed to examine effects of NGM282 on colonic transit, stool frequency and consistency, hepatic BA synthesis (fasting serum C4), fecal fat, and BA in functional constipation (FC).

METHODS

Two-dose NGM282 (1 and 6 mg, subcutaneously daily), parallel-group, randomized, placebo-controlled, 14-day study in patients with FC (Rome III criteria) and baseline colonic transit 24 h geometric center (GC) <3.0. We explored treatment interaction with SNPs in genes KLB, FGFR4, and TGR5 (GPBAR1).

METHODS

overall ANCOVA at α = 0.025 (baseline as covariate where available), with three pairwise comparisons among the three groups (α = 0.008).

RESULTS

Overall, NGM282 altered bowel function (number of bowel movements, looser stool form, and increased ease of passage) and significantly accelerated gastric and colonic transit. Dose-related effects were seen with GC 24 h, but not with gastric emptying (GE) and GC 48 h. There were no differences in fecal fat or weight, but there was reduced fecal total BA excretion with NGM282. The most common adverse events were increased appetite (n = 0 with placebo, 2 with 1 mg, 9 with 6 mg), injection site reaction (n = 2 placebo, 4 with 1 mg, 8 with 6 mg), and diarrhea (n = 1 with 1 mg and 4 with 6 mg NGM282). There was treatment interaction with KLB SNP, with greater increase in colonic transit in participants with the minor A allele (p = 0.056).

CONCLUSIONS

NGM282 significantly impacts GE and colonic transit, consistent with the observed clinical symptoms. The specific mechanism of prokinetic activity requires further research.

TGR5 (also known as G protein-coupled bile acid receptor 1, GPBAR1) is a G protein-coupled bile acid receptor that is expressed in many diverse tissues. TGR5 is involved in various metabolic processes, including glucose metabolism and energy expenditure; however, TGR5's function in skeletal muscle is not fully understood. Using both gain- and loss-of-function mouse models, we demonstrate here that Tgr5 activation promotes muscle cell differentiation and muscle hypertrophy. Both young and old transgenic mice with muscle-specific Tgr5 expression exhibited increased muscle strength. Moreover, we found that Tgr5 expression is increased by the unfolded protein response (UPR), which is an adaptive response required for maintenance of endoplasmic reticulum (ER) homeostasis. Both ER stress response element (ERSE)- and unfolded protein response element (UPRE)-like sites are present in the 5' upstream region of the Tgr5 gene promoter and are essential for Tgr5 expression by Atf6α (activating transcription factor 6α), a well known UPR-activated transcriptional regulator. We observed that in the skeletal muscle of mice, exercise-induced UPR increases Tgr5 expression, an effect that was abrogated in Atf6α KO mice, indicating that Atf6α is essential for this response. These findings indicate that the bile acid receptor Tgr5 contributes to improved muscle function and provide an additional explanation for the beneficial effects of exercise on skeletal muscle activity.

Our previous studies indicated that the G-protein-coupled bile acid receptor, Gpbar1 (TGR5), inhibits inflammation by inhibiting the NF-κB signalling pathway, eventually attenuating diabetic nephropathy (DN). Gentiopicroside (GPS), the main active secoiridoid glycoside of Gentiana manshurica Kitagawa, has been demonstrated to inhibit inflammation in various diseases via inhibiting the inflammatory signalling pathways. However, whether GPS inhibits the NF-κB signalling pathway by activating TGR5 and regulates the pathological progression of diabetic renal fibrosis requires further investigation. In this study, we found that GPS significantly reversed the downregulation of TGR5 and inhibited the overproduction of fibronectin (FN), transforming growth factor β1 (TGF-β1), intercellular adhesion molecule-1 (ICAM-1) and vascular adhesion molecule-1 (VCAM-1) in glomerular mesangial cells (GMCs) exposed to high glucose (HG). Additionally, GPS prevented the phosphorylation and degradation of IκBα, and subsequently inhibited the activation of the NF-κB signalling pathway. Further investigation found that GPS enhanced the stabilization of IκBα by promoting the interaction of β-arrestin2 with IκBα via TGR5 activation, which contributed to the inhibition of NF-κB signalling pathway. Importantly, the depletion of TGR5 blocked the inhibition of the NF-κB signalling pathway and reversed the downregulation of FN, ICAM-1, VCAM-1 and TGF-β1 by GPS in HG-induced GMCs. Moreover, GPS increased the TGR5 protein levels and promoted the interaction between IκBα and β-arrestin2, thereby inhibiting the reduction of IκBα and blocked NF-κB p65 nuclear translocation in the kidneys of STZ-induced diabetic mice. Collectively, these data suggested that GPS regulates the TGR5-β-arrestin2-NF-κB signalling pathway to prevent inflammation in the kidneys of diabetic mice, and ultimately ameliorates the pathological progression of diabetic renal fibrosis.

Bile acids are produced in the liver by the cholesterol breakdown and further metabolized by the intestinal microbiota to generate a group of chemically heterogeneous steroids that bind and activate a family of cells surface and nuclear receptors, collectively known as the bile acid-activated receptors (BARs). The two best characterized members of this family are the farnesoid-x-receptor (FXR) and G protein Bile Acid Receptor (GPBAR1). Both receptors are expressed by cells of innate immunity including liver-resident and intestinal-resident macrophages and monocytes-derived macrophages. Because FXR and GPBAR1 knockout mice are biased toward a pro-inflammatory phenotype, it appears the both receptors might have a role in the development and maintenance of a tolerogenic phenotype. FXR and GPBAR1 ligands have been proven effective in the treatment in inflammatory and metabolic disorders and ligands for these receptors are currently under development for the treatment of non-alcoholic steato-hepatitis and diabetes.

OBJECTIVE

Bile acids (BAs) regulate energy expenditure by activating G-protein Coupled Bile Acid Receptor Gpbar1/TGR5 by cAMP-dependent mechanisms. Cholecystectomy (XGB) increases BAs recirculation rates resulting in increased tissue exposure to BAs during the light phase of the diurnal cycle in mice. We aimed to determine: 1) the effects of XGB on basal metabolic rate (BMR) and 2) the roles of TGR5 on XGB-dependent changes in BMR.

METHODS

BMR was determined by indirect calorimetry in wild type and Tgr5 deficient (Tgr5-/-) male mice. Bile flow and BAs secretion rates were measured by surgical diversion of biliary duct. Biliary BAs and cholesterol were quantified by enzymatic methods. BAs serum concentration and specific composition was determined by liquid chromatography/tandem mass spectrometry. Gene expression was determined by qPCR analysis.

RESULTS

XGB increased biliary BAs and cholesterol secretion rates, and elevated serum BAs concentration in wild type and Tgr5-/- mice during the light phase of the diurnal cycle. BMR was ~25% higher in cholecystectomized wild type mice (p <0.02), whereas no changes were detected in cholecystectomized Tgr5-/- mice compared to wild-type animals.

CONCLUSIONS

XGB increases BMR by TGR5-dependent mechanisms in mice.

GPBAR1/TGR5 is a G protein-coupled receptor of bile acids. TGR5 is known to regulate the BA homeostasis and energy metabolism. Recent studies highlight an important role of TGR5 in alleviating obesity and improving glucose regulation, however, the mechanism of which is still unclear. Here we report that TGR5 is involved in mediating the anti-obesity and anti-hyperglycemia effect of a natural compound, oleanolic acid. By comparing the miRNA profiles between wild type and TGR5-/- livers after OA treatment, we identified miR-26a as a novel downstream target gene of TGR5 activation. The expression of miR-26a in the liver was induced in a TGR5-dependent manner after feeding the mice with a bile acid diet. TGR5 activation strongly increased the expression of miR-26a in macrophages, including the Kupffer cells in the liver. We further demonstrated that JNK pathway was required for miR-26a induction by TGR5 activation. Interestingly, we located the TGR5-responsive DNA element to a proximal region of miR-26's promoter, which was independent of the transcription of its host genes. These results unravel a new mechanism by which bile acid receptor TGR5 activates a miRNA gene expression.

Chenodeoxycholic acid (CDCA), 3α,7α-dihydroxy-5β-cholan-24-oic acid, is a primary bile acid generated in the liver from cholesterol. In liver cells CDCA is conjugated with glycine or taurine to form two bile salts, Glyco-CDCA and Tauro-CDCA, before being released into the bile ducts. In the intestine, CDCA is further metabolized to generate a 7β epimer, i.e., the ursodeoxycholic acid (UDCA), or dehydroxylate to generate lithocolic acid (LCA). In humans, CDCA is the physiological ligand for the bile acid sensor farnesoid X receptor (FXR), while LCA is a potent agonist for a G protein-coupled receptor, known as GPBAR1 (TGR5). Along with UDCA, CDCA has been clinically used for the dissolution of gallbladder stones at doses ranging from 375 to 750 mg/day, with a success rate of 8 to 18%. Because the efficacy of CDCA was significantly lower than that of UDCA and 18-30% of patients developed significant side effects, the most frequent being diarrhea and a reversible increase in aminotransferases plasma levels, this application has lost its therapeutic relevance. Additionally, the combination of CDCA with UDCA, generally at doses of 5-10 mg/kg each, has failed to provide significant advantages over UDCA alone. In 2017, CDCA has been approved as an orphan indication for the treatment of patients with cerebrotendinous xanthomatosis (CTX), a rare autosomal recessive disorder caused by mutations of sterol 27-hydroxylase (CYP27A1) gene. Since CYP27A1 is essential for cholesterol breakdown, CTX patients develop abnormal lipid storage with increased plasma and tissue levels of cholestanol and very low/absent production of CDCA. CDCA is a potent inhibitor of CYP27A1, and early initiation of CDCA therapy, at doses up to 750 mg/day, is considered the standard medical therapy for CTX resulting in decreased plasma levels of cholestanol and stabilization of neurologic symptoms. Studies in CTX patients have also shown that CDCA might suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in the liver. Furthermore, CDCA promotes the release of glucagon-like peptide-1 (GLP-1) in diabetic patients, likely by activating GPBAR1.

Bile acids are signaling molecules interacting with nuclear receptors and membrane G-protein-coupled receptors. Among these receptors, the farnesoid X receptor (FXR) and the membrane G-coupled receptor (GPBAR1) have gained increasing consideration as druggable receptors and their exogenous dual regulation represents an attractive strategy in the treatment of enterohepatic and metabolic disorders. However, the therapeutic use of dual modulators could be associated to severe side effects and therefore the discovery of selective GPBAR1 and FXR agonists is an essential step in the medicinal chemistry optimization of bile acid scaffold. In this study, a new series of 6-ethylcholane derivatives modified on the tetracyclic core and on the side chain has been designed and synthesized and their in vitro activities on FXR and GPBAR1 were assayed. This speculation resulted in the identification of compound 7 as a potent and selective GPBAR1 agonist and of several derivatives showing potent dual agonistic activity.

TGR5 (Gpbar1) is a G protein-coupled receptor responsive to bile acids (BAs), which is expressed in different non-parenchymal cells of the liver, including biliary epithelial cells, liver-resident macrophages, sinusoidal endothelial cells (LSECs), and activated hepatic stellate cells (HSCs). Mice with targeted deletion of TGR5 are more susceptible towards cholestatic liver injury induced by cholic acid-feeding and bile duct ligation, resulting in a reduced proliferative response and increased liver injury. Conjugated lithocholic acid (LCA) represents the most potent TGR5 BA ligand and LCA-feeding has been used as a model to rapidly induce severe cholestatic liver injury in mice. Thus, TGR5 knockout (KO) mice and wildtype (WT) littermates were fed a diet supplemented with 1% LCA for 84 h. Liver injury and gene expression changes induced by the LCA diet revealed an enrichment of pathways associated with inflammation, proliferation, and matrix remodeling. Knockout of TGR5 in mice caused upregulation of endothelin-1 (ET-1) expression in the livers. Analysis of TGR5-dependent ET-1 signaling in isolated LSECs and HSCs demonstrated that TGR5 activation reduces ET-1 expression and secretion from LSECs and triggers internalization of the ET-1 receptor in HSCs, dampening ET-1 responsiveness. Thus, we identified two independent mechanisms by which TGR5 inhibits ET-1 signaling and modulates portal pressure.

The G-protein-coupled bile acid receptor Gpbar1 (TGR5) has been demonstrated to be able to negatively regulate hepatic inflammatory response. In this study, we aimed to determine the methylation status of TGR5 promoter in patients with acute-on-chronic hepatitis B liver failure (ACHBLF) and its predictive value for prognosis. We enrolled 76 consecutive ACHBLF patients, 80 chronic hepatitis B (CHB) patients and 30 healthy controls (HCs). Methylation status of TGR5 promoter in peripheral mononuclear cell (PBMC) was detected by methylation-specific polymerase chain reaction (MSP). The mRNA level of TGR5 was determined by quantitative real-time polymerase chain reaction (RT-qPCR). We found that the frequency of TGR5 promoter methylation was significantly higher in ACHBLF (35/76, 46.05%) than CHB patients (5/80, 6.25%; χ(2) = 32.38, P < 0.01) and HCs (1/30, 3.33%; χ(2) = 17.50, P < 0.01). TGR5 mRNA level was significantly lower (Z = -9.12, P < 0.01) in participants with aberrant methylation than those without. TGR5 methylation showed a sensitivity of 46.05% (35/76), specificity of 93.75% (75/80), positive predictive value (PPV) of 87.5% (35/40) and negative predictive value (NPV) of 64.66% (75/116) in discriminating ACHBLF from CHB patients. ACHBLF patients with methylated TGR5 showed significantly poor survival than those without (P < 0.01). When used to predict 3-month mortality of ACHBLF, TGR5 methylation [area under the receiver operating characteristic curve (AUC) = 0.75] performed significantly better than model for end-stage liver diseases (MELD) score (AUC = 0.65; P < 0.05). Therefore, our study demonstrated that aberrant TGR5 promoter methylation occurred in ACHBLF and might be a potential prognostic marker for the disease.

Drug-induced liver injury caused by acetaminophen (acetyl-para-aminophenol [APAP]) is the main cause of acute liver failure and liver transplantation in several Western countries. Whereas direct toxicity exerted by APAP metabolites is a key determinant for early hepatocytes injury, the recruitment of cells of innate immunity exerts a mechanistic role in disease progression, determining the clinical outcomes. GPBAR1 is a G protein-coupled receptor for secondary bile acids placed at the interface between liver sinusoidal cells and innate immunity. In this report, using genetic and pharmacological approaches, we demonstrate that whereas Gpbar1 gene deletion worsens the severity of liver injury, its pharmacological activation by 6β-ethyl-3a,7b-dihydroxy-5b-cholan-24-ol rescues mice from liver injury caused by APAP. This protective effect was supported by a robust attenuation of liver recruitment of monocyte-derived macrophages and their repolarization toward an anti-inflammatory phenotype. Macrophage depletion by gadolinium chloride pretreatment abrogated disease development, whereas their reconstitution by spleen-derived macrophage transplantation restored the sensitivity to APAP in a GPBAR1-dependent manner. RNA sequencing analyses demonstrated that GPBAR1 agonism modulated the expression of multiple pathways, including the chemokine CCL2 and its receptor, CCR2. Treating wild-type mice with an anti-CCL2 mAb attenuated the severity of liver injury. We demonstrated that negative regulation of CCL2 production by GPBAR1 agonism was promoter dependent and involved FOXO1. In conclusion, we have shown that GPBAR1 is an upstream modulator of CCL2/CCR2 axis at the sinusoidal cell/macrophage interface, providing a novel target in the treatment of liver damage caused by APAP.

It is well documented that incidence of fertility problems is high in lactating cows but not in heifers of the same genetic merit. Understanding the metabolic and molecular differences between fertile heifers and relatively infertile lactating cows will help us understand the pathogenesis of infertility in dairy cows. Follicular waves in lactating cows (30-50 days in milk; n = 12) and heifers (n = 10) were synchronized by ultrasound-guided follicle ablation. Follicular fluid and granulosa cells of the dominant follicle were collected by ultrasound-guided aspiration along with blood sampling on Day 6 after synchronization. Dominant and subordinate follicles were larger in lactating cows than in heifers. Metabolic stress in lactating cows was evidenced by lower glucose and higher ß-hydroxy butyric acid compared with heifers. Insulin-like growth factor 1 signaling was reduced in the dominant follicle in lactating cows through reduced insulin-like growth factor 1 concentrations in plasma and follicular fluid of the dominant follicle, and reduced expression of pregnancy-associated plasma protein A (PAPPA) in their granulosa cells. We also found increased levels of total bile acids in the follicular fluid of the dominant follicle of lactating cows compared with heifers. Granulosa cells of the dominant follicle had higher expression of SLC10A2 and GPBAR1 (bile acid transporter and receptor, respectively) in lactating cows. These novel data are indicative of increased bile acid signaling within the dominant follicles of lactating cows compared with heifers. Overall, we demonstrate in the present study the metabolic, endocrine, and molecular differences within the microenvironment of the dominant follicles in lactating cows and heifers. These differences in follicular microenvironment may contribute toward abnormal ovarian function in lactating dairy cows.

Cirrhosis is a end-stage disease of the liver in which fibrogenesis, angiogenesis and distortion of intrahepatic microcirculation lead to increased intrahepatic resistance to portal blood flow, a condition known as portal hypertension. Portal hypertension is maintained by a variety of molecular mechanisms including sinusoidal endothelial cells (LSECs) hyporeactivity, activation of hepatic stellate cells (HSCs), reduction in hepatic endothelial nitric oxide synthase (eNOS) activity along with increased eNOS-derived NO generation in the splanchnic and systemic circulations. A reduction of the expression/function of the two major hydrogen sulfide (H2S)-producing enzymes, cystathionine γ-lyase (CSE) and cystathionine β-synthase (CBS), has also been demonstrated. A deficit in the transsulfuration pathway leading to the accumulation of homocysteine might contribute to defective generation of H2S and endothelial hyporeactivity. Bile acids are ligands for nuclear receptors, such as farnesoid X receptor (FXR), and G-protein-coupled receptors (GPCRs), such as the G-protein bile acid receptor 1 (GPBAR1). FXR and GPBAR1 ligands regulate the expression/activity of CSE by both genomic and non-genomic effects and have been proved effective in protecting against endothelial dysfunction observed in rodent models of cirrhosis. GPBAR1, a receptor for secondary bile acids, is selectively expressed by LSECs and its activation increases the expression of CSE and attenuates the production of endotelin-1, a potent vasoconstrictor agent. In vivo GPBAR1 ligand attenuates the imbalance between vasodilatory and vaso-constricting agents, making GPBAR1 a promising target in the treatment of portal hypertension.

Background & aims: Bile-acid metabolism and the intestinal microbiota are impaired in alcohol-related liver disease. Activation of the bile-acid receptor TGR5 (or GPBAR1) controls both biliary homeostasis and inflammatory processes. We examined the role of TGR5 in alcohol-induced liver injury in mice.

Methods: We used TGR5-deficient (TGR5-KO) and wild-type (WT) female mice, fed alcohol or not, to study the involvement of liver macrophages, the intestinal microbiota (16S sequencing), and bile-acid profiles (high-performance liquid chromatography coupled to tandem mass spectrometry). Hepatic triglyceride accumulation and inflammatory processes were assessed in parallel.

Results: TGR5 deficiency worsened liver injury, as shown by greater steatosis and inflammation than in WT mice. Isolation of liver macrophages from WT and TGR5-KO alcohol-fed mice showed that TGR5 deficiency did not increase the pro-inflammatory phenotype of liver macrophages but increased their recruitment to the liver. TGR5 deficiency induced dysbiosis, independently of alcohol intake, and transplantation of the TGR5-KO intestinal microbiota to WT mice was sufficient to worsen alcohol-induced liver inflammation. Secondary bile-acid levels were markedly lower in alcohol-fed TGR5-KO than normally fed WT and TGR5-KO mice. Consistent with these results, predictive analysis showed the abundance of bacterial genes involved in bile-acid transformation to be lower in alcohol-fed TGR5-KO than WT mice. This altered bile-acid profile may explain, in particular, why bile-acid synthesis was not repressed and inflammatory processes were exacerbated.

Conclusions: A lack of TGR5 was associated with worsening of alcohol-induced liver injury, a phenotype mainly related to intestinal microbiota dysbiosis and an altered bile-acid profile, following the consumption of alcohol.

Lay summary: Excessive chronic alcohol intake can induce liver disease. Bile acids are molecules produced by the liver and can modulate disease severity. We addressed the specific role of TGR5, a bile-acid receptor. We found that TGR5 deficiency worsened alcohol-induced liver injury and induced both intestinal microbiota dysbiosis and bile-acid pool remodelling. Our data suggest that both the intestinal microbiota and TGR5 may be targeted in the context of human alcohol-induced liver injury.

Keywords: ALD, alcohol-related liver diseases; ALT, alanine aminotransferase; Alc, alcohol; Alcoholic liver disease; BA, bile acids; BHI, brain heart infusion; Bile acid; C57, conventional mice; C57C57, conventional mice transplanted with their own IM; CA, cholic acid; CCL, CC motif chemokine ligands; CDCA, chenodeoxycholic acid; Col1a1, collagen type-I alpha-1 chain; DCA, deoxycholic acid; Dysbiosis; FDR, false-discovery rate; FXR, farnesoid X receptor; Gut-liver axis; IM, intestinal microbiota; Inflammation; KC, Kupffer cells; KO, knockout; Kupffer cells; LCA, lithocholic acid; LDA, linear discriminative analysis; LEfsE, LDA effect size; MCA, muricholic acid; MO, monocytes/macrophages; Microbiome; NFkB, nuclear factor-kappa B; OTU, operational taxonomic unit; PCA, principal component analysis; PCoA, principal coordinate analysis; PICRUSt, phylogenetic investigation of communities by reconstruction of unobserved states; RIN, RNA integrity number; TBA, total bile acids; TG, triglycerides; TGF, transforming growth factor; TIMP1, tissue inhibitor of metalloproteinase 1; TNF, tumour necrosis factor; UDCA, ursodeoxycholic acid; WT, wild-type; WTKO, WT mice transplanted with the IM of TGR5-KO mice; alpha-SMA, alpha-smooth muscle actin; mMMP9, matrix metallopeptidase 9.

Bile acids (BAs) improve metabolism and exert anti-obesity effects through the activation of the Takeda G protein-coupled receptor 5 (TGR5) in peripheral tissues. TGR5 is also found in the brain hypothalamus, but whether hypothalamic BA signaling is implicated in body weight control and obesity pathophysiology remains unknown. Here we show that hypothalamic BA content is reduced in diet-induced obese mice. Central administration of BAs or a specific TGR5 agonist in these animals decreases body weight and fat mass by activating the sympathetic nervous system, thereby promoting negative energy balance. Conversely, genetic downregulation of hypothalamic TGR5 expression in the mediobasal hypothalamus favors the development of obesity and worsens established obesity by blunting sympathetic activity. Lastly, hypothalamic TGR5 signaling is required for the anti-obesity action of dietary BA supplementation. Together, these findings identify hypothalamic TGR5 signaling as a key mediator of a top-down neural mechanism that counteracts diet-induced obesity.

Keywords: GPBAR1; TGR5; bile acids; body weight; diet; energy expenditure; food intake; hypothalamus; obesity; sympathetic nervous system.

Treatment options for gallbladder carcinoma (GBC) are limited and GBC prognosis remains poor. There is no well-accepted targeted therapy to date, so effective biomarkers of GBC are urgently needed. Here we investigated the expression and correlations of fibroblast growth factor receptors (FGFR1-4) and 18 fibroblast growth factors (FGFs) in two independent patient cohorts and evaluated their prognostic significance. Consequently, we demonstrated that both FGF19 and FGFR4 were unfavorable prognostic biomarkers, and their co-expression was a more sensitive predictor. By analyzing the correlations between all 18 FGFs and FGFR4, we showed that FGF19 expression was significantly associated with FGFR4 and promoted GBC progression via stimulating FGFR4. With experiments using GBC cells, GPBAR1^-/- mice models, and human subjects, we demonstrated that elevated bile acids (BAs) could increase the transcription and expression of FGF19 and FGFR4 by activating GPBAR1-cAMP-EGR1 pathway. FGF19 secreted from GBC cells promoted GBC progression by stimulating FGFR4 and downstream ERK in an autocrine manner with bile as a potential carrier. Patients with GBC had significantly higher FGF19 in serum and bile, compared to patients with cholelithiasis. BLU9931 inhibited FGFR4 and attenuated its oncogenic effects in GBC cell line. In conclusion, upregulation of BAs elevated co-expression of FGF19 and FGFR4 by activating GPBAR1-cAMP-EGR1 pathway. Co-expression of FGF19 and FGFR4 was a sensitive and unfavorable prognostic marker. GBC cells secreted FGF19 and facilitated progression by activating FGFR4 with bile as a potential carrier in an autocrine pathway.

Abnormal loss of components of the extracellular matrix (ECM) including type II collagen and aggrecan caused by proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) is an important pathophysiological characteristic of osteoarthritis (OA). G-protein-coupled bile acid receptor, Gpbar1 (TGR5), is an important member of the bile acid receptor subclass of G Protein-Coupled Receptors (GPCRs). Little information regarding the effects of TGR5 in the pathological development of OA has been reported before. In the current study, we showed that TGR5 is expressed in human primary chondrocytes and human chondrosarcoma SW1353 cells. Interestingly, expression of TGR5 was reduced in response to TNF-α treatment in SW1353 cells. Our results indicate that activation of TGR5 using its specific agonist INT-777 reduced TNF-α-induced degradation of the articular ECM, including type II collagen and aggrecan, by inhibiting expression of matrix metalloproteinase-3 (MMP-3), MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs- 4 (ADAMTS-4) and ADAMTS-5. We also found that INT-777 treatment inhibited phosphorylation of p38 and activation of the IκB kinase/inhibitory κBα/nuclear factor- κB (IKK/IκBα/NF-κB) signaling pathway. Notably, knockdown of TGR5 abolished the protective effects of INT-777 against ECM degradation, suggesting the involvement of TGR5. Our findings implicate that TGR5 might be considered as a potential therapeutic target for the treatment of OA.

The G-protein bile acid receptor 1 (GPBAR1) has emerged in the last decade as prominent target for the treatment of metabolic and inflammatory diseases including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. To date numerous bile acid derivatives have been identified as GPBAR1 agonists, however their clinical application is hampered by the lack of selectivity toward the other bile acid receptors. Therefore, non-steroidal GPBAR1 ligands able to selectively activate the receptor are urgently needed. With this aim, we here designed, synthesized and biologically evaluated ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl) urea derivatives as novel potent GPBAR1 agonists. Particularly, compounds 9 and 10 induce the mRNA expression of the GPBAR1 target gene pro-glucagon and show high selectivity over the other bile acid receptors FXR, LXRα, LXRβ and PXR, and the related receptors PPARα and PPARγ. Computational studies elucidated the binding mode of 10 to GPBAR1, providing important structural insights for the design of non-steroidal GPBAR1 agonists. The pharmacokinetic properties of 9 and 10 suggest that the ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureydil scaffold might be exploited to achieve effective drug candidates to treat GPBAR1 related disorders.

Farnesoid X receptor (FXR) has become a particularly attractive target for the discovery of drugs for the treatment of liver and metabolic diseases. Obeticholic acid (INT-747), a FXR agonist, has advanced into clinical phase III trials in patients with nonalcoholic steatohepatitis (NASH), but adverse effects (e.g., pruritus, LDL increase) were observed. Pruritus might be induced by Takeda G-protein-coupled receptor 5 (TGR5, GPBAR1), and there are chances to develop FXR agonists with higher selectivity over TGR5. In this letter, novel bile acids bearing different modifications on ring A and side chain of INT-747 are reported and discussed. Our results indicated that the side chain of INT-747 is amenable to a variety of chemical modifications with good FXR potency in vitro. Especially, compound 18 not only showed promising FXR potency and excellent pharmacokinetic properties, but also proved superior pharmacological efficacy in the HFD + CCl4 model.

Uniparental disomy (UPD) is an unusual situation wherein two homologous chromosomes are inherited from the same parent. UPDs can cause clinical abnormalities owing to the aberrant dosage of genes regulated by epigenetic imprinting or homozygosity of variants for recessive phenotypes. The aim of this study was to identify the genetic cause of the obesity and developmental delay phenotype in a 3-year-old Chinese boy.

Chromosomal microarray analysis (CMA) was used for detecting potential copy number variations (CNVs) and homozygous segments. Whole-exome sequencing (WES) identified sequence variants. Sanger sequencing further confirmed the variants in GPBAR1 and CAPN10 both in the patient and the parents.

No clinically significant CNVs were identified by CMA but a complete UPD of chromosome 2 (UPD2) was revealed in the patient. WES identified a total of 13 rare homozygous single-nucleotide variants (SNVs) on chromosome 2. Among the 13 SNVs, a nonsense variation in GPBAR1 (c.753T>G; p.Y251*) and a missense variation in CAPN10 (c.413C>T; p.S138F) were evaluated as candidate disease-causing variants based on their functional impacts to their respective protein and the biological relevance of the genes to the clinical presentation of our patient. Both GPBAR1 and CAPN10 variants were detected in the patient's mother in a heterozygous state, indicating that the patient had maternal UPD2. No other clinically relevant variants were identified.

Homozygosity of rare recessive variations caused by UPD2 likely contributed to the phenotypes of our patient. Based on emerging evidence, the nonsense variation in GPBAR1 and the missense variation in CAPN10 are considered as causally related to our patient's phenotype, that is, obesity and delayed development, respectively. The present study further supports the role of GPBAR1 in obesity and the role of calpain-10 in neurological function.

The G protein-coupled bile acid receptor (GPBAR1) has been recognized as a promising new target for the treatment of diverse diseases, including obesity, type 2 diabetes, fatty liver disease and atherosclerosis. The identification of novel and potent GPBAR1 agonists is highly relevant, as these diseases are on the rise and pharmacological unmet therapeutic needs are pervasive. Therefore, the aim of this study was to develop a proficient workflow for the in silico prediction of GPBAR1 activating compounds, primarily from natural sources. A protocol was set up, starting with a comprehensive collection of structural information of known ligands. This information was used to generate ligand-based pharmacophore models in LigandScout 4.08 Advanced. After theoretical validation, the two most promising models, namely BAMS22 and TTM8, were employed as queries for the virtual screening of natural product and synthetic small molecule databases. Virtual hits were progressed to shape matching experiments and physicochemical clustering. Out of 33 diverse virtual hits subjected to experimental testing using a reporter gene-based assay, two natural products, farnesiferol B (27) and microlobidene (28), were confirmed as GPBAR1 activators reaching more than 50% receptor activation at 20 μM with EC50s of 13.53 μM and 13.88 μM, respectively. This activity is comparable to that of the endogenous ligand lithocholic acid (1). Seven further virtual hits showed activity reaching at least 15% receptor activation either at 5 or 20 μM, including new scaffolds from natural and synthetic origin.