Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Chemogenetics defines receptor-mediated functions of short chain free fatty acids

Nature Chemical Biology volume 15, pages 489–498 (2019)Cite this article

Subjects

Abstract

Differentiating actions of short chain fatty acids (SCFAs) at free fatty acid receptor 2 (FFA2) from other free fatty acid-responsive receptors and from non-receptor-mediated effects has been challenging. Using a novel chemogenetic and knock-in strategy, whereby an engineered variant of FFA2 (FFA2-DREADD) that is unresponsive to natural SCFAs but is instead activated by sorbic acid replaced the wild-type receptor, we determined that activation of FFA2 in differentiated adipocytes and colonic crypt enteroendocrine cells of mouse accounts fully for SCFA-regulated lipolysis and release of the incretin glucagon-like peptide-1 (GLP-1), respectively. In vivo studies confirmed the specific role of FFA2 in GLP-1 release and also demonstrated a direct role for FFA2 in accelerating gut transit. Thereby, we establish the general principle that such a chemogenetic knock-in strategy can successfully define novel G-protein-coupled receptor (GPCR) biology and provide both target validation and establish therapeutic potential of a ‘hard to target’ GPCR.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: G protein interaction patterns and signal regulation by specific agonists of hFFA2 and hFFA2-DREADD are indistinguishable.
Fig. 2: Initial characterization of hFFA2-DREADD-HA expressing mice.
Fig. 3: Immunocytochemical detection of expression of hFFA2-DREADD-HA.
Fig. 4: Activation of hFFA2-DREADD-HA promotes release of GLP-1.
Fig. 5: Activation of hFFA2-DREADD-HA is anti-lipolytic.
Fig. 6: In vivo activation of hFFA2-DREADD-HA reveals roles in GLP-1 release and gut transit.

Similar content being viewed by others

Data availability

All data is available from the corresponding authors or is available through the University of Glasgow online data repository.

Code availability

Concentration-response curves analyzed using GraphPad Prism v.5.02.

References

  1. Barratt, M. J., Lebrilla, C., Shapiro, H. Y. & Gordon, J. I. The gut microbiota, food science, and human nutrition: a timely marriage. Cell Host Microbe 22, 134–141 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. McKenzie, C., Tan, J., Macia, L. & Mackay, C. R. The nutrition-gut microbiome-physiology axis and allergic diseases. Immunol. Rev. 278, 277–295 (2017).

    Article  CAS  PubMed  Google Scholar 

  3. Stoddart, L. A., Smith, N. J. & Milligan, G. International union of pharmacology. LXXI. free fatty acid receptors FFA1, -2, and -3: pharmacology and pathophysiological functions. Pharmacol. Rev. 60, 405–417 (2008).

    Article  CAS  PubMed  Google Scholar 

  4. Bolognini, D., Tobin, A. B., Milligan, G. & Moss, C. E. The pharmacology and function of receptors for short-chain fatty acids. Mol. Pharmacol. 89, 388–398 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Bindels, L. B., Dewulf, E. M. & Delzenne, N. M. GPR43/FFA2: physiopathological relevance and therapeutic prospects. Trends Pharmacol. Sci. 34, 226–232 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Milligan, G., Shimpukade, B., Ulven, T. & Hudson, B. D. Complex pharmacology of free fatty acid receptors. Chem. Rev. 117, 67–110 (2017).

    Article  CAS  PubMed  Google Scholar 

  7. Milligan, G., Bolognini, D. & Sergeev, E. Ligands at the free fatty acid receptors 2/3 (GPR43/GPR41). Handb. Exp. Pharmacol. 236, 17–32 (2017).

    Article  CAS  PubMed  Google Scholar 

  8. Sergeev, E. et al. A single extracellular amino acid in free fatty acid receptor 2 defines antagonist species selectivity and G protein selection bias. Sci. Rep. 7, 13741 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Namour, F. et al. Safety, pharmacokinetics and pharmacodynamics of GLPG0974, a potent and selective FFA2 antagonist, in healthy male subjects. Br. J. Clin. Pharmacol. 82, 139–148 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S. & Roth, B. L. Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand. Proc. Natl Acad. Sci. USA 104, 5163–5168 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Urban, D. J. & Roth, B. L. DREADDs (designer receptors exclusively activated by designer drugs): chemogenetic tools with therapeutic utility. Annu. Rev. Pharmacol. Toxicol. 55, 399–417 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Smith, K. S., Bucci, D. J., Luikart, B. W. & Mahler, S. V. DREADDS: use and application in behavioral neuroscience. Behav. Neurosci. 130, 137–155 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  13. Thiel, G., Kaufmann, A. & Rössler, O. G. G-protein-coupled designer receptors—new chemical-genetic tools for signal transduction research. Biol. Chem. 394, 1615–1622 (2013).

    Article  CAS  PubMed  Google Scholar 

  14. Roth, B. L. DREADDs for neuroscientists. Neuron 89, 683–694 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Bradley, S. J., Tobin, A. B. & Prihandoko, R. The use of chemogenetic approaches to study the physiological roles of muscarinic acetylcholine receptors in the central nervous system. Neuropharmacol. 136, 421–426 (2018).

    Article  CAS  Google Scholar 

  16. Hudson, B. D. et al. Chemically engineering ligand selectivity at the free fatty acid receptor 2 based on pharmacological variation between species orthologs. FASEB J. 26, 4951–4965 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ballesteros, J. A. & Weinstein, H. Integrated methods for the construction of three-dimensional models and computational probing of structure-function relations in G protein-coupled receptors. Meth. Neurosci. 255, 366–428 (2013).

    Google Scholar 

  18. Hudson, B. D. et al. Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor. J. Biol. Chem. 288, 17296–17312 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Sergeev, E. et al. Non-equivalence of key positively charged residues of the free fatty acid 2 receptor in the recognition and function of agonist versus antagonist ligands. J. Biol. Chem. 291, 303–317 (2016).

    Article  CAS  PubMed  Google Scholar 

  20. Pizzonero, M. et al. Discovery and optimization of an azetidine chemical series as a free fatty acid receptor 2 (FFA2) antagonist: from hit to clinic. J. Med. Chem. 57, 10044–10057 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Brown, A. J. et al. The orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. J. Biol. Chem. 278, 11312–11319 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Karamitri, A. et al. Type 2 diabetes-associated variants of the MT2 melatonin receptor affect distinct modes of signaling. Sci. Signal. 11, pii eaan6622 (2018).

    Article  CAS  Google Scholar 

  23. Lee, S. U. et al. β-Arrestin 2 mediates G protein-coupled receptor 43 signals to nuclear factor-κB. Biol. Pharm. Bull. 36, 1754–1759 (2013).

    Article  CAS  PubMed  Google Scholar 

  24. Ang, Z., Xiong, D., Wu, M. & Ding, J. L. FFAR2-FFAR3 receptor heteromerization modulates short-chain fatty acid sensing. FASEB J. 32, 289–303 (2018).

    Article  CAS  PubMed  Google Scholar 

  25. Zaibi, M. S. et al. Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids. FEBS Lett. 584, 2381–2386 (2010).

    Article  CAS  PubMed  Google Scholar 

  26. Bolognini, D. et al. A novel allosteric activator of free fatty acid 2 receptor displays unique Gi-functional bias. J. Biol. Chem. 291, 18915–18931 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Tolhurst, G. et al. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2. Diabetes 61, 364–371 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Nøhr, M. K. et al. GPR41/FFAR3 and GPR43/FFAR2 as cosensors for short-chain fatty acids in enteroendocrine cells vs FFAR3 in enteric neurons and FFAR2 in enteric leukocytes. Endocrinology 154, 3552–3564 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. Li, Y., Kokrashvili, Z., Mosinger, B. & Margolskee, R. F. Gustducin couples fatty acid receptors to GLP-1 release in colon. Am. J. Physiol. Endocrinol. Metab. 304, E651–E660 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Engelstoft, M. S. et al. Research resource: a chromogranin A reporter for serotonin and histamine secreting enteroendocrine cells. Mol. Endocrinol. 29, 1658–1671 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Psichas, A. et al. The short chain fatty acid propionate stimulates GLP-1 and PYY secretion via free fatty acid receptor 2 in rodents. Int. J. Obes. 39, 424–429 (2015).

    Article  CAS  Google Scholar 

  32. Richardson, A., Delbridge, A. T., Brown, N. J., Rumsey, R. D. & Read, N. W. Short chain fatty acids in the terminal ileum accelerate stomach to caecum transit time in the rat. Gut 32, 266–269 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tough, I. R., Forbes, S. & Cox, H. M. Signaling of free fatty acid receptors 2 and 3 differs in colonic mucosa following selective agonism or coagonism by luminal propionate. Neurogastroenterol. Motil. 30, e13454 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hauser, A. S. et al. Trends in GPCR drug discovery: new agents, targets and indications. Nat. Rev. Drug Discov. 16, 829–842 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Roth, B. L., Irwin, J. J. & Shoichet, B. K. Discovery of new GPCR ligands to illuminate new biology. Nat. Chem. Biol. 13, 1143–1151 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Milligan, G. G protein-coupled receptors not currently in the spotlight: free fatty acid receptor 2 and GPR35. Br. J. Pharmacol. 175, 2543–2553 (2018).

    Article  CAS  PubMed  Google Scholar 

  37. Viskaitis, P. et al. Modulation of SF1 neuron activity co-ordinately regulates both feeding behaviour and associated emotional states. Cell Rep. 21, 3559–3572 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Christiansen, C. B. et al. The impact of short chain fatty acids on GLP-1 and PYY secretion from the isolated perfused rat colon. Am. J. Physiol. Gastrointest. Liver Physiol. 315, G53–G65 (2018).

    Article  CAS  PubMed  Google Scholar 

  39. Ge, H. et al. Activation of G protein-coupled receptor 43 in adipocytes leads to inhibition of lipolysis and suppression of plasma free fatty acids. Endocrinology 149, 4519–4526 (2008).

    Article  CAS  PubMed  Google Scholar 

  40. Lee, T. et al. Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2. Mol. Pharmacol. 74, 1599–1609 (2008).

    Article  CAS  PubMed  Google Scholar 

  41. Tang, C. & Offermanns, S. FFA2 and FFA3 in metabolic regulation. Handb. Exp. Pharmacol. 236, 205–220 (2017).

    Article  CAS  PubMed  Google Scholar 

  42. Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622 (2009).

    Article  CAS  PubMed  Google Scholar 

  43. Smith, P. M. et al. The microbial metabolites, short-chain fatty acids, regulate colonic Treg cell homeostasis. Science 341, 569–573 (2013).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

These studies were funded by Biotechnology and Biosciences Research Council grant nos. BB/L027887/1 (Milligan) and BB/L02781X/1 (Tobin) and a CIHR Foundation grant no. FDN-148431 (Bouvier). S.J.B. is funded through a University of Glasgow Lord Kelvin Adam Smith Fellowship and an MRC project grant (no. MR/P019366/1). We are also grateful to M. Hogue and V. Lukasheva for the design, construction and characterization of the GRK2-based biosensor. We acknowledge the BSU facilities at the Cancer Research UK Beatson Institute (C596/A17196) and the Biological Services at the University of Glasgow.

Author information

Authors and Affiliations

  1. Centre for Translational Pharmacology, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, UK

    Daniele Bolognini, Natasja Barki, Brian D. Hudson, Eugenia Sergeev, Colin Molloy, Catherine E. Moss, Sophie J. Bradley, Andrew B. Tobin & Graeme Milligan

  2. UK Dementia Research Institute at the University of Cambridge, Cambridge, UK

    Adrian J. Butcher

  3. Department of Biochemistry and Molecular Medicine, Institute for Research in Immunology and Cancer, Université de Montreal, Montreal, Quebec, Canada

    Christian Le Gouill & Michel Bouvier

Authors
  1. Daniele Bolognini
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Natasja Barki
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Adrian J. Butcher
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Brian D. Hudson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Eugenia Sergeev
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Colin Molloy
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Catherine E. Moss
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Sophie J. Bradley
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Christian Le Gouill
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Michel Bouvier
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Andrew B. Tobin
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Graeme Milligan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.M. and A.B.T. devised the program of work. G.M., A.B.T. and D.B. wrote the paper with assistance from all other authors. G.M., A.B.T., D.B. and N.B. designed the experiments. D.B. (genetic characterization, lipolysis, GLP-1 release, immunostaining, pharmacological characterization), A.J.B. (biosensor studies), E.S. (ligand binding studies), C.L.G. (biosensor studies), C.E.M. (GLP-1 release) and N.B. (GLP-1 release and gut transit) performed experiments. B.D.H. developed the hFFA2-DREADD receptor and performed initial characterization. M.B. directed the biosensor studies. S.J.B. and C.M. oversaw the animal-based studies.

Corresponding authors

Correspondence to Andrew B. Tobin or Graeme Milligan.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figs. 1–10, Supplementary Table 1

Reporting Summary

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Bolognini, D., Barki, N., Butcher, A.J. et al. Chemogenetics defines receptor-mediated functions of short chain free fatty acids. Nat Chem Biol 15, 489–498 (2019). https://doi.org/10.1038/s41589-019-0270-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-019-0270-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing