Abstract
Bile acids (BAs) are signalling molecules that mediate various cellular responses in both physiological and pathological processes. Several studies report that BAs can be detected in the brain1, yet their physiological role in the central nervous system is still largely unknown. Here we show that postprandial BAs can reach the brain and activate a negative-feedback loop controlling satiety in response to physiological feeding via TGR5, a G-protein-coupled receptor activated by multiple conjugated and unconjugated BAs2 and an established regulator of peripheral metabolism3,4,5,6,7,8. Notably, peripheral or central administration of a BA mix or a TGR5-specific BA mimetic (INT-777) exerted an anorexigenic effect in wild-type mice, while whole-body, neuron-specific or agouti-related peptide neuronal TGR5 deletion caused a significant increase in food intake. Accordingly, orexigenic peptide expression and secretion were reduced after short-term TGR5 activation. In vitro studies demonstrated that activation of the Rho–ROCK–actin-remodelling pathway decreases orexigenic agouti-related peptide/neuropeptide Y (AgRP/NPY) release in a TGR5-dependent manner. Taken together, these data identify a signalling cascade by which BAs exert acute effects at the transition between fasting and feeding and prime the switch towards satiety, unveiling a previously unrecognized role of physiological feedback mediated by BAs in the central nervous system.
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Data availability
All the datasets generated during the current study are available upon request to the corresponding author. Source data are provided with this paper.
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Acknowledgements
We thank A. Fouassier, S. Bichet, T. Clerc, P. Colin, A. Aebi, T. Teav (Metabolomics Platform), the Histology facility and the Phenotyping Unit (UDP) for technical assistance, the Mouse Clinical Institute in Strasbourg for the generation of the TGR5:GFP mouse strain, and the Bordeaux Imaging Center (CNRS-INSERM and Bordeaux University; ANR-10-INBS-04) for confocal microscopy. This work was funded by the Swiss National Science Foundation (SNSF nos. 310030_189178 and CRSII5_180317/1, the Kristian Gerhard Jebsen Foundation and the Ecole Polytechnique Fédérale de Lausanne (EPFL; to K.S.), the National Institutes of Health (DK097566 to S.D.) and INSERM, ANR-17-CE14-0007, ANR-10-EQX-008-1 OPTOPATH (to D.C.). L.A.V.-V. and A.P. were supported by a postdoctoral fellowship from the AXA Research Fund.
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Contributions
A.P., L.A.V.-V. and K.S. conceived and designed the project. B.S., S.D. and D.C. provided critical expertise. A.G. and R.P. provided chemical compounds. A.P., L.A.V.-V., N.B., Y.S., Q.H., V.F., A.C.-J., P.Z., G.B., S.J. and A.B. performed experiments. A.P., L.A.V.-V., N.B., V.F., A.C.-J., P.Z., G.B., S.J. and J.I. analysed the data. A.P., L.A.V.-V. and K.S. wrote the manuscript. All the authors read and approved the manuscript.
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A.G. and R.P. are cofounders of TES Pharma. R.P. is the president and CEO of TES Pharma. The other authors declare no competing interests.
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Peer review information Nature Metabolism thanks Richard Palmiter and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Christoph Schmitt.
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Extended data
Extended Data Fig. 1 BA mix and INT-777 reach the hypothalamus after oral or icv administration, respectively, and reduce food intake by activating TGR5.
a, Conjugated and unconjugated BA species measured in the hypothalamus 30- and 60-min after oral administration of a BA mix at three different doses or vehicle in wild-type mice (n = 8 animals). Bars represent the mean from 8 replicates. b, One-hour cumulative food intake of wild-type mice after oral administration of a BA mix at three different doses or vehicle. n = 8 (Vehicle and 1 mg/Kg) and n = 10 (10 mg/Kg and 100 mg/Kg) animals. c, Gpbar1 mRNA levels in arcuate nucleus (ARC)-enriched hypothalamic punches of TGR5 wild-type (Gpbar1+/+) and germline TGR5 knockout (Gpbar1-/-) mice. n = 8 animals. d, One-hour (1 h) food intake measured after the first i.c.v injection (d1 – 1 h) of vehicle (n = 8 animals) or INT-777 (n = 9 animals) in wild-type mice. e, Two-hour (2 h) food intake measured after the first i.c.v injection (d1 – 2 h) of vehicle (n = 8 animals) or INT-777 (n = 9 animals) in wild-type mice. f, Food intake measured after a daily (right before the dark phase) i.c.v injection of vehicle (n = 8 animals) or INT-777 (n = 9 animals) in wild-type mice at the indicated time points. g, Body weight change (expressed as a percentage of the initial body weight) of vehicle (n = 8 animals) or INT-777 (n = 9 animals) in wild-type mice at the indicated time points. Results represent mean (a) or mean ± SEM (b-g). n represents biologically independent replicates. Two-tailed Student t-test (c, d and e) or two-way ANOVA followed by Bonferroni post-hoc correction (b) vs Vehicle (b, d-e) or Gpbar1+/+ (c) were used for statistical analysis. P values (exact value, * P ≤ 0.05 or **** P ≤ 0.0001) are indicated in the figure.
Extended Data Fig. 2 TGR5 activation in neurons reduces food intake.
a, Quantification of AgRP neurons in the arcuate nucleus of over-night fasted TGR5 wild-type (Gpbar1+/+, 4 animals) and TGR5 knockout (Gpbar1-/-, 3 animals) mice. b, Body weight curve of 8-week-old Gpbar1+/+ and Gpbar1-/- mice over an 8-week period. n = 8 animals. c, Body composition (lean and fat mass expressed as a percentage over the body weight (BW)) of Gpbar1+/+ and Gpbar1-/- mice. n = 10 animals. d, Validation of the TGR5:GFP knock-in mouse model (n = 4 animals) in which the mouse Gpbar1 gene was substituted with a construct containing the human GPBAR1, the F2a sequence and the Gfp. Brains from wild-type mice (n = 3 animals) were used as controls. e, Gpbar1 mRNA levels in arcuate nucleus (ARC)-enriched hypothalamic punches of control mice (Gpbar1fl/fl) and neuron-specific TGR5 knockout (SynCre;Gpbar1fl/fl) mice. n = 8 animals. f, Body weight curve of 8-week-old of control mice (Gpbar1fl/fl, 10 animals) and neuron-specific TGR5 knockout (SynCre;Gpbar1fl/fl, 9 animals) mice over an 8-week period. g, Body composition (lean and fat mass expressed as percentage over body weight (BW)) of Gpbar1fl/fl (n = 10 animals) and SynCre;Gpbar1fl/fl (n = 6 animals) mice. h, 12-hour food intake of Gpbar1fl/fl (n = 7 animals) and SynCre;Gpbar1fl/fl (n = 8 animals) mice. i, Pomc mRNA levels in ARC-enriched hypothalamic punches of Gpbar1fl/fl and SynCre;Gpbar1fl/fl mice. n = 7 (SynCre;Gpbar1fl/fl fasting) and n = 8 (all other groups) animals. j, 12-hour cumulative food intake of control mice (Gpbar1fl/fl) and neuron-specific TGR5 knockout (SynCre;Gpbar1fl/fl) mice after oral administration of INT-777 (100 mg/Kg) or vehicle. n = 5 (Gpbar1fl/fl), n = 8 (SynCre;Gpbar1fl/fl INT-777) and n = 9 (SynCre;Gpbar1fl/fl vehicle) animals. k, Total food intake of Gpbar1fl/fl and SynCre;Gpbar1fl/fl during night and day. n = 6 (Gpbar1fl/fl INT-777 night), n = 7 (Gpbar1fl/fl other groups), n = 8 (SynCre;Gpbar1fl/fl other groups) and n = 9 (SynCre;Gpbar1fl/fl vehicle day) animals. l, 12-hour cumulative food intake of control mice (Gpbar1+/+) and germline TGR5 knock-out (Gpbar1-/-) mice after oral administration of the TGR5 specific BA agonist INT-777 (100 mg/Kg) or vehicle. n = 6 (Gpbar1+/+ vehicle), n = 7 (Gpbar1-/- vehicle) and n = 8 (all other groups) animals. m, Total food intake of Gpbar1+/+ and Gpbar1-/- mice during night and day. n = 7 (Gpbar1-/- vehicle night) and n = 8 (all other groups) animals. Results represent mean ± SEM. n represents biologically independent replicates. Two-tailed Student t-test (d, e, h and i), one-way ANOVA (k and m) or two-way ANOVA (j and l) followed by Bonferroni post-hoc corrections vs. Wild-type (d), Gpbar1fl/fl (e, h and i), Gpbar1fl/fl Vehicle (j and k) or Gpbar1+/+ Vehicle (l and m) were used for statistical analysis. P values (exact value, * P ≤ 0.05 or **** P ≤ 0.0001) are indicated in the figure.
Extended Data Fig. 3 Disruption of TGR5 in AgRP or POMC neurons in CD-fed mice does not change body weight and composition.
a, Gpbar1 mRNA levels in arcuate nucleus (ARC)-enriched hypothalamic punches of control mice (Gpbar1fl/fl) and POMC neuron-specific TGR5 knockout (PomcCre;Gpbar1fl/fl) mice. n = 6 animals. b, 12-hours food intake of Gpbar1fl/fl and PomcCre;Gpbar1fl/fl mice. n = 7 animals. c, Body weight curve of 8-week-old of control mice (Gpbar1fl/fl, n = 7 animals) and AgRP neuron-specific TGR5 knockout (AgRPCre;Gpbar1fl/fl, n = 9 animals) mice over an 8-week period. d, Body composition (lean and fat mass expressed as percentage over body weight (BW)) of Gpbar1fl/fl and AgRPCre;Gpbar1fl/fl mice. n = 8 animals. e, Body weight curve of 8-week-old of control mice (Gpbar1fl/fl) and POMC neuron-specific TGR5 knockout (PomcCre;Gpbar1fl/fl) mice over an 8-week period. n = 10 animals. f, Body composition (lean and fat mass expressed as percentage over body weight (BW)) of Gpbar1fl/fl (n = 7 animals) and PomcCre;Gpbar1fl/fl (n = 8 animals) mice. g-h, Representative images (g) and quantification (h) of Fos immunoreactivity (in red) in AgRP/NPY neurons (in green) and merge (in orange) in the hypothalamus of fed or fasted AgRP/NPY:GFP reporter mice. Arrows in images indicate colocalization of Fos and GFP. Scale bar = 100 μm. n = 4 animals. Results represent mean ± SEM. n represents biologically independent replicates. Two-tailed Student t-test vs. Gpbar1fl/fl (a) or fed AgRP/NPY-GFP (h) groups was used for statistical analysis. P values are indicated in the figure.
Extended Data Fig. 4 TGR5 inhibits AgRP secretion through activation of the Rho/ROCK/actin signalling axis.
a, Gamma-aminobutyric acid (GABA) release in ex vivo hypothalamic explants from TGR5 wild-type (Gpbar1+/+) and germline TGR5 knockout (Gpbar1-/-) mice after starvation or starvation followed by 10- and 30-minutes stimulation with TGR5 agonist INT-777 or vehicle. n = 4 (all other groups) and n = 8 (Gpbar1+/+ vehicle 10 min) animals. b-c, GABA (b) or alpha-melanocyte-stimulating hormone (αMSH) (c) release in ex vivo hypothalamic explants from Gpbar1+/+ and Gpbar1-/- mice after high-glucose (15 mM) solution to mimic fed conditions, followed by 10- and 30-minutes stimulation with TGR5 agonist INT-777 or vehicle. n = 4 (b), n = 4 (all other groups in c) and n = 8 (Gpbar1+/+ and Gpbar1-/- fed, c) animals. d, Gpbar1 mRNA levels in mouse embryonic hypothalamic N41 cell line (mHypoE-N41) after Gpbar1 silencing using transient transfection of sh-Gpbar1 or control vector (sh-Ctrl). n = 12 samples. e, Agrp and Npy mRNA levels in mHypoE-N41 cells after 60 min starvation conditions followed by 60 min stimulation with INT-777 or vehicle. n = 3 samples. f, AgRP release after starvation followed by short-term (5, 10 and 15 min) stimulation with TGR5 agonist INT-777 or vehicle in mHypoE-N41 cells. n = 3 samples. g, AgRP release after starvation followed by 10 min stimulation with INT-777 or vehicle in mHypoE-N41 Gpbar1-silenced cells using transient transfection of sh-Gpbar1 or control vector (sh-Ctrl). n = 3 samples. (h and i) Quantitative densitometry of phosphorylated vs total ROCK signalling targets (MLC and COFILIN) from the cells described in Fig. 4f. n = 4 samples. j, Representative images of phalloidin staining to detect actin fibres (in green) and DAPI staining to detect nuclei (in blue), phosphorylated COFILIN (p-COFILIN) immunodetection (in red) and merge, after 10 min stimulation with INT-777, BA mix or vehicle in mHypoE-N41 cells. Scale bar = 10μm and digital zoom. n = 4 samples. Results represent mean ± SEM. n represents biologically independent replicates. Two-tailed Student t-test (d-f) or one-way ANOVA followed by Bonferroni post-hoc correction (g, h and i) vs. sh-Ctrl (d) or vehicle (e-i) groups were used for statistical analysis. P values (exact value or **** P ≤ 0.0001) are indicated in the figure.
Supplementary information
Supplementary Table 1
Elution gradient for chromatographic separation of BAs.
Supplementary Table 2
Oligonucleotide sequences.
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Perino, A., Velázquez-Villegas, L.A., Bresciani, N. et al. Central anorexigenic actions of bile acids are mediated by TGR5. Nat Metab 3, 595–603 (2021). https://doi.org/10.1038/s42255-021-00398-4
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DOI: https://doi.org/10.1038/s42255-021-00398-4
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