Journal: The Journal of Biological Chemistry

Article Title: Farnesoid X receptor induces Takeda G-protein receptor 5 cross-talk to regulate bile acid synthesis and hepatic metabolism

doi: 10.1074/jbc.M117.784322

Figure Lengend Snippet: Effect of INT-767, OCA, and INT-777 on GLP-1 secretion, cellular [Ca2+]c, and cAMP activity in L cells. A, effects of agonists and L-type Ca2+ channel inhibitor phloretin on glucose-stimulated GLP-1 secretion in Glutag cells. Glutag cells were maintained in 10% FBS and 1% antibiotic media in 24-well plates prior to the experiment. Cells were incubated without glucose media with FXR or TGR5 (10 μm each) agonist or vehicle (DMSO) as described under “Experimental procedures.” GLP-1 was normalized to the amount of protein in the cell and indicated as (pmol/μg). Results were expressed as means ± S.E. Student's t test was used for statistical analysis. * indicates statistically significant difference of serum GLP-1 between ligand-treated versus vehicle (p ≤ 0.05). # represents significant difference of serum GLP-1 level between ligand-treated versus phloretin-treated. B, effects of INT-767, OCA, and INT-777 on cellular [Ca2+] in Glutag cells. Glutag cells were maintained in 96-well plates and incubated with Hanks' balanced salt solution with loading dye as described under “Experimental procedures” using a Ca2+ fluorescence kit. Initial fluorescence readings were obtained without addition of ligands, designated as F0, followed by addition of 10 μm each of OCA, INT-767, INT-777, or 8-Br-cAMP (as a positive control) treatment for 20 s, designated as (F). The results were represented as the ratio F/F0 versus time in seconds. The assay was performed in quadruplet. * indicates statistically significant difference in F/F0-treated versus vehicle (p ≤ 0.05). C, CREB reporter assay in CHO cells. CHO cells stably expressing CREB/luciferase reporter were transfected with 0.1 μg of hTGR5 plasmid and treated with different concentrations of FXR agonist indicated or DMSO as vehicle control. The firefly reporter activity (RLU) was normalized with β-gal activity. The assay was performed in quadruplicate. The results were represented by linear regression analysis. D, PKA activity assay. Glutag cells were treated with 10 μm each agonist for 2 h. Total PKA activity ELISA was used to quantify PKA activity. * indicates statistically significant difference in PKA activity in treated versus vehicle (p ≤ 0.05). E, GLP-1 secretion assay of Glutag cells. Left panel, glucose-induced GLP-1 secretion from Glutag cells deficient in TGR5 (siTGR5) compared with siControl. Middle panel, glucose-induced GLP-1 secretion from Glutag cells deficient in FXR (shFXR) compared with shControl. Right panel, glucose induced GLP-1 secretion from Glutag cells deficient in FXR and TGR5 (shFXR and siTGP5) compared with shControl siTGR5. * indicates statistically significant difference in GLP-1 in treated versus vehicle control (p ≤ 0.05). Upper inset shows immunoblotting analysis of TGR5 and FXR in Fxr and Tgr5-deficient Glutag cells. Student's t test was used for statistical analysis.

Article Snippet: Glutag cells were maintained in DMEM in 24-well plates and were treated with 10 μ m each of OCA, FEX, INT-767, or INT-777 for 2 h. Cells were harvested, lysed, and analyzed with a PKA kinase activity assay kit (Enzo Life Sciences, Farmingdale, NY), according to the manufacturer's protocol.

Techniques: Activity Assay, Incubation, Fluorescence, Positive Control, Reporter Assay, Stable Transfection, Expressing, Luciferase, Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Farnesoid X receptor induces Takeda G-protein receptor 5 cross-talk to regulate bile acid synthesis and hepatic metabolism

doi: 10.1074/jbc.M117.784322

Figure Lengend Snippet: Mechanisms of intestinal FXR and TGR5 cross-talk in the regulation of GLP-1 secretion, hepatic bile aid synthesis, glucose and lipid metabolism, and insulin and glucose sensitivity. In the liver, the classic bile acid synthesis is initiated by cholesterol 7α-hydroxylase (Cyp7a1 and Cyp8b1 catalyze cholic acid synthesis, and sterol 27-hydroxylase catalyzes steroid side-chain oxidation to synthesize CA and CDCA, which are conjugated to taurine (T)). The alternative pathway is initiated by Cyp27a1 and oxysterol 7α-hydroxylase (Cyp7b1) to synthesize mainly CDCA. INT-767 activates FXR/SHP in hepatocytes to repress Cyp7a1 and Cyp8b1 and stimulate Cyp7b1 and Cyp27a1 expression to improve glucose and lipid metabolism. In intestinal L cells, INT-767 activates FXR, which stimulates TGR5 and prohormone convertase 1/3 (PC1/3) expression. FXR also induces FGF15, which stimulates energy metabolism in brown adipocytes. FGF15 activates hepatic FGFR4/βKlotho signaling to inhibit Cyp7a1 via JNK/ERK1/2 pathway. TGR5 activation stimulates adenylyl cyclase (AC) to increase cAMP, which activates protein kinase A (PKA) and phosphorylates and activates CREB to increase PC1/3 transcription. PC1/3 splices pre-proglucagon to GLP-1. INT-767 increases intracellular [Ca2+] and cAMP activity. INT-767 activation of FXR may depolarize membrane potential by inhibiting KATP channel to stimulate Ca2+ uptake through Ca2+ channels. Ca2+ stimulates GLP-1 secretion from L cells. GLP-1 and FXR stimulate insulin synthesis and secretion from pancreatic β cells.

Article Snippet: Glutag cells were maintained in DMEM in 24-well plates and were treated with 10 μ m each of OCA, FEX, INT-767, or INT-777 for 2 h. Cells were harvested, lysed, and analyzed with a PKA kinase activity assay kit (Enzo Life Sciences, Farmingdale, NY), according to the manufacturer's protocol.

Techniques: Expressing, Activation Assay, Activity Assay