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The bile acid membrane receptor TGR5 as an emerging target in metabolism and inflammation

Open AccessPublished:December 09, 2010DOI:https://doi.org/10.1016/j.jhep.2010.12.004
      Bile acids (BAs) are amphipathic molecules that facilitate the uptake of lipids, and their levels fluctuate in the intestine as well as in the blood circulation depending on food intake. Besides their role in dietary lipid absorption, bile acids function as signaling molecules capable to activate specific receptors. These BA receptors are not only important in the regulation of bile acid synthesis and their metabolism, but also regulate glucose homeostasis, lipid metabolism, and energy expenditure. These processes are important in diabetes and other facets of the metabolic syndrome, which represents a considerable increasing health burden. In addition to the function of the nuclear receptor FXRα in regulating local effects in the organs of the enterohepatic axis, increasing evidence points to a crucial role of the G-protein coupled receptor (GPCR) TGR5 in mediating systemic actions of BAs. Here we discuss the current knowledge on BA receptors, with a strong focus on the cell membrane receptor TGR5, which emerges as a valuable target for intervention in metabolic diseases.

      Keywords

      Introduction

      BAs are multifunctional in metabolism

      Bile acids (BAs) are a component of bile, which also contains phosphatidylcholine, bilirubin, and cholesterol as main constituents. An important physiological role of BAs is to facilitate the uptake of lipids together with the fat-soluble vitamins A, D, E, and K from the intestine. BAs facilitate these absorptive processes through their detergent properties, which allow the emulsification of lipids [
      • Russell D.W.
      Fifty years of advances in bile acid synthesis and metabolism.
      ]. BAs also play a major role in influencing the intestinal microbial flora, as well as in the elimination of cholesterol from the body [
      • Russell D.W.
      • Setchell K.D.
      Bile acid biosynthesis.
      ,
      • Inagaki T.
      • Moschetta A.
      • Lee Y.K.
      • Peng L.
      • Zhao G.
      • Downes M.
      • et al.
      Regulation of antibacterial defense in the small intestine by the nuclear bile acid receptor.
      ]. More recently, BAs are increasingly being appreciated for their signaling properties to transmit information to cells and organs concerning the fasting/feeding state thereby regulating processes ranging from bile acid and lipid metabolism to glucose and energy homeostasis [
      • Houten S.M.
      • Watanabe M.
      • Auwerx J.
      Endocrine functions of bile acids.
      ,
      • Thomas C.
      • Pellicciari R.
      • Pruzanski M.
      • Auwerx J.
      • Schoonjans K.
      Targeting bile-acid signalling for metabolic diseases.
      ]. This is in fact not too surprising given the central role BAs play in dietary lipid absorption. The scope of this review is to provide an update on the most recent developments in the field of BA signaling, and their pharmaceutical implications to treat metabolic diseases, such as diabetes and other risk factors of the metabolic syndrome.

      Signaling pathways activated by BAs

      Nuclear receptor signaling pathways

      Figure thumbnail fx2
      Figure thumbnail fx3
      Figure thumbnail fx4
      Figure thumbnail gr1
      Fig. 1Simplified overview of classical bile acid synthesis pathway in mice. CYP7A1 and CYP8B1 catalyze the first and third reaction, respectively. Dotted lines represent several steps in the synthesis of BAs that involve multiple enzymes. AKR1D1 is an abbreviation for Δ4-3-oxosteroid 5β-reductase.
      In addition to FXRα, other nuclear receptors directly activated by LCA are the pregnane X receptor (PXR) and the vitamin D receptor (VDR) [
      • Xie W.
      • Radominska-Pandya A.
      • Shi Y.
      • Simon C.M.
      • Nelson M.C.
      • Ong E.S.
      • et al.
      An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids.
      ,
      • Staudinger J.L.
      • Goodwin B.
      • Jones S.A.
      • Hawkins-Brown D.
      • MacKenzie K.I.
      • LaTour A.
      • et al.
      The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity.
      ,
      • Makishima M.
      • Lu T.T.
      • Xie W.
      • Whitfield G.K.
      • Domoto H.
      • Evans R.M.
      • et al.
      Vitamin D receptor as an intestinal bile acid sensor.
      ] (see Fig. 2). PXR is not only activated by LCA, but also by certain bile acid precursors, such as 7α-OH-4-cholesten-3-one [
      • Goodwin B.
      • Gauthier K.C.
      • Umetani M.
      • Watson M.A.
      • Lochansky M.I.
      • Collins J.L.
      • et al.
      Identification of bile acid precursors as endogenous ligands for the nuclear xenobiotic pregnane X receptor.
      ]. PXR is like FXRα expressed in the liver and intestine [
      • Xie W.
      • Radominska-Pandya A.
      • Shi Y.
      • Simon C.M.
      • Nelson M.C.
      • Ong E.S.
      • et al.
      An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids.
      ,
      • Staudinger J.L.
      • Goodwin B.
      • Jones S.A.
      • Hawkins-Brown D.
      • MacKenzie K.I.
      • LaTour A.
      • et al.
      The nuclear receptor PXR is a lithocholic acid sensor that protects against liver toxicity.
      ]. One of the physiological functions of PXR is to induce phase I detoxification metabolism through induction of CYP3A expression, which explains the finding that PXR transgenic mice are protected against LCA-induced liver toxicity in a model of cholestasis [
      • Xie W.
      • Radominska-Pandya A.
      • Shi Y.
      • Simon C.M.
      • Nelson M.C.
      • Ong E.S.
      • et al.
      An essential role for nuclear receptors SXR/PXR in detoxification of cholestatic bile acids.
      ]. Like PXR, VDR plays a role in the detoxification of BAs through upregulation of CYP3A [
      • Makishima M.
      • Lu T.T.
      • Xie W.
      • Whitfield G.K.
      • Domoto H.
      • Evans R.M.
      • et al.
      Vitamin D receptor as an intestinal bile acid sensor.
      ]. In addition to their roles in detoxifying BAs, both nuclear receptors inhibit BA synthesis. VDR is not expressed in hepatocytes [
      • Gascon-Barre M.
      • Demers C.
      • Mirshahi A.
      • Neron S.
      • Zalzal S.
      • Nanci A.
      The normal liver harbors the vitamin D nuclear receptor in nonparenchymal and biliary epithelial cells.
      ]. However, activation of VDR in the intestine by vitamin D widely inhibits BA synthesis via the FGF19-CYP7A1 pathway [
      • Schmidt D.R.
      • Holmstrom S.R.
      • Fon Tacer K.
      • Bookout A.L.
      • Kliewer S.A.
      • Mangelsdorf D.J.
      Regulation of bile acid synthesis by fat-soluble vitamins A and D.
      ], and activation of PXR with rifampicin, an agonist of human PXR, inhibits BA synthesis through a pathway involving HNF4α [
      • Li T.
      • Chiang J.Y.
      Mechanism of rifampicin and pregnane X receptor inhibition of human cholesterol 7 alpha-hydroxylase gene transcription.
      ]. It should be noted that relative high concentrations of LCA (ranging from 30 to 100 μM) are needed to induce activation of VDR and PXR, which could raise questions about the physiological relevance of the direct regulation of their activity by BAs.
      Figure thumbnail gr2
      Fig. 2Representation of signaling pathways that are modulated by BAs. Genomic and non-genomic actions of BAs are mediated by nuclear receptors and G-protein coupled receptors. The BAs that modulate the activity of the receptor are indicated in the arrow, and are sorted by decreasing potency (top to bottom). Arrows in green indicate agonistic actions, red indicates antagonistic/inhibitory actions and blue indicates a modulatory effect, either agonistic or antagonistic. Receptors for which only a limited number of BAs were tested are indicated with dashed arrows. Only conjugated forms of LCA and DCA are reported to activate the muscarinic receptors.

      Kinase-signaling pathways regulated by BAs

      BAs also modulate kinase-signaling pathways, such as the JNK pathway, which has been demonstrated to be involved in the downregulation of CYP7A1 [
      • Gupta S.
      • Stravitz R.T.
      • Dent P.
      • Hylemon P.B.
      Down-regulation of cholesterol 7alpha-hydroxylase (CYP7A1) gene expression by bile acids in primary rat hepatocytes is mediated by the c-Jun N-terminal kinase pathway.
      ]. Another study has coupled JNK activation indirectly to BAs as the mechanism of the FGF19-FGFR4 signaling pathway to suppress CYP7A1 [
      • Holt J.A.
      • Luo G.
      • Billin A.N.
      • Bisi J.
      • McNeill Y.Y.
      • Kozarsky K.F.
      • et al.
      Definition of a novel growth factor-dependent signal cascade for the suppression of bile acid biosynthesis.
      ]. In addition to JNK, also the p38 mitogen activated protein kinases (p38MAPK), the extracellular signal-regulated kinase (ERK) pathway, as well as Akt are activated by BAs [
      • Qiao D.
      • Stratagouleas E.D.
      • Martinez J.D.
      Activation and role of mitogen-activated protein kinases in deoxycholic acid-induced apoptosis.
      ,
      • Dent P.
      • Fang Y.
      • Gupta S.
      • Studer E.
      • Mitchell C.
      • Spiegel S.
      • et al.
      Conjugated bile acids promote ERK1/2 and AKT activation via a pertussis toxin-sensitive mechanism in murine and human hepatocytes.
      ]. Although the precise mechanisms involved are not fully clear, a study has indicated that phosphorylation of ERK1, ERK2, and Akt by BAs is induced through both radical oxygen species (ROS)-dependent, as well as through GPCR-dependent pathways [
      • Dent P.
      • Fang Y.
      • Gupta S.
      • Studer E.
      • Mitchell C.
      • Spiegel S.
      • et al.
      Conjugated bile acids promote ERK1/2 and AKT activation via a pertussis toxin-sensitive mechanism in murine and human hepatocytes.
      ]. The activation of these phosphorylation cascades by BAs is, besides the suppression of CYP7A1, also involved in the regulation of apoptosis and cytoprotective effects [
      • Qiao D.
      • Stratagouleas E.D.
      • Martinez J.D.
      Activation and role of mitogen-activated protein kinases in deoxycholic acid-induced apoptosis.
      ,
      • Qiao L.
      • Han S.I.
      • Fang Y.
      • Park J.S.
      • Gupta S.
      • Gilfor D.
      • et al.
      Bile acid regulation of C/EBPbeta, CREB, and c-Jun function, via the extracellular signal-regulated kinase and c-Jun NH2-terminal kinase pathways, modulates the apoptotic response of hepatocytes.
      ]. It is also tempting to speculate that some of these signaling pathways may be involved in the enhanced lifespan, observed in yeast exposed to BAs, as yeast do not express either the nuclear or membrane BA receptors [
      • Goldberg A.A.
      • Richard V.R.
      • Kyryakov P.
      • Bourque S.D.
      • Beach A.
      • Burstein M.T.
      • et al.
      Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes.
      ].

      GPCR signaling pathways modulated by BAs

      Most relevant to this review is that BAs also bind and modulate the activity of specific G-protein coupled receptors (GPCRs) (see Fig. 2). The total GPCR family comprises over 800 receptors, which are divided into three subgroups [
      • Pierce K.L.
      • Premont R.T.
      • Lefkowitz R.J.
      Seven-transmembrane receptors.
      ]. There are currently three known GPCRs whose activities are modulated by BAs. Based on their sequence homology, these BA-modulated GPCRs are classified in the class A or rhodopsin-like receptor class, which represents the largest subgroup of GPCRs. BAs are able to modulate the activity of the muscarinic receptors (also designated as acetylcholine receptors) [
      • Raufman J.P.
      • Zimniak P.
      • Bartoszko-Malik A.
      Lithocholyltaurine interacts with cholinergic receptors on dispersed chief cells from guinea pig stomach.
      ,
      • Raufman J.P.
      • Chen Y.
      • Zimniak P.
      • Cheng K.
      Deoxycholic acid conjugates are muscarinic cholinergic receptor antagonists.
      ], and inhibit the activity of the formyl-peptide receptors (FPRs) [
      • Le Y.
      • Murphy P.M.
      • Wang J.M.
      Formyl-peptide receptors revisited.
      ,
      • Ferrari C.
      • Macchiarulo A.
      • Costantino G.
      • Pellicciari R.
      Pharmacophore model for bile acids recognition by the FPR receptor.
      ]. FPR receptors have been described to be expressed in neutrophils and monocytes, and are activated by N-formyl groups, present on bacterial and mitochondrial proteins. This induces cell chemotaxis, believed to direct phagocytes to sites of tissue damage or infections. It is possible that the inhibition of activity of these receptors by BAs contributes to anti-inflammatory properties of BAs, as CDCA inhibited FPR-induced human leukocyte chemotaxis. [
      • Chen X.
      • Yang D.
      • Shen W.
      • Dong H.F.
      • Wang J.M.
      • Oppenheim J.J.
      • et al.
      Characterization of chenodeoxycholic acid as an endogenous antagonist of the G-coupled formyl peptide receptors.
      ]. The five muscarinic receptors (designated M1–M5) regulate numerous physiological processes by mediating parasympathetic innervation via acetylcholine sensing. A dysfunction in their activity may contribute to several diseases, including metabolic diseases [
      • Wess J.
      • Eglen R.M.
      • Gautam D.
      Muscarinic acetylcholine receptors: mutant mice provide new insights for drug development.
      ]. Muscarinic receptors are expressed in the central nervous system as well as in peripheral organs. For example, the M3 muscarinic receptors, which are predominantly expressed in gastrointestinal tissues, control smooth-muscle contractility and glandular secretion. Also the pancreatic β-islet cells express M3 muscarinic receptors, and induce insulin release during the pre-absorptive phase of feeding [
      • Gautam D.
      • Han S.J.
      • Hamdan F.F.
      • Jeon J.
      • Li B.
      • Li J.H.
      • et al.
      A critical role for beta cell M3 muscarinic acetylcholine receptors in regulating insulin release and blood glucose homeostasis in vivo.
      ]. These data suggest that these receptors could potentially contribute to a broad range of effects observed by BAs, although it should be mentioned that high millimolar concentrations of BAs are necessary to induce activation of muscarinic receptors. The relevance has not been analyzed so far in humans.
      The GPCR that has been most studied in relation to BAs is TGR5, also known as M-BAR, GPBAR, or GPR131. This cell-surface BA receptor was discovered in 2002 [
      • Maruyama T.
      • Miyamoto Y.
      • Nakamura T.
      • Tamai Y.
      • Okada H.
      • Sugiyama E.
      • et al.
      Identification of membrane-type receptor for bile acids (M-BAR).
      ], and first characterized in 2003 [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. TGR5 is encoded by a single-exon gene, and its conservation among several vertebrate species underlines the relevance of this GPCR in vertebrate physiology [
      • Maruyama T.
      • Miyamoto Y.
      • Nakamura T.
      • Tamai Y.
      • Okada H.
      • Sugiyama E.
      • et al.
      Identification of membrane-type receptor for bile acids (M-BAR).
      ]. Human TGR5 is activated by multiple BAs, with LCA being the most potent natural agonist with an EC50 of 0.53 μM [
      • Maruyama T.
      • Miyamoto Y.
      • Nakamura T.
      • Tamai Y.
      • Okada H.
      • Sugiyama E.
      • et al.
      Identification of membrane-type receptor for bile acids (M-BAR).
      ,
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. Next to LCA, other bile acids that activate TGR5 include conjugated and unconjugated forms of DCA, CDCA, and CA with an EC50 of 1.0, 4.4, and 7.7 μM, respectively [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. Human and mouse TGR5 show basically the same affinity for the different BAs, although certain synthetic TGR5 agonists reveal a large difference between mouse and human TGR5 with regard to their efficacy to activate the receptor [
      • Herbert M.R.
      • Siegel D.L.
      • Staszewski L.
      • Cayanan C.
      • Banerjee U.
      • Dhamija S.
      • et al.
      Synthesis and SAR of 2-aryl-3-aminomethylquinolines as agonists of the bile acid receptor TGR5.
      ].

      The bile acid receptor TGR5

      Expression and signaling of TGR5

      TGR5 is expressed in many different organs and cells with varying degrees of expression. For example, high expression levels of TGR5 have been detected in gallbladder epithelium [
      • Vassileva G.
      • Golovko A.
      • Markowitz L.
      • Abbondanzo S.J.
      • Zeng M.
      • Yang S.
      • et al.
      Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation.
      ,
      • Keitel V.
      • Cupisti K.
      • Ullmer C.
      • Knoefel W.T.
      • Kubitz R.
      • Haussinger D.
      The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders.
      ] and in the intestine, mainly in the ileum and colon [
      • Maruyama T.
      • Miyamoto Y.
      • Nakamura T.
      • Tamai Y.
      • Okada H.
      • Sugiyama E.
      • et al.
      Identification of membrane-type receptor for bile acids (M-BAR).
      ,
      • Maruyama T.
      • Tanaka K.
      • Suzuki J.
      • Miyoshi H.
      • Harada N.
      • Nakamura T.
      • et al.
      Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice.
      ]. TGR5 was also highly expressed in human monocytes as compared to other human leukocyte subsets examined [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. In line with these findings, rabbit spleen and rabbit alveolar macrophages were also found to express relatively high levels of TGR5 versus other tissues [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. TGR5 is detected in several liver cells, including rat liver sinusoidal endothelial cells, as well as in rat Kupffer cells, resident macrophages of the liver [
      • Keitel V.
      • Reinehr R.
      • Gatsios P.
      • Rupprecht C.
      • Gorg B.
      • Selbach O.
      • et al.
      The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells.
      ,
      • Keitel V.
      • Donner M.
      • Winandy S.
      • Kubitz R.
      • Haussinger D.
      Expression and function of the bile acid receptor TGR5 in Kupffer cells.
      ]. TGR5 is also expressed in BAT, skeletal muscle, and selected areas of the central nervous system [
      • Watanabe M.
      • Houten S.M.
      • Mataki C.
      • Christoffolete M.A.
      • Kim B.W.
      • Sato H.
      • et al.
      Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.
      ,
      • Poole D.P.
      • Godfrey C.
      • Cattaruzza F.
      • Cottrell G.S.
      • Kirkland J.G.
      • Pelayo J.C.
      • et al.
      Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system.
      ]. Furthermore, TGR5 is ubiquitously expressed in many more different organs and cells with varying degrees of expression [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ,
      • Vassileva G.
      • Golovko A.
      • Markowitz L.
      • Abbondanzo S.J.
      • Zeng M.
      • Yang S.
      • et al.
      Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation.
      ].
      In resting conditions, GPCRs, including TGR5, are in a so-called low-affinity state. In response to binding of BAs to the ligand-binding pocket of the receptor, a complex is released from TGR5 consisting of G-protein-αs, β, and γ [
      • Pierce K.L.
      • Premont R.T.
      • Lefkowitz R.J.
      Seven-transmembrane receptors.
      ,
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. GDP is subsequently released from the G-protein and replaced by GTP, leading to dissociation of the G-protein complexes into G-protein-αs and βγ dimers. G-protein-αs subsequently activates adenylyl cyclase resulting in the induction of cAMP and activation of protein kinase A (PKA), which in turn induces further downstream signaling [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ] (see Fig. 3). Whether TGR5 may also bind to other G-proteins, which have distinct downstream effector molecules, is still an open question [
      • Pierce K.L.
      • Premont R.T.
      • Lefkowitz R.J.
      Seven-transmembrane receptors.
      ].
      Figure thumbnail gr3
      Fig. 3Simplified overview of the TGR5-signaling pathway leading to downstream signaling via cAMP induction.

      TGR5 in BA homeostasis and metabolism

      To explore the biological role of TGR5, several groups have independently generated TGR5−/− mice [
      • Vassileva G.
      • Golovko A.
      • Markowitz L.
      • Abbondanzo S.J.
      • Zeng M.
      • Yang S.
      • et al.
      Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation.
      ,
      • Maruyama T.
      • Tanaka K.
      • Suzuki J.
      • Miyoshi H.
      • Harada N.
      • Nakamura T.
      • et al.
      Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice.
      ,
      • Thomas C.
      • Gioiello A.
      • Noriega L.
      • Strehle A.
      • Oury J.
      • Rizzo G.
      • et al.
      TGR5-mediated bile acid sensing controls glucose homeostasis.
      ]. Interestingly, the total bile acid pool size in TGR5−/− mice was decreased as compared to that of wild-type mice [
      • Maruyama T.
      • Tanaka K.
      • Suzuki J.
      • Miyoshi H.
      • Harada N.
      • Nakamura T.
      • et al.
      Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice.
      ], which was also observed by our laboratory (JA & KS, unpublished data). The cause of the decreased BA pool in TGR5−/− mice is currently unknown, but appears not to be caused by changed fecal excretion of BAs, which was similar between TGR5−/− and control mice [
      • Maruyama T.
      • Tanaka K.
      • Suzuki J.
      • Miyoshi H.
      • Harada N.
      • Nakamura T.
      • et al.
      Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice.
      ].
      In addition to changes in the BA pool size, TGR5−/− mice fed a lithogenic diet were protected against cholesterol gallstone formation [
      • Vassileva G.
      • Hu W.
      • Hoos L.
      • Tetzloff G.
      • Yang S.
      • Liu L.
      • et al.
      Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice.
      ]. Hydrophobic bile salts decrease gallbladder smooth muscle function potentially via stimulation of TGR5, which could be a contributing factor in the manifestation of gallstone disease [
      • Lavoie B.
      • Balemba O.B.
      • Godfrey C.
      • Watson C.A.
      • Vassileva G.
      • Corvera C.U.
      • et al.
      Hydrophobic bile salts inhibit gallbladder smooth muscle function via stimulation of GPBAR1 receptors and activation of KATP channels.
      ]. Additionally, TGR5 is expressed in human gallbladder epithelial cells and cholangiocytes and was shown to play a role in bile composition, via the induction of chloride secretion [
      • Keitel V.
      • Cupisti K.
      • Ullmer C.
      • Knoefel W.T.
      • Kubitz R.
      • Haussinger D.
      The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders.
      ,
      • Keitel V.
      • Ullmer C.
      • Haussinger D.
      The membrane-bound bile acid receptor TGR5 (Gpbar-1) is localized in the primary cilium of cholangiocytes.
      ]. Taken together, the physiological changes observed in mice lacking TGR5 with regard to BA homeostasis, combined with the expression of TGR5 in human tissues relevant to BA homeostasis hint toward a role of TGR5 in bile formation and homeostasis in man [
      • Keitel V.
      • Cupisti K.
      • Ullmer C.
      • Knoefel W.T.
      • Kubitz R.
      • Haussinger D.
      The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders.
      ,
      • Keitel V.
      • Ullmer C.
      • Haussinger D.
      The membrane-bound bile acid receptor TGR5 (Gpbar-1) is localized in the primary cilium of cholangiocytes.
      ].

      Effect of TGR5 on body weight

      TGR5 activation induces a significant reduction of the body weight of mice fed a high fat diet. We first demonstrated that dietary supplementation of BAs significantly reduces body weight gain in C57Bl/6J mice fed a high fat diet [
      • Watanabe M.
      • Houten S.M.
      • Mataki C.
      • Christoffolete M.A.
      • Kim B.W.
      • Sato H.
      • et al.
      Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.
      ]. CA administration completely prevented high fat diet-induced changes in adipose mass and morphology, and reversed 120 days of diet-induced weight gain within 30 days, without toxicity [
      • Watanabe M.
      • Houten S.M.
      • Mataki C.
      • Christoffolete M.A.
      • Kim B.W.
      • Sato H.
      • et al.
      Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.
      ]. The weight reducing effects were not due to reduced caloric intake, but were the consequence of enhanced energy expenditure. Using deiodinase 2 (D2)-deficient mice, the effect of CA on energy expenditure was shown to require the induction of deiodinase 2 (D2) through a TGR5-cAMP-mediated pathway that was active in murine BAT and in human skeletal muscle myoblasts. Importantly, this effect of BAs on energy expenditure was independent of FXR, as the FXR agonist GW4064, did not increase cAMP levels in BAT, and increased diet-induced obesity in mice. D2 is able to increase mitochondrial oxidative phosphorylation and energy expenditure in brown adipose tissue and muscle via the conversion of inactive thyroxine (T4) into active 3,5,3′-tri-iodothyronine (T3), which subsequently binds and activates the thyroid hormone receptor, thereby inducing energy expenditure [
      • Watanabe M.
      • Houten S.M.
      • Mataki C.
      • Christoffolete M.A.
      • Kim B.W.
      • Sato H.
      • et al.
      Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.
      ]. It should be mentioned that postprandial plasma BA levels of 15 μM are capable of activating TGR5 [
      • Watanabe M.
      • Houten S.M.
      • Mataki C.
      • Christoffolete M.A.
      • Kim B.W.
      • Sato H.
      • et al.
      Bile acids induce energy expenditure by promoting intracellular thyroid hormone activation.
      ]. The latter effects were subsequently confirmed in a study using the semi-synthetic BA, 6-ethyl-23(S)methyl-cholic acid (6EMCA or INT-777), which acts as a specific TGR5, but not FXR, agonist [
      • Thomas C.
      • Gioiello A.
      • Noriega L.
      • Strehle A.
      • Oury J.
      • Rizzo G.
      • et al.
      TGR5-mediated bile acid sensing controls glucose homeostasis.
      ].
      In agreement with our data, Maruyama et al. observed that female TGR5−/− mice have increased body weight [
      • Maruyama T.
      • Tanaka K.
      • Suzuki J.
      • Miyoshi H.
      • Harada N.
      • Nakamura T.
      • et al.
      Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice.
      ]. The latter effect of TGR5 was already observed in heterozygous female mice. In agreement with this observation, female TGR5−/− mice had a higher fat content, while the lean body weight was unaffected as compared to wild-type mice. Also the body composition of the male TGR5−/− mice showed a tendency toward increased fat content [
      • Maruyama T.
      • Tanaka K.
      • Suzuki J.
      • Miyoshi H.
      • Harada N.
      • Nakamura T.
      • et al.
      Targeted disruption of G protein-coupled bile acid receptor 1 (Gpbar1/M-Bar) in mice.
      ]. Interestingly, in the TGR5−/− mice line that we have generated in our laboratory, we also observed a significant increase in body weight in males ([
      • Thomas C.
      • Gioiello A.
      • Noriega L.
      • Strehle A.
      • Oury J.
      • Rizzo G.
      • et al.
      TGR5-mediated bile acid sensing controls glucose homeostasis.
      ] and KS & JA unpublished data). These two studies are, however, in contrast to the data of Vassileva et al. [
      • Vassileva G.
      • Hu W.
      • Hoos L.
      • Tetzloff G.
      • Yang S.
      • Liu L.
      • et al.
      Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice.
      ], which did not observe an effect on body weight in the absence of TGR5. This difference may very well be explained by different diets used, or by other differences in animal experimental conditions applied in this specific study. Furthermore, it needs to be underscored, that the mere absence of a receptor, as in the TGR5−/− mice, should not necessary translate in the opposite phenotype as observed after receptor activation by exogeneous ligands.
      A recent study assessed BA levels in humans with respect to energy expenditure [
      • Brufau G.
      • Bahr M.J.
      • Staels B.
      • Claudel T.
      • Ockenga J.
      • Boker K.H.
      • et al.
      Plasma bile acids are not associated with energy metabolism in humans.
      ]. In this study, patients suffering from cirrhosis displaying elevated plasma BA levels and healthy controls were compared with respect to energy expenditure. It was concluded that no correlations between BA levels and energy expenditure exist. It should, however, be underlined that the many symptoms observed in cirrhosis are not controlled, and more than likely confound the study outcome. A better understanding of the impact of BA on energy expenditure in humans will require further studies specifically designed to address this question.

      Effects of TGR5 on glucose metabolism and insulin sensitivity

      High circulating levels of BAs have been linked with beneficial effects on glucose metabolism, such as improved insulin sensitivity and postprandial glycemic control [
      • Shaham O.
      • Wei R.
      • Wang T.J.
      • Ricciardi C.
      • Lewis G.D.
      • Vasan R.S.
      • et al.
      Metabolic profiling of the human response to a glucose challenge reveals distinct axes of insulin sensitivity.
      ,
      • Patti M.E.
      • Houten S.M.
      • Bianco A.C.
      • Bernier R.
      • Larsen P.R.
      • Holst J.J.
      • et al.
      Serum bile acids are higher in humans with prior gastric bypass: potential contribution to improved glucose and lipid metabolism.
      ]. We have demonstrated that BAs regulate glucose homeostasis through activation of TGR5 [
      • Thomas C.
      • Auwerx J.
      • Schoonjans K.
      Bile acids and the membrane bile acid receptor TGR5-connecting nutrition and metabolism.
      ]. In agreement with the report that TGR5 induces GLP-1 secretion in cultured mouse enteroendocrine STC-1 cells [
      • Katsuma S.
      • Hirasawa A.
      • Tsujimoto G.
      Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1.
      ], the semi-synthetic BA, 6-ethyl-23(S)methyl-cholic acid (6EMCA or INT-777), which is a specific TGR5 agonist, induces GLP-1 secretion in both STC-1 cells as well as in human intestinal NCI-H716 cells, and contributes as such to the effects of TGR5 in glucose homeostasis. Silencing of TGR5 in STC-1 cells using short hairpin RNA prevented the secretion of GLP-1, illustrating the involvement of TGR5 in this response. Although the mechanism underlying TGR5-induced GLP-1 secretion is not yet completely established, stimulation of oxidative phosphorylation may be involved in triggering this process. The resulting increase in ATP/ADP ratio can subsequently induce membrane depolarization and Ca2+ mobilization in a way reminiscent to the cascade of events that precedes insulin release in pancreatic β-cells [
      • Thomas C.
      • Gioiello A.
      • Noriega L.
      • Strehle A.
      • Oury J.
      • Rizzo G.
      • et al.
      TGR5-mediated bile acid sensing controls glucose homeostasis.
      ]. Using obese and insulin resistant mouse models, we have shown that mice with a gain-of-function of TGR5 became more glucose tolerant, whereas TGR5−/− mice showed a delayed glucose clearance compared to their wild-type littermates. This effect was correlated with a healthier pancreatic islet phenotype, and is at least partly explained by the tonic increase of GLP-1 secretion by TGR5 [
      • Thomas C.
      • Auwerx J.
      • Schoonjans K.
      Bile acids and the membrane bile acid receptor TGR5-connecting nutrition and metabolism.
      ]. It was recently reported by Poole et al. that TGR5 is also expressed in inhibitory motor neurons and modulates intestinal motility [
      • Poole D.P.
      • Godfrey C.
      • Cattaruzza F.
      • Cottrell G.S.
      • Kirkland J.G.
      • Pelayo J.C.
      • et al.
      Expression and function of the bile acid receptor GpBAR1 (TGR5) in the murine enteric nervous system.
      ]. This effect of TGR5 could be related to GLP-1 induction, which is also known to inhibit intestinal motility [
      • Tolessa T.
      • Gutniak M.
      • Holst J.J.
      • Efendic S.
      • Hellstrom P.M.
      Inhibitory effect of glucagon-like peptide-1 on small bowel motility. Fasting but not fed motility inhibited via nitric oxide independently of insulin and somatostatin.
      ]. In apparent contrast to these observations are the findings in a recent study with independently generated TGR5−/− mice, demonstrating that female and male chow fed TGR5−/− mice show improved insulin sensitivity [
      • Vassileva G.
      • Hu W.
      • Hoos L.
      • Tetzloff G.
      • Yang S.
      • Liu L.
      • et al.
      Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice.
      ]. However, it was also shown in this study that male TGR5−/− mice fed with a high fat diet display impaired insulin sensitivity, which is in agreement with our findings. Other evidence that TGR5 activation is beneficial with regard to diabetes comes from the observation that the triterpenoid, oleanolic acid, a natural TGR5 agonist, also improves glucose homeostasis [
      • Sato H.
      • Genet C.
      • Strehle A.
      • Thomas C.
      • Lobstein A.
      • Wagner A.
      • et al.
      Anti-hyperglycemic activity of a TGR5 agonist isolated from Olea europaea.
      ].

      TGR5 modulates the immune response

      One of the initial studies on TGR5 concerns its role in immune cells and has linked TGR5 to the immunomodulatory properties of BAs [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. This action of TGR5 is very relevant, as low-grade inflammation is suggested to contribute to the development of the metabolic syndrome [
      • Olefsky J.M.
      • Glass C.K.
      Macrophages, inflammation, and insulin resistance.
      ]. TGR5 is highly expressed in monocytes and macrophages, an observation derived from the finding that TGR5 expression is present in human spleen and human CD14+ monocytes, as well as in rabbit alveolar macrophages [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ,
      • Keitel V.
      • Donner M.
      • Winandy S.
      • Kubitz R.
      • Haussinger D.
      Expression and function of the bile acid receptor TGR5 in Kupffer cells.
      ]. In accordance with a report that cAMP inhibits LPS-induced cytokine secretion [
      • Yoshimura T.
      • Kurita C.
      • Nagao T.
      • Usami E.
      • Nakao T.
      • Watanabe S.
      • et al.
      Inhibition of tumor necrosis factor-alpha and interleukin-1-beta production by beta-adrenoceptor agonists from lipopolysaccharide-stimulated human peripheral blood mononuclear cells.
      ], BAs capable of activating TGR5 were found to increase cAMP production in alveolar macrophages [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. In addition, BAs reduce the phagocytic activity of these cells and inhibit LPS-induced production of several pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), Interleukin (IL)-1α, IL-1β, IL-6, and IL-8 [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. Human monocytic leukemia THP-1 cells transfected with TGR5 exhibit increased cAMP production and a reduction in LPS-induced TNF-α expression. These effects were not observed in untransfected THP-1 cells, which express low levels of TGR5, demonstrating that these effects of bile acids are mediated through activation of TGR5 [
      • Kawamata Y.
      • Fujii R.
      • Hosoya M.
      • Harada M.
      • Yoshida H.
      • Miwa M.
      • et al.
      A G protein-coupled receptor responsive to bile acids.
      ]. Furthermore, stimulation of isolated rat Kuppfer cells with taurolithocholic acid (TLC) or other TGR5 agonists, such as oleanolic acid, as well as cAMP stimulation resulted in reduced expression of IL-1α, IL-1β, IL-6, and TNF-α following LPS treatment [
      • Keitel V.
      • Donner M.
      • Winandy S.
      • Kubitz R.
      • Haussinger D.
      Expression and function of the bile acid receptor TGR5 in Kupffer cells.
      ]. Although the inhibition of inflammation through TGR5 activation can be considered beneficial for atherosclerosis, steatosis, and obesity, the anti-inflammatory action of TGR5 in macrophages might perhaps also explain observations that obstructive cholangitis is associated with enhanced susceptibility for infections due to decreased clearance of intrabiliary bacteria [
      • Scott-Conner C.E.
      • Grogan J.B.
      The pathophysiology of biliary obstruction and its effect on phagocytic and immune function.
      ]. Recently, association of 22 single nucleotide polymorphisms (SNPs) within the TGR5 genes were assessed with regard to primary sclerosing cholangitis (PSC). A weak association of one of these SNPs, rs11554825, was observed with PSC as well as with ulcerative colitis, although it could not be excluded that SNPs in neighboring genes that are in linkage disequilibrium with rs11554825 are responsible for this effect [
      • Hov J.R.
      • Keitel V.
      • Laerdahl J.K.
      • Spomer L.
      • Ellinghaus E.
      • ElSharawy A.
      • et al.
      Mutational characterization of the bile acid receptor TGR5 in primary sclerosing cholangitis.
      ].

      TGR5 in liver function

      Administration of the specific TGR5 agonist, INT-777 to high fat diet-fed mice reduces liver steatosis and associated hepatocyte damage, as measured by plasma liver enzymes LDH, ASAT, and ALAT [
      • Thomas C.
      • Gioiello A.
      • Noriega L.
      • Strehle A.
      • Oury J.
      • Rizzo G.
      • et al.
      TGR5-mediated bile acid sensing controls glucose homeostasis.
      ]. This is correlated with decreased plasma triglyceride and nonesterified fatty acid levels. The latter is consistent with the report by Vassileva et al. showing a pronounced hepatosteatosis in male TGR5−/− mice fed a high fat diet for 8 weeks [
      • Vassileva G.
      • Hu W.
      • Hoos L.
      • Tetzloff G.
      • Yang S.
      • Liu L.
      • et al.
      Gender-dependent effect of Gpbar1 genetic deletion on the metabolic profiles of diet-induced obese mice.
      ]. These data suggest that activation of TGR5 may prevent non-alcoholic fatty liver disease (NAFLD), although the precise regulatory mechanisms by which TGR5 induces this effect on liver triglycerides remains to be dissected. Although TGR5 seems not expressed in hepatocytes, it is detected in many cell types of the liver where it could directly or indirectly modulate liver function and triglyceride metabolism. TGR5 is for instance highly expressed in Kupffer cells, which are resident liver macrophages [
      • Keitel V.
      • Donner M.
      • Winandy S.
      • Kubitz R.
      • Haussinger D.
      Expression and function of the bile acid receptor TGR5 in Kupffer cells.
      ]. It has become clear that these cells, together with their pro-inflammatory cytokine secretion, are critically involved in the progression of NAFLD [
      • Baffy G.
      Kupffer cells in non-alcoholic fatty liver disease: the emerging view.
      ]. The anti-inflammatory action of TGR5 in these cells could, therefore, contribute to the protective effects of TGR5 on steatosis. TGR5 has also been shown to modulate microcirculation and fluid secretion in the endothelial and biliary epithelial cells of the liver [
      • Keitel V.
      • Reinehr R.
      • Gatsios P.
      • Rupprecht C.
      • Gorg B.
      • Selbach O.
      • et al.
      The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells.
      ,
      • Keitel V.
      • Ullmer C.
      • Haussinger D.
      The membrane-bound bile acid receptor TGR5 (Gpbar-1) is localized in the primary cilium of cholangiocytes.
      ]. Although the increased energy expenditure and GLP-1 secretion following TGR5 activation may very well explain the significant improvement in liver steatosis, it will be challenging to examine whether any of these other cell types in the liver contribute to the protective effects of TGR5 activation against steatosis. Cell-type specific TGR5−/− mouse models will be extremely valuable tools to address these questions.

      BA signaling as a target for intervention in the metabolic syndrome

      BA signaling as target for intervention

      The significance of BAs in human triglyceride metabolism is underlined by findings that bile-acid binding resins induce the production of VLDL and that treatment of cholesterol gallstones in humans with CDCA reduces hypertriglyceridemia [
      • Grundy S.M.
      • Ahrens Jr., E.H.
      • Salen G.
      Interruption of the enterohepatic circulation of bile acids in man: comparative effects of cholestyramine and ileal exclusion on cholesterol metabolism.
      ,
      • Nestel P.J.
      • Poyser A.
      Changes in cholesterol synthesis and excretion when cholesterol intake is increased.
      ,
      • Angelin B.
      • Einarsson K.
      • Hellstrom K.
      Effect of cholestyramine on bile acid kinetics in patients with portal cirrhosis of the liver. Evidence of a selective defect in the formation of cholic acid.
      ]. In addition to the effects of BAs on human triglyceride metabolism, BAs are correlated to increased insulin sensitivity in humans [
      • Shaham O.
      • Wei R.
      • Wang T.J.
      • Ricciardi C.
      • Lewis G.D.
      • Vasan R.S.
      • et al.
      Metabolic profiling of the human response to a glucose challenge reveals distinct axes of insulin sensitivity.
      ]. Furthermore, patients after bariatric surgery to correct for obesity have higher circulating levels of BAs, which are positively correlated to peak GLP-1 levels [
      • Patti M.E.
      • Houten S.M.
      • Bianco A.C.
      • Bernier R.
      • Larsen P.R.
      • Holst J.J.
      • et al.
      Serum bile acids are higher in humans with prior gastric bypass: potential contribution to improved glucose and lipid metabolism.
      ]. The latter observation was recently confirmed in obese patients, who have a decreased postprandial BA response and suboptimal GLP-1 secretion in comparison to normal weight subjects [
      • Glicksman C.
      • Pournaras D.J.
      • Wright M.
      • Roberts R.
      • Mahon D.
      • Welbourn R.
      • et al.
      Postprandial plasma bile acid responses in normal weight and obese subjects.
      ].
      A few studies report on the positive effects of bile-acid binding resins on glucose homeostasis [
      • Kobayashi M.
      • Ikegami H.
      • Fujisawa T.
      • Nojima K.
      • Kawabata Y.
      • Noso S.
      • et al.
      Prevention and treatment of obesity, insulin resistance, and diabetes by bile acid-binding resin.
      ,
      • Yamakawa T.
      • Takano T.
      • Utsunomiya H.
      • Kadonosono K.
      • Okamura A.
      Effect of colestimide therapy for glycemic control in type 2 diabetes mellitus with hypercholesterolemia.
      ,
      • Fonseca V.A.
      • Rosenstock J.
      • Wang A.C.
      • Truitt K.E.
      • Jones M.R.
      Colesevelam HCl improves glycemic control and reduces LDL cholesterol in patients with inadequately controlled type 2 diabetes on sulfonylurea-based therapy.
      ]. The positive effects of bile acid sequestrants on glucose homeostasis could be the consequence of improved general metabolism subsequent to the elimination of cholesterol. It might, however, also raise the intriguing possibilities that bile-acid binding resins through still unknown mechanisms trigger TGR5 activation and GLP-1 secretion, hence contributing to the beneficial effects on glucose homeostasis.
      The biological properties of TGR5 described in this review (see Table 1), mostly observed in animal models, strongly indicate that TGR5 is linked to the beneficial properties of BAs in humans. This is underlined by the findings that the improvements of BAs on metabolic homeostasis are among others linked to GLP-1, for which its regulation by TGR5 is well documented [
      • Thomas C.
      • Gioiello A.
      • Noriega L.
      • Strehle A.
      • Oury J.
      • Rizzo G.
      • et al.
      TGR5-mediated bile acid sensing controls glucose homeostasis.
      ,
      • Katsuma S.
      • Hirasawa A.
      • Tsujimoto G.
      Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1.
      ]. Furthermore, a recent human genetic study indicates a potential association of TGR5 with some aspects of the metabolic syndrome. This study addressed the link between the single nucleotide polymorphism (SNP) rs3731859 within the human TGR5 gene and the risk for type 2 diabetes [
      • Mussig K.
      • Staiger H.
      • Machicao F.
      • Machann J.
      • Schick F.
      • Schafer S.A.
      • et al.
      Preliminary report: genetic variation within the GPBAR1 gene is not associated with metabolic traits in white subjects at an increased risk for type 2 diabetes mellitus.
      ]. Although no correlation was found between the onset of diabetes and the investigated SNP, nominal associations with body mass index, waist circumference, intramyocellular lipids, and fasting GLP-1 levels were identified. This interesting result warrants further studies to explore the effect of this (and other) SNPs in the TGR5 gene in different populations and calls for further mechanistic studies that address whether this SNP, located at the 5′-untranslated region, influences TGR5 expression levels.
      Table 1Cellular actions described for TGR5 in different cell types.
      ∗Macrophages include alveolar macrophages, Kupffer cells and THP-1 cells.

      Pharmacological targeting of TGR5

      BAs bind and activate both TGR5, as well as other (nuclear) receptors, among which is FXR. The binding pocket for the membrane BA receptor TGR5 and the nuclear BA receptor FXRα is only partially conserved and minor structural modifications on the steroid side chain of BAs can dictate selectivity of the ligand toward TGR5 [
      • Pellicciari R.
      • Sato H.
      • Gioiello A.
      • Costantino G.
      • Macchiarulo A.
      • Sadeghpour B.M.
      • et al.
      Nongenomic actions of bile acids. Synthesis and preliminary characterization of 23- and 6,23-alkyl-substituted bile acid derivatives as selective modulators for the G-protein coupled receptor TGR5.
      ,
      • Macchiarulo A.
      • Gioiello A.
      • Thomas C.
      • Massarotti A.
      • Nuti R.
      • Rosatelli E.
      • et al.
      Molecular field analysis and 3D-quantitative structure–activity relationship study (MFA 3D-QSAR) unveil novel features of bile acid recognition at TGR5.
      ]. This unique ligand-binding pocket of TGR5, therefore, allows the design of receptor selective ligands, leading to drugs that are able to target TGR5 exclusively.
      TGR5 may be targeted by natural compounds as well as by synthetic agonists. Such TGR5 agonists include natural BAs, semi-synthetic BAs, such as 6-ethyl-23(S)methyl-cholic acid [
      • Pellicciari R.
      • Gioiello A.
      • Macchiarulo A.
      • Thomas C.
      • Rosatelli E.
      • Natalini B.
      • et al.
      Discovery of 6alpha-ethyl-23(S)-methylcholic acid (S-EMCA, INT-777) as a potent and selective agonist for the TGR5 receptor, a novel target for diabesity.
      ], bile alcohols, and triterpenoid compounds of plant origin, such as oleanolic acid and betulinic acid [
      • Sato H.
      • Genet C.
      • Strehle A.
      • Thomas C.
      • Lobstein A.
      • Wagner A.
      • et al.
      Anti-hyperglycemic activity of a TGR5 agonist isolated from Olea europaea.
      ,
      • Sato H.
      • Macchiarulo A.
      • Thomas C.
      • Gioiello A.
      • Une M.
      • Hofmann A.F.
      • et al.
      Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure–activity relationships, and molecular modeling studies.
      ,
      • Genet C.
      • Strehle A.
      • Schmidt C.
      • Boudjelal G.
      • Lobstein A.
      • Schoonjans K.
      • et al.
      Structure–activity relationship study of betulinic acid, a novel and selective TGR5 agonist, and its synthetic derivatives: potential impact in diabetes.
      ,
      • Genet C.
      • Schmidt C.
      • Strehle A.
      • Schoonjans K.
      • Auwerx J.
      • Saladin R.
      • et al.
      Redefining the TGR5 triterpenoid binding pocket at the C-3 position.
      ]. Also certain steroid hormones potently activate TGR5 [
      • Sato H.
      • Macchiarulo A.
      • Thomas C.
      • Gioiello A.
      • Une M.
      • Hofmann A.F.
      • et al.
      Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure–activity relationships, and molecular modeling studies.
      ], an observation that has been recently confirmed [
      • Keitel V.
      • Gorg B.
      • Bidmon H.J.
      • Zemtsova I.
      • Spomer L.
      • Zilles K.
      • et al.
      The bile acid receptor TGR5 (Gpbar-1) acts as a neurosteroid receptor in brain.
      ]. Significant progress has also been made with the search for synthetic TGR5 agonists, as 3-aryl-4-isoxazolecarboxamides were recently identified to activate TGR5, and found to induce GLP-1 secretion in canines [
      • Evans K.A.
      • Budzik B.W.
      • Ross S.A.
      • Wisnoski D.D.
      • Jin J.
      • Rivero R.A.
      • et al.
      Discovery of 3-aryl-4-isoxazolecarboxamides as TGR5 receptor agonists.
      ]. Also specific 2-aryl-3-aminomethylquinones were recently identified as TGR5 agonists and observed to induce GLP-1 secretion and to improve glucose homeostasis in diet-induced obese mice [
      • Herbert M.R.
      • Siegel D.L.
      • Staszewski L.
      • Cayanan C.
      • Banerjee U.
      • Dhamija S.
      • et al.
      Synthesis and SAR of 2-aryl-3-aminomethylquinolines as agonists of the bile acid receptor TGR5.
      ]. Furthermore, many drug companies have active TGR5 programs, which have already resulted in the publication of several patents that describe additional TGR5 compounds. Further biological studies using these novel selective and potent synthetic TGR5 agonists are clearly awaited. Studies in animal models with tissue-specific deficiencies in TGR5 and this steadily increasing battery of TGR5 agonists will result in a better understanding of TGR5 biology and pharmacology, and perhaps new treatment options for different aspects of the metabolic syndrome.

      Not all that shines is gold – issues with TGR5

      Recently several properties of TGR5 have been described, that require further investigation as they could potentially underlie some side effects. For example, in cell culture models TGR5 has also been linked to epidermal growth factor receptor (EGFR) and c-Jun N-terminal kinase (JNK) signaling pathways, which modulate cell proliferation and apoptosis [
      • Yang J.I.
      • Yoon J.H.
      • Myung S.J.
      • Gwak G.Y.
      • Kim W.
      • Chung G.E.
      • et al.
      Bile acid-induced TGR5-dependent c-Jun-N terminal kinase activation leads to enhanced caspase 8 activation in hepatocytes.
      ,
      • Yasuda H.
      • Hirata S.
      • Inoue K.
      • Mashima H.
      • Ohnishi H.
      • Yoshiba M.
      Involvement of membrane-type bile acid receptor M-BAR/TGR5 in bile acid-induced activation of epidermal growth factor receptor and mitogen-activated protein kinases in gastric carcinoma cells.
      ]. This may suggest that TGR5 has a potential role in cancer development, but unfortunately evidence of such an effect was only ascertained in cultured cells and further studies in vivo are definitely required [
      • Yang J.I.
      • Yoon J.H.
      • Myung S.J.
      • Gwak G.Y.
      • Kim W.
      • Chung G.E.
      • et al.
      Bile acid-induced TGR5-dependent c-Jun-N terminal kinase activation leads to enhanced caspase 8 activation in hepatocytes.
      ,
      • Yasuda H.
      • Hirata S.
      • Inoue K.
      • Mashima H.
      • Ohnishi H.
      • Yoshiba M.
      Involvement of membrane-type bile acid receptor M-BAR/TGR5 in bile acid-induced activation of epidermal growth factor receptor and mitogen-activated protein kinases in gastric carcinoma cells.
      ]. TGR5 agonists, including certain steroids [
      • Sato H.
      • Macchiarulo A.
      • Thomas C.
      • Gioiello A.
      • Une M.
      • Hofmann A.F.
      • et al.
      Novel potent and selective bile acid derivatives as TGR5 agonists: biological screening, structure–activity relationships, and molecular modeling studies.
      ], were also reported to stimulate the generation of reactive oxygen species in cultured astrocytes [
      • Keitel V.
      • Gorg B.
      • Bidmon H.J.
      • Zemtsova I.
      • Spomer L.
      • Zilles K.
      • et al.
      The bile acid receptor TGR5 (Gpbar-1) acts as a neurosteroid receptor in brain.
      ]. The impact of this potential liability also requires further investigation in vivo. TGR5 activation has been suggested to influence cardiomyocytes as the TGR5-active bile acids tauro-CDCA and LCA activated AKT and inhibited glycogen synthase kinase-3β in these cells [
      • Desai M.S.
      • Shabier Z.
      • Taylor M.
      • Lam F.
      • Thevananther S.
      • Kosters A.
      • et al.
      Hypertrophic cardiomyopathy and dysregulation of cardiac energetics in a mouse model of biliary fibrosis.
      ]. In view of the pleiotropic effects of BAs, it will be of interest to assess these effects in TGR5−/− mice. In addition to these cellular observations, it has been reported that TGR5−/− mice have reduced pancreatitis upon direct exposure of the pancreas to high concentrations of taurolithocholic acid 3-sulfate sodium salt (TLCS) [
      • Perides G.
      • Laukkarinen J.M.
      • Vassileva G.
      • Steer M.L.
      Biliary acute pancreatitis in mice is mediated by the G-protein-coupled cell surface bile acid receptor Gpbar1.
      ]. In this study, the fact that the pancreas is exposed to very high concentrations of TLCS, which are normally never reached even under pathological conditions, may raise questions about its physiological relevance. Finally, the fact that TGR5−/− mice were protected against cholelithiasis [
      • Vassileva G.
      • Golovko A.
      • Markowitz L.
      • Abbondanzo S.J.
      • Zeng M.
      • Yang S.
      • et al.
      Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation.
      ] could imply that TGR5 agonism predisposes to this condition; there are, however, no data to support a negative effect of TGR5 agonists on cholelithiasis. It needs to be stressed that many of these potential unwanted side effects were either observed in cultured cells or in animal models where extremely high, non-physiological concentrations of BAs were used. Further detailed studies using conditions closer to physiology are, therefore, necessary to evaluate whether such effects are also relevant for the clinical setting.

      Conclusions and future perspectives

      The metabolic studies described above suggest that targeting TGR5 could provide an exciting new therapeutic approach to improve several aspects of the metabolic syndrome. Multiple studies reveal that TGR5 has beneficial effects on body weight in high-fat diet-fed mice. In addition, TGR5 activation improves glucose homeostasis and reduces hepatic steatosis. Also beneficial effects of TGR5 on macrophage-driven inflammation, as evidenced by the reduction of pro-inflammatory cytokines, may contribute to a potential positive effect of TGR5 with regard to the metabolic syndrome. These properties of TGR5 clearly suggest that activation of this membrane receptor is valuable to combat multiple aspects of the metabolic syndrome in humans. In view of the established role of BA or BA like molecules (dafachronic acids) to promote longevity in yeast [
      • Goldberg A.A.
      • Richard V.R.
      • Kyryakov P.
      • Bourque S.D.
      • Beach A.
      • Burstein M.T.
      • et al.
      Chemical genetic screen identifies lithocholic acid as an anti-aging compound that extends yeast chronological life span in a TOR-independent manner, by modulating housekeeping longevity assurance processes.
      ] and in the worm Caenorhabditis elegans [
      • Motola D.L.
      • Cummins C.L.
      • Rottiers V.
      • Sharma K.K.
      • Li T.
      • Li Y.
      • et al.
      Identification of ligands for DAF-12 that govern dauer formation and reproduction in C. elegans.
      ,
      • Gerisch B.
      • Rottiers V.
      • Li D.
      • Motola D.L.
      • Cummins C.L.
      • Lehrach H.
      • et al.
      A bile acid-like steroid modulates Caenorhabditis elegans lifespan through nuclear receptor signaling.
      ], it is furthermore plausible that the various strategies to modulate BA-signaling described in this review could increase lifespan through their potent hormonal activities that improve metabolism and reduce inflammation, two important contributors that determine healthspan [
      • Houtkooper R.H.
      • Williams R.W.
      • Auwerx J.
      Metabolic networks of longevity.
      ].
      The development of several novel natural, semi-synthetic, and synthetic TGR5 agonists is likely to further advance this receptor as a target for the metabolic syndrome. In addition, localized or tissue-specific gene targeting will shed light on ways to increase the efficacy and specificity of drugs that target this BA receptor. Despite the fact that we are convinced that targeting BA signaling pathways through TGR5 holds great promise for the treatment of metabolic diseases, still much work needs to be done, especially to make sure that such compounds are safe.

      Disclosure

      Dr. Auwerx consults for Intercept pharmaceuticals, a company that develops bile acid based therapeutics.

      Acknowledgments

      We thank Swiss National Science Foundation , the ERC , the NIH , Nestlé and the Ecole Polytechnique Fédérale de Lausanne for funding. T.W.H.P. is supported by a long-term fellowship from FEBS, L.G.N. is supported by a CONACYT fellowship, and M.N. is supported by a fellowship from AXA. We acknowledge Charles Thomas for instructive discussions.

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