2018/07/05 by Shinobu Miyazaki‐Anzai, Shinobu Miyazaki-Anzai, Masashi Masuda +5 · 26 citations
Medicine · #Drug Transport and Resistance Mechanisms #Cholesterol and Lipid Metabolism #Pregnancy and Medication Impact
paper · pdf · doi:10.1194/jlr.m087239
Simultaneous activation of bile acid receptors farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5) by INT-767 significantly reduces atherosclerotic formation. In this study, we investigated the effect of simultaneous inactivation of these bile acid receptors in atherosclerosis and which bile acid receptor mediates the anti-atherogenic effect of INT-767. To investigate the role of simultaneous inactivation of FXR and TGR5 in vivo, we generated LDL receptor knockout (LDLR) KO mice with FXR and TGR5 dual deficiency, which exhibited severe atherosclerosis and aortic inflammation through nuclear factor κΒ activation. The lipid-lowering effects of INT-767 were completely blocked by FXR single deficiency but not TGR5 single deficiency. INT-767 was able to block atherosclerotic formation and decrease levels of aortic cytokines and chemokines in LDLR KO mice under either FXR or TGR5 single deficiency. Dual deficiency of FXR and TGR5 completely blocked the anti-atherogenic and anti-inflammatory effects of INT-767 in LDLR KO mice. We demonstrated that 1) FXR and TGR5 dual deficiency exacerbated the development of atherosclerosis and 2) the anti-atherogenic effect of INT-767 requires the anti-inflammatory effect but not the lipid-lowering effect through the simultaneous activation of FXR and TGR5. Our results indicate that dual activation of FXR and TGR5 is a promising strategy for treating atherosclerosis. Simultaneous activation of bile acid receptors farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5) by INT-767 significantly reduces atherosclerotic formation. In this study, we investigated the effect of simultaneous inactivation of these bile acid receptors in atherosclerosis and which bile acid receptor mediates the anti-atherogenic effect of INT-767. To investigate the role of simultaneous inactivation of FXR and TGR5 in vivo, we generated LDL receptor knockout (LDLR) KO mice with FXR and TGR5 dual deficiency, which exhibited severe atherosclerosis and aortic inflammation through nuclear factor κΒ activation. The lipid-lowering effects of INT-767 were completely blocked by FXR single deficiency but not TGR5 single deficiency. INT-767 was able to block atherosclerotic formation and decrease levels of aortic cytokines and chemokines in LDLR KO mice under either FXR or TGR5 single deficiency. Dual deficiency of FXR and TGR5 completely blocked the anti-atherogenic and anti-inflammatory effects of INT-767 in LDLR KO mice. We demonstrated that 1) FXR and TGR5 dual deficiency exacerbated the development of atherosclerosis and 2) the anti-atherogenic effect of INT-767 requires the anti-inflammatory effect but not the lipid-lowering effect through the simultaneous activation of FXR and TGR5. Our results indicate that dual activation of FXR and TGR5 is a promising strategy for treating atherosclerosis. INT-767 is a potent dual activator for farnesoid X receptor (FXR) and G protein-coupled bile acid receptor 1 (TGR5), which are two major receptors responsible for bile acid signaling (1.Rizzo G. Passeri D. De Franco F. Ciaccioli G. Donadio L. Orlandi S. Sadeghpour B. Wang X.X. Jiang T. Levi M. et al.Functional characterization of the semisynthetic bile acid derivative INT-767, a dual farnesoid X receptor and TGR5 agonist.Mol. Pharmacol. 2010; 78: 617-630Crossref PubMed Scopus (148) Google Scholar). A number of recent reports showed beneficial effects of INT-767 on cardiovascular and metabolic diseases (2.Baghdasaryan A. Claudel T. Gumhold J. Silbert D. Adorini L. Roda A. Vecchiotti S. Gonzalez F.J. Schoonjans K. Strazzabosco M. et al.Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2−/− (Abcb4−/−) mouse cholangiopathy model by promoting biliary HCO output.Hepatology. 2011; 54: 1303-1312Crossref PubMed Scopus (188) Google Scholar, 3.McMahan R.H. Wang X.X. Cheng L.L. Krisko T. Smith M. El Kasmi K. Pruzanski M. Adorini L. Golden-Mason L. Levi M. et al.Bile acid receptor activation modulates hepatic monocyte activity and improves nonalcoholic fatty liver disease.J. Biol. Chem. 2013; 288: 11761-11770Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar, 5.Beli E. Yan Y. Moldovan L. Vieira C.P. Gao R. Duan Y. Prasad R. Bhatwadekar A. White F.A. Townsend S. et al.Restructuring of the gut microbiome by intermittent fasting prevents retinopathy and prolongs survival in db/db mice.Diabetes. 2018; (Epub ahead of print. April 30, 2018; doi: 10.2337/db18-0158)Crossref PubMed Scopus (162) Google Scholar, 6.Roth J.D. Feigh M. Veidal S.S. Fensholdt L.K. Rigbolt K.T. Hansen H.H. Chen L.C. Petitjean M. Friley W. Vrang N. et al.INT-767 improves histopathological features in a diet-induced ob/ob mouse model of biopsy-confirmed non-alcoholic steatohepatitis.World J. Gastroenterol. 2018; 24: 195-210Crossref PubMed Scopus (45) Google Scholar, 7.Jadhav K. Xu Y. Xu Y. Li Y. Xu J. Zhu Y. Adorini L. Lee Y.K. Kasumov T. Yin L. et al.Reversal of metabolic disorders by pharmacological activation of bile acid receptors TGR5 and FXR.Mol. Metab. 2018; 9: 131-140Crossref PubMed Scopus (72) Google Scholar, 8.Wang X.X. Wang D. Luo Y. Myakala K. Dobrinskikh E. Rosenberg A.Z. Levi J. Kopp J.B. Field A. Hill A. et al.FXR/TGR5 dual agonist prevents progression of nephropathy in diabetes and obesity.J. Am. Soc. Nephrol. 2018; 29: 118-137Crossref PubMed Scopus (89) Google Scholar). Because administration of several FXR-specific or TGR5-specifc molecules showed similar beneficial effects on atherosclerosis (9.Hanniman E.A. Lambert G. McCarthy T.C. Sinal C.J. Loss of functional farnesoid X receptor increases atherosclerotic lesions in apolipoprotein E-deficient mice.J. Lipid Res. 2005; 46: 2595-2604Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 10.Hartman H.B. Gardell S.J. Petucci C.J. Wang S. Krueger J.A. Evans M.J. Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice.J. Lipid Res. 2009; 50: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 11.Mencarelli A. Renga B. Distrutti E. Fiorucci S. Antiatherosclerotic effect of farnesoid X receptor.Am. J. Physiol. Heart Circ. Physiol. 2009; 296: H272-H281Crossref PubMed Scopus (154) Google Scholar, 12.Pols T.W. Nomura M. Harach T. Lo Sasso G. Oosterveer M.H. Thomas C. Rizzo G. Gioiello A. Adorini L. Pellicciari R. et al.TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading.Cell Metab. 2011; 14: 747-757Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar), we studied whether simultaneous activation of both FXR and TGR5 is required for the beneficial effects of INT-767. FXR is selectively expressed in the liver, kidneys, and intestines (13.Bookout A.L. Mangelsdorf D.J. Quantitative real-time PCR protocol for analysis of nuclear receptor signaling pathways.Nucl. Recept. Signal. 2003; 1: e012Crossref PubMed Google Scholar), while TGR5 is mainly expressed in the intestines and immune cells, including macrophages (14.Kawamata Y. Fujii R. Hosoya M. Harada M. Yoshida H. Miwa M. Fukusumi S. Habata Y. Itoh T. Shintani Y. et al.A G protein-coupled receptor responsive to bile acids.J. Biol. Chem. 2003; 278: 9435-9440Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar). Many lines of evidence have demonstrated that activation of FXR by FXR-specific agonists inhibits the development of atherosclerosis, possibly through a potent lipid-lowering effect (10.Hartman H.B. Gardell S.J. Petucci C.J. Wang S. Krueger J.A. Evans M.J. Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice.J. Lipid Res. 2009; 50: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 11.Mencarelli A. Renga B. Distrutti E. Fiorucci S. Antiatherosclerotic effect of farnesoid X receptor.Am. J. Physiol. Heart Circ. Physiol. 2009; 296: H272-H281Crossref PubMed Scopus (154) Google Scholar, 15.Hambruch E. Miyazaki-Anzai S. Hahn U. Matysik S. Boettcher A. Perovic-Ottstadt S. Schluter T. Kinzel O. Krol H.D. Deuschle U. et al.Synthetic farnesoid X receptor agonists induce high-density lipoprotein-mediated transhepatic cholesterol efflux in mice and monkeys and prevent atherosclerosis in cholesteryl ester transfer protein transgenic low-density lipoprotein receptor (−/−) mice.J. Pharmacol. Exp. Ther. 2012; 343: 556-567Crossref PubMed Scopus (87) Google Scholar). TGR5 activation by TGR5-specific agonists such as INT-777 also significantly blocked atherosclerotic formation in LDLR KO mice through the inhibition of NF-κB-mediated macrophage inflammation (12.Pols T.W. Nomura M. Harach T. Lo Sasso G. Oosterveer M.H. Thomas C. Rizzo G. Gioiello A. Adorini L. Pellicciari R. et al.TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading.Cell Metab. 2011; 14: 747-757Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). Contrarily, either FXR or TGR5 deficiency did not significantly aggravate atherosclerosis in LDLR KO mice, suggesting that loss of one bile receptor was compensated for by the other receptor (12.Pols T.W. Nomura M. Harach T. Lo Sasso G. Oosterveer M.H. Thomas C. Rizzo G. Gioiello A. Adorini L. Pellicciari R. et al.TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading.Cell Metab. 2011; 14: 747-757Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 16.Zhang Y. Wang X. Vales C. Lee F.Y. Lee H. Lusis A.J. Edwards P.A. FXR deficiency causes reduced atherosclerosis in Ldlr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2316-2321Crossref PubMed Scopus (141) Google Scholar). We previously reported that INT-767 administration significantly blocked atherosclerotic formation in ApoE KO and LDLR KO mice (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar), which was recently confirmed by another group (7.Jadhav K. Xu Y. Xu Y. Li Y. Xu J. Zhu Y. Adorini L. Lee Y.K. Kasumov T. Yin L. et al.Reversal of metabolic disorders by pharmacological activation of bile acid receptors TGR5 and FXR.Mol. Metab. 2018; 9: 131-140Crossref PubMed Scopus (72) Google Scholar). In addition, INT-767 treatment showed potent lipid-lowering effects and anti-inflammatory effects (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). In this study, we examined 1) which bile acid receptor mediates the anti-atherogenic, lipid-lowering, and anti-inflammatory effects of INT-767, 2) whether the lipid-lowering effect, anti-inflammatory effect, or both is required for the anti-atherogenic effect of INT-767, and 3) whether simultaneous inhibition of FXR and TGR5 aggravates atherosclerosis in LDLR KO mice. FXR−/− and LDLR−/− mice on a C57BL/6J background were obtained from the Jackson Laboratory (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar, 17.Sinal C.J. Tohkin M. Miyata M. Ward J.M. Lambert G. Gonzalez F.J. Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis.Cell. 2000; 102: 731-744Abstract Full Text Full Text PDF PubMed Scopus (1422) Google Scholar). TGR5−/− mice on a C57BL/6J background were kindly provided by Dr. Galya Vassileva (Merck) (18.Vassileva G. Golovko A. Markowitz L. Abbondanzo S.J. Zeng M. Yang S. Hoos L. Tetzloff G. Levitan D. Murgolo N.J. et al.Targeted deletion of Gpbar1 protects mice from cholesterol gallstone formation.Biochem. J. 2006; 398: 423-430Crossref PubMed Scopus (220) Google Scholar). Male FXR+/−; TGR5+/−; LDLR−/− mice were mated to obtain LDLR−/− (LDLR KO), FXR−/−; LDLR−/− (FXR-DKO), TGR5−/−; LDLR−/− (TGR5-DKO), FXR−/−; TGR5−/−; LDLR−/− (TKO) mice. Male animals were fed a Western diet (TD88137) containing INT-767 for The of INT-767 was on and was in animals group were for animals were by a fasting were by the and of the of INT-767 was kindly provided by and in the aortic were as we previously (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). analysis for and in the aortic was a as we previously (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). were (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). analysis was as previously (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). The activity of nuclear was to the protocol from the with was by of was from the and with for was to with the to The was to a under and was by The was and to to the of the The was by of with and the of the was confirmed and PCR were that are were obtained from the and previously (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). were from two and reported as the or with a was for was We previously reported that a potent FXR and TGR5 dual INT-767, blocked the development of atherosclerosis in ApoE KO and LDLR KO mice (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). A number of have reported that either FXR or TGR5 single activation by agonists was to the development of atherosclerosis in mouse such as ApoE KO and LDLR KO mice (10.Hartman H.B. Gardell S.J. Petucci C.J. Wang S. Krueger J.A. Evans M.J. Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice.J. Lipid Res. 2009; 50: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 11.Mencarelli A. Renga B. Distrutti E. Fiorucci S. Antiatherosclerotic effect of farnesoid X receptor.Am. J. Physiol. Heart Circ. Physiol. 2009; 296: H272-H281Crossref PubMed Scopus (154) Google Scholar, 15.Hambruch E. Miyazaki-Anzai S. Hahn U. Matysik S. Boettcher A. Perovic-Ottstadt S. Schluter T. Kinzel O. Krol H.D. Deuschle U. et al.Synthetic farnesoid X receptor agonists induce high-density lipoprotein-mediated transhepatic cholesterol efflux in mice and monkeys and prevent atherosclerosis in cholesteryl ester transfer protein transgenic low-density lipoprotein receptor (−/−) mice.J. Pharmacol. Exp. Ther. 2012; 343: 556-567Crossref PubMed Scopus (87) Google Scholar). FXR and TGR5 single deficiency, was not to aggravate atherosclerosis in LDLR KO mice (12.Pols T.W. Nomura M. Harach T. Lo Sasso G. Oosterveer M.H. Thomas C. Rizzo G. Gioiello A. Adorini L. Pellicciari R. et al.TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading.Cell Metab. 2011; 14: 747-757Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 16.Zhang Y. Wang X. Vales C. Lee F.Y. Lee H. Lusis A.J. Edwards P.A. FXR deficiency causes reduced atherosclerosis in Ldlr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2316-2321Crossref PubMed Scopus (141) Google Scholar). We have examined 1) whether the anti-atherogenic effect of INT-767 requires activation of both FXR and TGR5 or whether one receptor is and 2) whether of major bile acid signaling aggravates atherosclerosis in of atherosclerosis. We LDLR KO mice in this FXR and TGR5 dual deficiency ApoE KO mice not and INT-767 treatment was in atherosclerosis in ApoE KO and LDLR KO mice (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). To which bile acid receptor mediates the anti-atherogenic effect of INT-767, 1) LDLR single KO mice, 2) LDLR KO mice, 3) LDLR KO mice, and LDLR KO (TKO) mice were with INT-767. analysis showed that FXR and TGR5 simultaneous deficiency atherosclerotic lesions of LDLR KO mice with LDLR and with a Y. Wang X. Vales C. Lee F.Y. Lee H. Lusis A.J. Edwards P.A. FXR deficiency causes reduced atherosclerosis in Ldlr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2316-2321Crossref PubMed Scopus (141) Google Scholar), FXR single deficiency reduced atherosclerotic lesions TGR5 single deficiency did not atherosclerosis in LDLR KO mice In addition, either FXR or TGR5 single deficiency did not the anti-atherogenic effect of INT-767. INT-767 treatment reduced atherosclerotic lesions by and in LDLR and mice, the other FXR and TGR5 deficiency the anti-atherogenic effect of INT-767 that the anti-atherogenic effect of INT-767 through activation of both FXR and TGR5. with (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar), INT-767 the potent lipid-lowering effect in LDLR KO mice. of and were reduced by and in LDLR KO mice with INT-767 TGR5 deficiency did not levels of or the lipid-lowering effect of INT-767. INT-767 treatment reduced levels of and by and and mice significantly levels of and with LDLR KO mice In addition, FXR single and dual deficiency completely blocked the lipid-lowering effects of INT-767 that INT-767 reduced through FXR activation. We previously reported that INT-767 reduced aortic and of mice through the of activation (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). In this study, we examined which bile acid receptor mediates the anti-inflammatory effect of INT-767. FXR and TGR5 single deficiency did not levels of and FXR and TGR5 dual deficiency levels of aortic and by and INT-767 treatment significantly reduced levels of aortic and in LDLR and mice, FXR and TGR5 dual deficiency completely blocked the effect of INT-767 with PCR analysis showed that FXR and TGR5 simultaneous deficiency but not FXR or TGR5 single deficiency, significantly and in atherosclerotic lesions of LDLR KO mice with a (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar), INT-767 treatment reduced and in atherosclerotic lesions of LDLR KO mice. FXR and single deficiency did not and FXR and TGR5 simultaneous deficiency completely blocked the effect of INT-767 on macrophage and We previously showed that the anti-inflammatory effect of INT-767 through inhibition of the aortic (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar). INT-767 significantly reduced levels of in the of LDLR KO mice FXR and TGR5 simultaneous deficiency levels of aortic and also blocked the effect of INT-767 and TGR5 dual deficiency were with INT-767 for of analysis of in the of LDLR KO and mice with INT-767. of analysis of and in the of and mice with INT-767. activity in the of LDLR KO and mice with INT-767. with either or against completely blocked the of to the We and other previously reported that treatment with FXR and TGR5 dual bile acid receptor INT-767, reduced atherosclerotic lesions in such as LDLR KO and ApoE KO mice (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar, 7.Jadhav K. Xu Y. Xu Y. Li Y. Xu J. Zhu Y. Adorini L. Lee Y.K. Kasumov T. Yin L. et al.Reversal of metabolic disorders by pharmacological activation of bile acid receptors TGR5 and FXR.Mol. Metab. 2018; 9: 131-140Crossref PubMed Scopus (72) Google Scholar). In addition, INT-767 significantly reduced such as and the of aortic cytokines and chemokines through the inhibition of the treatment with either or TGR5-specific agonists was to the development of atherosclerosis in mouse of atherosclerosis (10.Hartman H.B. Gardell S.J. Petucci C.J. Wang S. Krueger J.A. Evans M.J. Activation of farnesoid X receptor prevents atherosclerotic lesion formation in LDLR−/− and apoE−/− mice.J. Lipid Res. 2009; 50: 1090-1100Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 11.Mencarelli A. Renga B. Distrutti E. Fiorucci S. Antiatherosclerotic effect of farnesoid X receptor.Am. J. Physiol. Heart Circ. Physiol. 2009; 296: H272-H281Crossref PubMed Scopus (154) Google Scholar, 12.Pols T.W. Nomura M. Harach T. Lo Sasso G. Oosterveer M.H. Thomas C. Rizzo G. Gioiello A. Adorini L. Pellicciari R. et al.TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading.Cell Metab. 2011; 14: 747-757Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar, 15.Hambruch E. Miyazaki-Anzai S. Hahn U. Matysik S. Boettcher A. Perovic-Ottstadt S. Schluter T. Kinzel O. Krol H.D. Deuschle U. et al.Synthetic farnesoid X receptor agonists induce high-density lipoprotein-mediated transhepatic cholesterol efflux in mice and monkeys and prevent atherosclerosis in cholesteryl ester transfer protein transgenic low-density lipoprotein receptor (−/−) mice.J. Pharmacol. Exp. Ther. 2012; 343: 556-567Crossref PubMed Scopus (87) Google Scholar), in this study, we examined which bile acid receptor activation mediates the effect, the lipid-lowering effect, and the anti-inflammatory effect of INT-767. Our demonstrated that FXR and TGR5 simultaneous activation is required for the effect of INT-767. In addition, other bile such as X and receptor B. D. A. Y. J. et nuclear receptor is a acid that protects against liver PubMed Scopus Google Scholar, M. W. H. Evans Mangelsdorf D.J. receptor as bile acid 296: PubMed Scopus Google Scholar, Chen Y. Cheng K. C. L. of bile with a of J. Pharmacol. PubMed Scopus Google Scholar), are not in the effect of INT-767. The effect of INT-767 was not required to for the lipid-lowering effect to FXR deficiency was to block the lipid-lowering effect of INT-767, INT-767 was able to atherosclerotic formation in mice, similar to LDLR KO mice. results that the of the anti-inflammatory effect of INT-767 is the effect in for the lipid-lowering effect of INT-767 to Our in and from have that the anti-inflammatory of bile is through TGR5 activation (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar, 12.Pols T.W. Nomura M. Harach T. Lo Sasso G. Oosterveer M.H. Thomas C. Rizzo G. Gioiello A. Adorini L. Pellicciari R. et al.TGR5 activation inhibits atherosclerosis by reducing macrophage inflammation and lipid loading.Cell Metab. 2011; 14: 747-757Abstract Full Text Full Text PDF PubMed Scopus (405) Google Scholar). TGR5 is expressed in such as macrophages (14.Kawamata Y. Fujii R. Hosoya M. Harada M. Yoshida H. Miwa M. Fukusumi S. Habata Y. Itoh T. Shintani Y. et al.A G protein-coupled receptor responsive to bile acids.J. Biol. Chem. 2003; 278: 9435-9440Abstract Full Text Full Text PDF PubMed Scopus (1097) Google Scholar), FXR is or in (4.Miyazaki-Anzai S. Masuda M. Levi M. Keenan A.L. Miyazaki M. Dual activation of the bile acid nuclear receptor FXR and G-protein-coupled receptor TGR5 protects mice against atherosclerosis.PLoS One. 2014; 9: e108270Crossref PubMed Scopus (81) Google Scholar, 16.Zhang Y. Wang X. Vales C. Lee F.Y. Lee H. Lusis A.J. Edwards P.A. FXR deficiency causes reduced atherosclerosis in Ldlr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2316-2321Crossref PubMed Scopus (141) Google Scholar, S. M.J. B. Gonzalez F.J. of FXR in formation and atherosclerosis 2006; PubMed Scopus Google Scholar). FXR or TGR5 single deficiency did not the anti-inflammatory effect of INT-767, FXR and TGR5 simultaneous deficiency completely blocked the anti-inflammatory Our that FXR and TGR5 activation both to the anti-inflammatory effect of INT-767 in Because both FXR and TGR5 did not induce atherosclerotic formation in LDLR KO mice not we to in which FXR and TGR5 activation is for the development of atherosclerosis and aortic The is the that simultaneous inhibition of FXR and TGR5 is required for atherosclerotic formation in LDLR KO mice. A number of have that either FXR or TGR5 activation is to the development of atherosclerosis. either FXR or TGR5 deficiency was not to aggravate atherosclerosis in several mouse including LDLR KO mice Y. Wang X. Vales C. Lee F.Y. Lee H. Lusis A.J. Edwards P.A. FXR deficiency causes reduced atherosclerosis in Ldlr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2316-2321Crossref PubMed Scopus (141) Google Scholar, S. M.J. B. Gonzalez F.J. of FXR in formation and atherosclerosis 2006; PubMed Scopus Google Scholar). In addition, FXR deficiency atherosclerotic formation in LDLR KO mice Y. Wang X. Vales C. Lee F.Y. Lee H. Lusis A.J. Edwards P.A. FXR deficiency causes reduced atherosclerosis in Ldlr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2006; 26: 2316-2321Crossref PubMed Scopus (141) Google Scholar). results that are and FXR and TGR5 In the FXR activation to activation of TGR5 through the of gut and bile acid X. J.M. S. Gonzalez F.J. farnesoid X receptor agonist and the gut bile acid signaling to 2018; (Epub ahead of print. 2018; doi: PubMed Scopus Google Scholar). results that and activation of TGR5 by FXR activation and required for the effects of and TGR5-specific agonists to and inflammation are major of atherosclerosis. mice showed severe and aortic as INT-767 inhibits atherosclerosis in the of in mice, the of aortic inflammation is for atherosclerosis by FXR and TGR5 simultaneous deficiency. Our major 1) major bile acid FXR and TGR5 aggravate aortic inflammation and atherosclerosis, 2) simultaneous activation of FXR and TGR5 is required for the and anti-inflammatory effects of INT-767 to 3) the lipid-lowering effect of INT-767 through the activation of FXR and the anti-inflammatory effect of INT-767 is the lipid-lowering effect for the effect of INT-767 to these that both FXR and TGR5 for treatment of atherosclerosis either receptor The L. Adorini and M. Pruzanski for the INT-767 and G Vassileva (Merck) for the TGR5 knockout mice. KO farnesoid X receptor LDL receptor nuclear G protein-coupled bile acid receptor 1 KO