TGR5 Expression Research Articles

Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic β cells

Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gαs and caused an increase in intracellular cAMP and Ca2+. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gαs inhibitor) or U73122 (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, U73122 or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on Gs/cAMP/Ca2+ pathway. 8-pCPT-2′-O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

Mo2082 Expression and Function of the Transmembrane Bile Acid Receptor TGR5 in Human and Murine Pancreatic β Cells

Bile acids act as signaling molecules that can stimulate nuclear farnesoid X receptor and G-protein–coupled TGR5 receptors to regulate lipid, glucose, and energy metabolism. TGR5 receptors are expressed in several tissues and shown to regulate energy. Recent studies in enteroendocrine cells have shown that activation of Gs-coupled bile acid receptor TGR5 causes release of glucagon-like peptide (GLP)-1 and peptide YY (PYY). GLP-1 causes insulin production and secretion from pancreatic β cells and improves glucose homeostasis.

Bile acids induce monocyte differentiation toward interleukin‐12 hypo‐producing dendritic cells via a TGR5‐dependent pathway

Dendritic cells (DCs) are known as antigen-presenting cells and play a central role in both innate and acquired immunity. Peripheral blood monocytes give rise to resident and recruited DCs in lymph nodes and non-lymphoid tissues. The ligands of nuclear hormone receptors can modulate DC differentiation and so influence various biological functions of DCs. The role of bile acids (BAs) as signalling molecules has recently become apparent, but the functional role of BAs in DC differentiation has not yet been elucidated. We show that DCs derived from human peripheral blood monocytes cultured with a BA produce lower levels of interleukin-12 (IL-12) and tumour necrosis factor-α in response to stimulation with commensal bacterial antigens. Stimulation through the nuclear receptor farnesoid X (FXR) did not affect the differentiation of DCs. However, DCs differentiated with the specific agonist for TGR5, a transmembrane BA receptor, showed an IL-12 hypo-producing phenotype. Expression of TGR5 could only be identified in monocytes and was rapidly down-regulated during monocyte differentiation to DCs. Stimulation with 8-bromoadenosine-cyclic AMP (8-Br-cAMP), which acts downstream of TGR5 signalling, also promoted differentiation into IL-12 hypo-producing DCs. These results indicate that BAs induce the differentiation of IL-12 hypo-producing DCs from monocytes via the TGR5-cAMP pathway.

TGR5 in the Biliary Tree

Background/Aims: TGR5 is a plasma membrane-bound, G-protein-coupled receptor for bile acids. TGR5 mRNA has been detected in a variety of tissues, including liver. The aim of the present study was to determine the localization and function of the receptor in biliary epithelial cells. Methods: Liver and gallbladder tissue from humans and rodents were analyzed for TGR5 expression and localization by real-time PCR, Western blot and immunofluorescence microscopy. Cholangiocytes and gallbladder epithelial cells were isolated from wild-type and TGR5 knockout mice. Cyclic AMP (cAMP) was measured using a radioimmunoassay and chloride concentrations were analyzed using the chloride-sensitive dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE). Cell proliferation was determined by bromodeoxyuridine incorporation. Results: TGR5 is localized in the apical membrane and the primary cilium of cholangiocytes and gallbladder epithelial cells. Activation of the receptor by bile acids led to a rise in intracellular cAMP concentrations and a decrease in intracellular chloride concentrations as measured by MQAE fluorescence, indicating increased chloride secretion. This effect could be abolished in the presence of an inhibitor of the cAMP-dependent chloride channel cystic fibrosis transmembrane conductance regulator. Furthermore, activation of TGR5 by bile acids induced cholangiocyte proliferation, which was not observed in cells derived from TGR5 knockout mice. Conclusion: In biliary epithelial cells, TGR5 acts as a bile acid sensor coupling biliary bile acid concentrations to bile formation. Furthermore, the receptor may play a role in bile acid-dependent cholangiocyte proliferation and may protect biliary epithelial cells from bile acid-induced cell death.

Expression and Function of Bile Acid Receptor TGR5 in Gastrointestinal Smooth Muscle

Bile acids, the main active constituents of bile, act, in addition to their role in nutrient absorption, as signaling molecules to exert genomic and non-genomic effects via nuclear receptors and the plasma membrane G protein-coupled receptor TGR5. TGR5 is expressed in epithelial cells and enteric neurons of the gastrointestinal tract and may mediate the effects of bile acids on motility and secretion. However, the mechanisms by which bile acids regulate smooth muscle function are not known. AIM To determine the expression of TGR5 in gastrointestinal smooth muscle and identify their signaling pathways. METHODS TGR5 expression was determined by RT-PCR and western blot. G protein activation in response to TGR5 selective ligand, oleanolic acid (OA) was measured as increase in Gα binding to [35S]GTPγS. OA-induced increase in cAMP was measured by ELISA and muscle relaxation by scanning micrometry. RESULTS TGR5 expression was detected by RT-PCR and western blot in rabbit gastric muscle and human intestinal muscle. OA activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3. Consistent with activation of Gαs, OA increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced muscle contraction. CONCLUSION Smooth muscle cells express membrane receptors (TGR5) for bile acids preferentially coupled to Gs. The receptors mediate stimulation of adenylyl cyclase activity and muscle relaxation.

TGR5: A Novel Target for Weight Maintenance and Glucose Metabolism

TGR5, an emerging G protein-coupled receptor, was identified as a membrane receptor for bile acids. The expression of TGR5 and its function are distinct from the previously identified nuclear bile acid receptor, farnesoid X receptor (FXR). These two bile acid receptors complement with each other for maintaining bile acid homeostasis and mediating bile acid signaling. Both receptors are also shown to play roles in regulating inflammation and glucose metabolism. An interesting finding for TGR5 is its role in energy metabolism. The discovery of TGR5 expression in brown adipocyte tissues (BATs) and the recent demonstration of BAT in adult human body suggest a potential approach to combat obesity by targeting TGR5 to increase thermogenesis. We summarize here the latest finding of TGR5 research, especially its role in energy metabolism and glucose homeostasis.

Bile Acids Have the Gall to Function as Hormones

In this issue of Cell Metabolism, Thomas et al. (2009) show that specific activation of the bile-acid-activated G protein-coupled receptor TGR5 improves pancreatic and hepatic function and impairs the development of obesity following administration of a high-fat diet.

The membrane-bound bile acid receptor TGR5 is localized in the epithelium of human gallbladders #

TGR5 (Gpbar-1) is a plasma membrane-bound, G protein-coupled receptor for bile acids. TGR5 messenger RNA (mRNA) has been detected in many tissues, including rat cholangiocytes and mouse gallbladder. A role for TGR5 in gallstone formation has been suggested, because TGR5 knockout mice did not develop gallstones when fed a lithogenic diet. In this study, expression and localization of TGR5 was studied in human gallbladders. TGR5 mRNA and protein were detected in all 19 gallbladders. Although TGR5 mRNA was significantly elevated in the presence of gallstones, no such relation was found for TGR5 protein levels. In order to study the localization of TGR5 in human gallbladders, a novel antibody was generated. The receptor was localized in the apical membrane and the rab11-positive recycling endosome of gallbladder epithelial cells. Furthermore, the TGR5 staining colocalized with the cyclic adenosine monophosphate-regulated chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) and the apical sodium-dependent bile salt uptake transporter, suggesting a functional coupling of TGR5 to bile acid uptake and chloride secretion. Stimulation with bile acids significantly increased cyclic adenosine monophosphate concentration in human gallbladder tissue. Incubation of gallbladder epithelial cells with a TGR5 agonist led to a rise of N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE)-fluorescence, suggestive of a decrease in intracellular chloride concentration. The TGR5 agonist-dependent increase in MQAE-fluorescence was absent in TGR5 knockout mice or in the presence of a CFTR inhibitor, indicating that TGR5 mediates chloride secretion via activation of CFTR. The presence of the receptor in both the plasma membrane and the recycling endosome indicate that TGR5 can be regulated by translocation. The data suggest a role for TGR5 in bile acid-induced fluid secretion in biliary epithelial cells.

Expression and function of the bile acid receptor TGR5 in Kupffer cells

Kupffer cells are resident macrophages in the liver and play a central role in the hepatic response to injury. Bile acids can impair macrophage function leading to decreased cytokine release. TGR5 is a novel, membrane-bound bile acid receptor, and it has been suggested that the immunosuppressive effect of bile acids can be mediated by TGR5. However, the function of TGR5 in Kupffer cells has not been studied and a direct link between TGR5 and cytokine production in macrophages has not been established. The present study demonstrates that TGR5 is localized in the plasma membrane of isolated Kupffer cells and is responsive to bile acids. Furthermore, bile acids inhibited LPS-induced cytokine expression in Kupffer cells via TGR5-cAMP dependent pathways. TGR5-immunoreactivity in Kupffer cells was increased in rat livers following bile-duct ligation, suggesting that TGR5 may play a protective role in obstructive cholestasis preventing excessive cytokine production thereby reducing liver injury.

Bile acid-induced TGR5-dependent c-Jun-N terminal kinase activation leads to enhanced caspase 8 activation in hepatocytes

TGR5 is a novel G protein-coupled cell-surface bile acid receptor. In cholestasis, bile acids induce hepatocyte apoptosis by primarily activating death receptor-mediated signaling. We examined if bile acid-induced TGR5 activation is participating in bile acid-induced hepatocyte apoptosis. TGR5 expression and its responsiveness to bile acid were confirmed in human hepatocytes. TGR5 inhibition attenuated bile acid-induced caspase 8 activation, which resulted from reduced bile acid-induced caspase 8 recruited to a death-inducing signaling complex (DISC). Bile acid-induced c-Jun-N terminal kinase (JNK) activation was dependent on bile acid activation of TGR5. JNK formed complexes with caspase 8, which were reduced following bile acid treatment, but this reduction was prevented when TGR5 or JNK was inhibited. In conclusion, bile acids activate TGR5, which leads to JNK activation and reduced complex formation of JNK with caspase 8, thus facilitating caspase 8 recruitment to DISC. These observations suggest therapeutic applications for TGR5 signaling blockage in cholestasis.

The G-protein coupled bile salt receptor TGR5 is expressed in liver sinusoidal endothelial cells

Sinusoidal endothelial cells (SEC) constitute a permeable barrier between hepatocytes and blood. SEC are exposed to high concentrations of bile salts from the enterohepatic circulation. Whether SEC are responsive to bile salts is unknown. TGR5, a G-protein-coupled bile acid receptor, which triggers cAMP formation, has been discovered recently in macrophages. In this study, rat TGR5 was cloned and antibodies directed against the C-terminus of rat TGR5 were developed, which detected TGR5 as a glycoprotein in transfected HepG2-cells. Apart from Kupffer cells, TGR5 was detected in SEC of rat liver. SEC expressed TGR5 over the entire acinus, whereas endothelial cells of the portal or central veins were not immunoreactive toward TGR5 antibodies. In isolated SEC, TGR5 mRNA and protein were detected by reverse transcription (RT) PCR, immunofluorescence microscopy, and Western blot analysis. Bile salts increased cAMP in isolated SEC and induced mRNA expression of endothelial NO synthase (eNOS), a known cAMP-dependent gene. In addition, bile acids activated eNOS by phosphorylation of eNOS at amino acid position 1177. In line with eNOS activation, bile acids induced NO production in liver slices. This is the first report on the expression of TGR5 in SEC. The data suggest that SEC are directly responsive toward specific bile salts. Regulation of eNOS in SEC by TGR5 connects bile salts with hepatic hemodynamics. This is of particular importance in cholestatic livers when bile salt concentrations are increased.

Bile acids promote glucagon-like peptide-1 secretion through TGR5 in a murine enteroendocrine cell line STC-1

Bile acids play essential roles in the absorption of dietary lipids and in the regulation of bile acid biosynthesis. Recently, a G protein-coupled receptor, TGR5, was identified as a cell-surface bile acid receptor. In this study, we show that bile acids promote glucagon-like peptide-1 (GLP-1) secretion through TGR5 in a murine enteroendocrine cell line STC-1. In STC-1 cells, bile acids promoted GLP-1 secretion in a dose-dependent manner. As STC-1 cells express TGR5 mRNA, we examined whether bile acids induce GLP-1 secretion through TGR5. RNA interference experiments showed that reduced expression of TGR5 resulted in reduced secretion of GLP-1. Furthermore, transient transfection of STC-1 cells with an expression plasmid containing TGR5 significantly enhanced GLP-1 secretion, indicating that bile acids promote GLP-1 secretion through TGR5 in STC-1 cells. Bile acids induced rapid and dose-dependent elevation of intracellular cAMP levels in STC-1 cells. An adenylate cyclase inhibitor, MDL12330A, significantly suppressed bile acid-promoted GLP-1 secretion, suggesting that bile acids induce GLP-1 secretion via intracellular cAMP production in STC-1 cells.