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• W3048662837 startingPage "101449" @default.
• W3048662837 abstract "•Propionate activates Gαi/o but no Gαq/11 signaling in STC-1 cells or colonic crypts•Acute Gαi/o signaling via FFA2 requires endocytosis and mediates GLP-1 release•FFA2 traffics to very early endosomes to control rapid recycling and Gαi/o signals•Propionate-induced internalization of FFA2 drives p38 signaling and GLP-1 release The ability of propionate, a short-chain fatty acid produced from the fermentation of non-digestible carbohydrates in the colon, to stimulate the release of anorectic gut hormones, such as glucagon like peptide-1 (GLP-1), is an attractive approach to enhance appetite regulation, weight management, and glycemic control. Propionate induces GLP-1 release via its G protein-coupled receptor (GPCR), free fatty acid receptor 2 (FFA2), a GPCR that activates Gαi and Gαq/11. However, how pleiotropic GPCR signaling mechanisms in the gut regulates appetite is poorly understood. Here, we identify propionate-mediated G protein signaling is spatially directed within the cell whereby FFA2 is targeted to very early endosomes. Furthermore, propionate activates a Gαi/p38 signaling pathway, which requires receptor internalization and is essential for propionate-induced GLP-1 release in enteroendocrine cells and colonic crypts. Our study reveals that intestinal metabolites engage membrane trafficking pathways and that receptor internalization could orchestrate complex GPCR pathways within the gut. The ability of propionate, a short-chain fatty acid produced from the fermentation of non-digestible carbohydrates in the colon, to stimulate the release of anorectic gut hormones, such as glucagon like peptide-1 (GLP-1), is an attractive approach to enhance appetite regulation, weight management, and glycemic control. Propionate induces GLP-1 release via its G protein-coupled receptor (GPCR), free fatty acid receptor 2 (FFA2), a GPCR that activates Gαi and Gαq/11. However, how pleiotropic GPCR signaling mechanisms in the gut regulates appetite is poorly understood. Here, we identify propionate-mediated G protein signaling is spatially directed within the cell whereby FFA2 is targeted to very early endosomes. Furthermore, propionate activates a Gαi/p38 signaling pathway, which requires receptor internalization and is essential for propionate-induced GLP-1 release in enteroendocrine cells and colonic crypts. Our study reveals that intestinal metabolites engage membrane trafficking pathways and that receptor internalization could orchestrate complex GPCR pathways within the gut. The consumption of dietary fiber, or non-digestible carbohydrates (NDCs), has been shown to protect against diet-induced obesity (Chambers et al., 2015Chambers E.S. Viardot A. Psichas A. Morrison D.J. Murphy K.G. Zac-Varghese S.E. Macdougall K. Preston T. Tedford C. Finlayson G.S. et al.Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults.Gut. 2015; 64: 1744-1754Crossref PubMed Scopus (711) Google Scholar). The protective effects of NDCs are largely attributed to short-chain fatty acids (SCFAs) that are produced in the colon by microbiota from the fermentation of NDCs (Chambers et al., 2015Chambers E.S. Viardot A. Psichas A. Morrison D.J. Murphy K.G. Zac-Varghese S.E. Macdougall K. Preston T. Tedford C. Finlayson G.S. et al.Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults.Gut. 2015; 64: 1744-1754Crossref PubMed Scopus (711) Google Scholar; Den Besten et al., 2013Den Besten G. Van Eunen K. Groen A.K. Venema K. Reijngoud D.J. Bakker B.M. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism.J. Lipid Res. 2013; 54: 2325-2340Abstract Full Text Full Text PDF PubMed Scopus (2476) Google Scholar; James et al., 2003James S.L. Muir J.G. Curtis S.L. Gibson P.R. Dietary fibre: a roughage guide.Intern. Med. J. 2003; 33: 291-296Crossref PubMed Scopus (76) Google Scholar). Acetate, propionate, and butyrate are the predominant SCFAs produced and, in addition to regulation of gastro-intestinal functions, are involved in energy and glucose homeostasis and immune responses (Den Besten et al., 2013Den Besten G. Van Eunen K. Groen A.K. Venema K. Reijngoud D.J. Bakker B.M. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism.J. Lipid Res. 2013; 54: 2325-2340Abstract Full Text Full Text PDF PubMed Scopus (2476) Google Scholar). Traditionally, roles of SCFAs in these metabolic processes were thought to be limited to their ability to act as an energy source or as a regulator of cholesterol synthesis; however, with the discovery and characterization of G protein-coupled receptors (GPCRs) activated by SCFAs, free fatty acid receptor 2 (FFA2, previously known as GPR43) and free fatty acid receptor 3 (FFA3, previously known as GPR41), it is now widely appreciated that many SCFA activities can be attributed to these receptors (Fuller et al., 2015Fuller M. Priyadarshini M. Gibbons S.M. Angueira A.R. Brodsky M. Hayes M.G. Kovatcheva-Datchary P. Bäckhed F. Gilbert J.A. Lowe Jr., W.L. et al.The short-chain fatty acid receptor, FFA2, contributes to gestational glucose homeostasis.Am. J. Physiol. Endocrinol. Metab. 2015; 309: E840-E851Crossref PubMed Scopus (52) Google Scholar; Li et al., 2018Li M. Van Esch B. Henricks P.a.J. Folkerts G. Garssen J. The anti-inflammatory effects of short chain fatty acids on lipopolysaccharide- or tumor necrosis factor alpha-stimulated endothelial cells via activation of GPR41/43 and inhibition of HDACs.Front. Pharmacol. 2018; 9: 533Crossref PubMed Scopus (64) Google Scholar; Pingitore et al., 2019Pingitore A. Gonzalez-Abuin N. Ruz-Maldonado I. Huang G.C. Frost G. Persaud S.J. Short chain fatty acids stimulate insulin secretion and reduce apoptosis in mouse and human islets in vitro: role of free fatty acid receptor 2.Diabetes Obes. Metab. 2019; 21: 330-339Crossref PubMed Scopus (53) Google Scholar; Tolhurst et al., 2012Tolhurst G. Heffron H. Lam Y.S. Parker H.E. Habib A.M. Diakogiannaki E. Cameron J. Grosse J. Reimann F. Gribble F.M. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.Diabetes. 2012; 61: 364-371Crossref PubMed Scopus (1342) Google Scholar; Bolognini et al., 2016Bolognini D. Moss C.E. Nilsson K. Petersson A.U. Donnelly I. Sergeev E. Konig G.M. Kostenis E. Kurowska-Stolarska M. Miller A. et al.A novel allosteric activator of free fatty acid 2 receptor displays unique Gi-functional bias.J. Biol. Chem. 2016; 291: 18915-18931Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Among the three SCFAs, propionate has been of particular translational interest owing to its ability to acutely suppress appetite via activation of FFA2 in enteroendocrine L cells and release of the anorectic gut hormones peptide YY (PYY) and incretin glucagon like peptide-1 (GLP-1) (Tolhurst et al., 2012Tolhurst G. Heffron H. Lam Y.S. Parker H.E. Habib A.M. Diakogiannaki E. Cameron J. Grosse J. Reimann F. Gribble F.M. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.Diabetes. 2012; 61: 364-371Crossref PubMed Scopus (1342) Google Scholar; Psichas et al., 2015Psichas A. Sleeth M.L. Murphy K.G. Brooks L. Bewick G.A. Hanyaloglu A.C. Ghatei M.A. Bloom S.R. Frost G. The short chain fatty acid propionate stimulates GLP-1 and PYY secretion via free fatty acid receptor 2 in rodents.Int. J. Obes. (Lond.). 2015; 39: 424-429Crossref PubMed Scopus (433) Google Scholar), contributing to its role in rapid weight loss and improved insulin sensitivity following Roux-en-Y gastric bypass (Liou et al., 2013Liou A.P. Paziuk M. Luevano Jr., J.M. Machineni S. Turnbaugh P.J. Kaplan L.M. Conserved shifts in the gut microbiota due to gastric bypass reduce host weight and adiposity.Sci. Transl. Med. 2013; 5: 178ra41Crossref PubMed Scopus (713) Google Scholar). Direct health benefits of propionate in humans have been recently demonstrated whereby increasing the colonic levels of propionate in overweight humans not only exhibited reduced weight gain but also reduced abdominal adiposity and improved insulin sensitivity (Chambers et al., 2015Chambers E.S. Viardot A. Psichas A. Morrison D.J. Murphy K.G. Zac-Varghese S.E. Macdougall K. Preston T. Tedford C. Finlayson G.S. et al.Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults.Gut. 2015; 64: 1744-1754Crossref PubMed Scopus (711) Google Scholar). Thus propionate, and its receptor-mediated actions, represents an attractive system to develop therapeutic strategies in obesity management. Although the role of SCFAs and their receptors in mediating the release of anorectic gut hormones has been demonstrated in rodent models and humans (Tolhurst et al., 2012Tolhurst G. Heffron H. Lam Y.S. Parker H.E. Habib A.M. Diakogiannaki E. Cameron J. Grosse J. Reimann F. Gribble F.M. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.Diabetes. 2012; 61: 364-371Crossref PubMed Scopus (1342) Google Scholar; Bolognini et al., 2016Bolognini D. Moss C.E. Nilsson K. Petersson A.U. Donnelly I. Sergeev E. Konig G.M. Kostenis E. Kurowska-Stolarska M. Miller A. et al.A novel allosteric activator of free fatty acid 2 receptor displays unique Gi-functional bias.J. Biol. Chem. 2016; 291: 18915-18931Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar; Psichas et al., 2015Psichas A. Sleeth M.L. Murphy K.G. Brooks L. Bewick G.A. Hanyaloglu A.C. Ghatei M.A. Bloom S.R. Frost G. The short chain fatty acid propionate stimulates GLP-1 and PYY secretion via free fatty acid receptor 2 in rodents.Int. J. Obes. (Lond.). 2015; 39: 424-429Crossref PubMed Scopus (433) Google Scholar; Chambers et al., 2015Chambers E.S. Viardot A. Psichas A. Morrison D.J. Murphy K.G. Zac-Varghese S.E. Macdougall K. Preston T. Tedford C. Finlayson G.S. et al.Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults.Gut. 2015; 64: 1744-1754Crossref PubMed Scopus (711) Google Scholar), our understanding of the molecular mechanisms by propionate, regulating the release of anorectic gut hormone from enteroendocrine L cells, remains limited. FFA2 is coupled to both the Gαi/o and Gαq/11 families of heterotrimeric G proteins (Brown et al., 2003Brown A.J. Goldsworthy S.M. Barnes A.A. Eilert M.M. Tcheang L. Daniels D. Muir A.I. Wigglesworth M.J. Kinghorn I. Fraser N.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. 2003; 278: 11312-11319Abstract Full Text Full Text PDF PubMed Scopus (1579) Google Scholar; Le Poul et al., 2003Le Poul E. Loison C. Struyf S. Springael J.Y. Lannoy V. Decobecq M.E. Brezillon S. Dupriez V. Vassart G. Van Damme J. et al.Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation.J. Biol. Chem. 2003; 278: 25481-25489Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar), although Gαq/11 is implicated in mediating gut hormone release via increases in calcium (Bolognini et al., 2016Bolognini D. Moss C.E. Nilsson K. Petersson A.U. Donnelly I. Sergeev E. Konig G.M. Kostenis E. Kurowska-Stolarska M. Miller A. et al.A novel allosteric activator of free fatty acid 2 receptor displays unique Gi-functional bias.J. Biol. Chem. 2016; 291: 18915-18931Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar; Tolhurst et al., 2012Tolhurst G. Heffron H. Lam Y.S. Parker H.E. Habib A.M. Diakogiannaki E. Cameron J. Grosse J. Reimann F. Gribble F.M. Short-chain fatty acids stimulate glucagon-like peptide-1 secretion via the G-protein-coupled receptor FFAR2.Diabetes. 2012; 61: 364-371Crossref PubMed Scopus (1342) Google Scholar). Models of GPCR signaling, however, have rapidly evolved over recent years from single receptors activating distinct G protein pathways at the plasma membrane, to high signal diversity that can be differentially activated by distinct ligands and exquisitely regulated at a spatial and temporal level. The spatiotemporal regulation of GPCRs can occur via a variety of processes, with membrane trafficking of GPCRs playing a central role. Membrane trafficking of GPCRs was classically viewed as a mechanism to control active cell surface receptor number by driving receptor internalization and post-endocytic sorting to divergent cellular fates. However, it is now understood that receptor internalization to endosomes provides additional intracellular signaling platforms including activation of heterotrimeric G protein signaling (Eichel and Von Zastrow, 2018Eichel K. Von Zastrow M. Subcellular organization of GPCR signaling.Trends Pharmacol. Sci. 2018; 39: 200-208Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar; Hanyaloglu, 2018Hanyaloglu A.C. Advances in membrane trafficking and endosomal signaling of G protein-coupled receptors.Int. Rev. Cell Mol. Biol. 2018; 339: 93-131Crossref PubMed Scopus (22) Google Scholar; Calebiro et al., 2009Calebiro D. Nikolaev V.O. Gagliani M.C. De Filippis T. Dees C. Tacchetti C. Persani L. Lohse M.J. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors.PLoS Biol. 2009; 7: e1000172Crossref PubMed Scopus (405) Google Scholar; Ferrandon et al., 2009Ferrandon S. Feinstein T.N. Castro M. Wang B. Bouley R. Potts J.T. Gardella T.J. Vilardaga J.P. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis.Nat. Chem. Biol. 2009; 5: 734-742Crossref PubMed Scopus (412) Google Scholar). Endosomal signaling of GPCRs exhibits distinct functions from signaling activated at the plasma membrane, demonstrating the integrated nature of trafficking and signaling and providing a mechanism for cells to achieve highly specific and diverse downstream responses to its dynamic extracellular environment (Thomsen et al., 2018Thomsen A.R.B. Jensen D.D. Hicks G.A. Bunnett N.W. Therapeutic targeting of endosomal G-protein-coupled receptors.Trends Pharmacol. Sci. 2018; 39: 879-891Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar; Caengprasath and Hanyaloglu, 2019Caengprasath N. Hanyaloglu A.C. Hardwiring wire-less networks: spatially encoded GPCR signaling in endocrine systems.Curr. Opin. Cell Biol. 2019; 57: 77-82Crossref PubMed Scopus (8) Google Scholar). Furthermore, we have previously shown that GPCRs are organized to distinct endosomal compartments to activate signaling (Sposini et al., 2017Sposini S. Jean-Alphonse F.G. Ayoub M.A. Oqua A. West C. Lavery S. Brosens J.J. Reiter E. Hanyaloglu A.C. Integration of GPCR signaling and sorting from very early endosomes via opposing APPL1 mechanisms.Cell Rep. 2017; 21: 2855-2867Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar; Jean-Alphonse et al., 2014Jean-Alphonse F. Bowersox S. Chen S. Beard G. Puthenveedu M.A. Hanyaloglu A.C. Spatially restricted G protein-coupled receptor activity via divergent endocytic compartments.J. Biol. Chem. 2014; 289: 3960-3977Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These discoveries over the past decade have rewritten the GPCR “signaling atlas,” offering new interpretations of faulty GPCR activity in disease and providing novel therapeutic strategies to target GPCR signaling (Thomsen et al., 2018Thomsen A.R.B. Jensen D.D. Hicks G.A. Bunnett N.W. Therapeutic targeting of endosomal G-protein-coupled receptors.Trends Pharmacol. Sci. 2018; 39: 879-891Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). However, the role of membrane trafficking for FFA2 and the distinct actions of propionate that activates pleiotropic G protein signal pathways within the gut remain unknown. In this study, we demonstrate internalization-dependent FFA2 signaling drives propionate-induced GLP-1 release from enteroendocrine cells. Furthermore, we provide evidence that G protein signaling activated by FFA2 is differentially regulated by membrane trafficking and that an unexpected Gαi/p38 signaling pathway is required for propionate-induced GLP-1 release. Although propionate is known to mediate anorectic gut hormone release via FFA2, the ability of this SCFA to activate both upstream Gαq/11 and Gαi/o signal pathways in enteroendocrine cells has yet to be fully demonstrated. FFA2 couples to both Gαi/o to inhibit adenylate cyclase and reduce intracellular levels of cAMP and Gαq/11 that activates phospholipase C resulting in increases in inositol 1,4,5-triphosphate (IP3) and diacylglycerol, leading to mobilization of calcium from intracellular stores. In both mouse enteroendocrine (STC-1) cells and colonic crypts, 1 mM propionate, a physiologically relevant dose that induced maximal responses in STC-1 cells and HEK 293 cells expressing FFA2 (Figures S1A and S1B) was able to inhibit forskolin-induced cAMP, which was significantly reversed by pre-treatment with Gαi/o inhibitor pertussis toxin (Ptx) (Figures 1A and 1B ). Surprisingly, 1 mM propionate did not induce either an increase in intracellular calcium (Figures 1C and 1D and Video S1) or IP1, a downstream metabolite of IP3, in either STC-1 cells or colonic crypts (Figures 1E and 1F) despite its ability to induce GLP-1 release (Figures 1G and 1H). Treatment with a higher dose of propionate (10 mM) in both STC-1 cells and crypts also did not significantly increase intracellular IP1 or calcium levels (Figure S2). In contrast, a previously described selective FFA2 synthetic allosteric ligand (Lee et al., 2008Lee T. Schwandner R. Swaminath G. Weiszmann J. Cardozo M. Greenberg J. Jaeckel P. Ge H. Wang Y. Jiao X. et al.Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2.Mol. Pharmacol. 2008; 74: 1599-1609Crossref PubMed Scopus (124) Google Scholar), 4-CTMB, and a previously characterized selective FFA2 synthetic orthosteric ligand, compound 1 (Hudson et al., 2013Hudson B.D. Due-Hansen M.E. Christiansen E. Hansen A.M. Mackenzie A.E. Murdoch H. Pandey S.K. Ward R.J. Marquez R. Tikhonova I.G. et al.Defining the molecular basis for the first potent and selective orthosteric agonists of the FFA2 free fatty acid receptor.J. Biol. Chem. 2013; 288: 17296-17312Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar) (Cmp1), activated both Gαi/o and Gαq/11 signaling in STC-1 cells and colonic crypts (Figures S1D and 1C–1F and Videos S2 and S3). Both compounds were also used at doses shown to induce maximal signal responses (Lee et al., 2008Lee T. Schwandner R. Swaminath G. Weiszmann J. Cardozo M. Greenberg J. Jaeckel P. Ge H. Wang Y. Jiao X. et al.Identification and functional characterization of allosteric agonists for the G protein-coupled receptor FFA2.Mol. Pharmacol. 2008; 74: 1599-1609Crossref PubMed Scopus (124) Google Scholar; Hudson et al., 2012Hudson B.D. Tikhonova I.G. Pandey S.K. Ulven T. Milligan G. Extracellular ionic locks determine variation in constitutive activity and ligand potency between species orthologs of the free fatty acid receptors FFA2 and FFA3.J. Biol. Chem. 2012; 287: 41195-41209Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar and Figure S1C), thus further demonstrating functional FFA2 in both cultures. The synthetic ligand-induced calcium responses were Gαq/11 mediated as they were significantly impaired by the pre-treatment of a selective Gαq/11 inhibitor, YM-254890 (Takasaki et al., 2004Takasaki J. Saito T. Taniguchi M. Kawasaki T. Moritani Y. Hayashi K. Kobori M. A novel Galphaq/11-selective inhibitor.J. Biol. Chem. 2004; 279: 47438-47445Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), in STC-1 cells (Figure S3). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyNDJkYWNkZDE5Y2E3ODExMGE4NGQxYjA3OTNlY2MyMyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NTk2MjIxfQ.k85ZtoNqWWSdleCtbuHytkMIOtFXmvaf_V4Jmu1OvyQSe1IzikfI773oAeT3qPUzFbt-QPJkncB3s9zwI6bbUQT9AMDcMIWrJVkmCW89acq4DJDa_kkrALWV-sUwDCmhV8Z3GSncDRGwlN_58NYvRMjPX5sajVboURDK0z35AySvJqXpGjOEI8zHtWK86yPDfI_pb_WjEhXivKv-R0jODq3hTdV3Mqb_0m27u6Nq9BsG5uBzISw4JYcUqvAeBAeV8DeEEflSOgKygg4a441x1A0_kmft8uMUa45I_9hvTc7afja09K20w-demDoWVk5TITIIAUuzVpfdb0SqGJgKiw Download .mp4 (0.87 MB) Help with .mp4 files Video S1. Propionate Is Unable to Increase Levels of Intracellular Calcium in Colonic Crypts, Related to Figure 1Crypts were labeled with calcium indicator dye (Fluo4-AM) and intracellular calcium mobilization measured in live colonic crypts via confocal microscopy. Cultures were treated with sodium propionate (Pro) (1 mM). eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyZjViOGNjN2MwNjIyMmUwN2E2YzYzMTQ4ZjlhZDYwZSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NTk2MjIxfQ.BfQCcw0roVOGXEp0Ok6diVYcrAyivFDKHDCB2Z2zKr8NpRBH1BK8I8gfkLBU6f7OS_rG_P5x0iBYcPfMDAtJbzYErZLc3qbADcF1A98Sm2mWXEjwATAImhyHHG-9A046qq5weAhB2xUYQ1nH2I9aT2Lo3s3LnEzpkgGcr6FJlHZKQGMrtqyK6lxApc8fH473k3fqaIZKpu1hm0vlYhIy_Vi1epnWvZd_an5naTR3CMNKNK_-NuO5ftFQPCHU15Y2mx08ejetWbPi9srd5O2SxonZ2zWIRZlbeYy91zm2Zxui1PiRzZ-aowxTdz8duLBE_TzOH9NmUb8H1ShZueSopg Download .mp4 (0.9 MB) Help with .mp4 files Video S2. The Vehicle DMSO Is Unable to Increase Levels of Intracellular Calcium in Colonic Crypts, Related to Figure 1Crypts were labeled with calcium indicator dye (Fluo4-AM), and intracellular calcium mobilization was measured in live colonic crypts via confocal microscopy. Cultures were treated with DMSO as a control for synthetic ligand Cmp1. eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiJiYmMwNjFkYzYzOGUyYzA1YmNhNzM0NmU3MGYyOWRkMyIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4NTk2MjIxfQ.g6LieOffXtTYw-SRTgaFLFnrpEgzUpqVQfaSRQSEbh5Zrd8KtOXYwi2euqlgYII7lw_uKZIyia99lZrp_qPGLURyza5QCown77XvldpT_G58RR8kB2iGdDuinVso3D25CiW3GfBggPK51cqYgvxQNiwDTIWO_nIR1VTL1_XFplmtZEl6P-PGkBSLFNy8pznUA2dzCNpjoQtn2oMkVb6nOQBZxE5J3kk9WlDvojgBIeK-XeCrLICsHPH6GX5E3nQYTaFDttWV12d6aDMp6OdBWoJ_tr35HlbaXrZ3muEFno71W06pQ7B1idffXfCWqJ5cmO3c0Tlng7cR4BA5s3eE6g Download .mp4 (1.68 MB) Help with .mp4 files Video S3. An FFA2-Selective Synthetic Ligand Increases Levels of Intracellular Calcium in Colonic Crypts, Related to Figure 1Crypts were labeled with calcium indicator dye (Fluo4-AM), and intracellular calcium mobilization was measured in live colonic crypts via confocal microscopy. Cultures were treated with Cmp1 (10 μM). Despite the lack of detectable Gαq/11 signaling, propionate-induced-Gαi/o signaling was dependent on FFA2 as the reduction of forskolin-induced cAMP in colonic crypts derived from FFA2 knockout mice (FFA2 −/−) was completely abolished (Figure 1I), consistent with our prior reports from the same mouse model that propionate-induced gut hormone release from the colon requires FFA2 (Psichas et al., 2015Psichas A. Sleeth M.L. Murphy K.G. Brooks L. Bewick G.A. Hanyaloglu A.C. Ghatei M.A. Bloom S.R. Frost G. The short chain fatty acid propionate stimulates GLP-1 and PYY secretion via free fatty acid receptor 2 in rodents.Int. J. Obes. (Lond.). 2015; 39: 424-429Crossref PubMed Scopus (433) Google Scholar). This loss of propionate-mediated Gαi/o signaling in the FFA2 −/− crypts was not due to alterations in FFA3 expression (Figure 1J). These data confirm that despite functional FFA2 responses, propionate activates Gαi/o signaling without detectable Gαq/11 responses in these cultures. To determine if the inability of propionate to activate Gαq/11 signaling via FFA2 was cellular context-specific, we stimulated HEK 293 cells expressing FLAG-FFA2. Treatment with 1 mM propionate significantly induced increases in intracellular calcium and IP1 (Figure S4) confirming activation of Gαq/11 signaling. Taken together, this demonstrates that, unlike synthetic FFA2 ligands, propionate is not able to signal via Gαq/11 in enteroendocrine cells, suggesting additional mechanisms beyond G protein activation are employed to induce propionate-mediated anorectic gut hormone secretion. We next determined if propionate/FFA2 activation is spatially regulated via membrane trafficking as a potential mechanism underlying its actions in the gut. Many GPCRs undergo ligand-induced internalization via a well-described β-arrestin- and clathrin-dependent mechanism, whereby the large GTPase dynamin regulates the latter steps of endocytosis that drive clathrin-coated vesicle scission. To inhibit FFA2 internalization, the ability of a potent inhibitor of dynamin GTPase activity, Dyngo-4a, known to block the internalization of many GPCRs (Mccluskey et al., 2013Mccluskey A. Daniel J.A. Hadzic G. Chau N. Clayton E.L. Mariana A. Whiting A. Gorgani N.N. Lloyd J. Quan A. et al.Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis.Traffic. 2013; 14: 1272-1289Crossref PubMed Scopus (161) Google Scholar; Eichel et al., 2016Eichel K. Jullie D. Von Zastrow M. β-Arrestin drives MAP kinase signalling from clathrin-coated structures after GPCR dissociation.Nat. Cell Biol. 2016; 18: 303-310Crossref PubMed Scopus (154) Google Scholar; Tsvetanova and Von Zastrow, 2014Tsvetanova N.G. Von Zastrow M. Spatial encoding of cyclic AMP signaling specificity by GPCR endocytosis.Nat. Chem. Biol. 2014; 10: 1061-1065Crossref PubMed Scopus (178) Google Scholar; Sposini et al., 2017Sposini S. Jean-Alphonse F.G. Ayoub M.A. Oqua A. West C. Lavery S. Brosens J.J. Reiter E. Hanyaloglu A.C. Integration of GPCR signaling and sorting from very early endosomes via opposing APPL1 mechanisms.Cell Rep. 2017; 21: 2855-2867Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), was first assessed in HEK 293 cells expressing FLAG-tagged FFA2 and imaged via confocal microscopy. Unexpectedly, FFA2 exhibited both constitutive and propionate-dependent internalization from the plasma membrane (Figure 2A), which was confirmed via flow cytometry (Figure S5A). In cells pre-treated with Dyngo-4a, a strong inhibition of both constitutive and propionate-induced FFA2 internalization was observed (Figure 2A), demonstrating that FFA2 constitutive and ligand-induced internalization occur in a dynamin-dependent manner. Under conditions where dynamin-dependent FFA2 internalization was inhibited in HEK 293 cells, the ability of propionate to inhibit forskolin-induced cAMP was impaired (Figure 2B). In contrast, FFA2-mediated Gαq/11 signaling, as measured by intracellular calcium responses (Figure 2C) or IP-1 accumulation (Figure S5B), was not significantly affected, suggesting a differential requirement of FFA2 internalization for FFA2-mediated signaling. These results were also confirmed in HEK 293 cells lacking β-arrestins 1 and 2 (Grundmann et al., 2018Grundmann M. Merten N. Malfacini D. Inoue A. Preis P. Simon K. Ruttiger N. Ziegler N. Benkel T. Schmitt N.K. et al.Lack of beta-arrestin signaling in the absence of active G proteins.Nat. Commun. 2018; 9: 341Crossref PubMed Scopus (217) Google Scholar) (Figure S5C). Interestingly, only ligand-induced, but not constitutive, FFA2 internalization was inhibited by lack of β-arrestins (Figures 2D and S5D). However, as in cells pre-treated with Dyngo-4a, propionate-dependent inhibition of forskolin-induced cAMP was significantly impaired in β-arrestin knockout cells compared with wild-type HEK 293 cells (Figure 2E). In contrast, propionate-induced calcium mobilization and IP1 accumulation was unperturbed (Figures 2F and S5E). The requirement of receptor internalization for Gαi/o signaling was also determined for the endogenous propionate-responsive receptors expressed in STC-1 cells. As specific antibodies are not available for these receptors, STC-1 cells were transfected with FLAG-tagged FFA2 to confirm required conditions to inhibit FFA2 internalization in these cells. Similar to HEK 293 cells, FFA2 internalization exhibited both constitutive and ligand-induced endocytic profiles, and both were inhibited by Dyngo-4a (Figure 2G). Consistent with our observations in HEK 293 cells, Dyngo-4a pre-treatment inhibited propionate-mediated activation of Gαi/o signaling (Figure 2H). Overall, these data demonstrate a requirement for ligand-induced FFA2 internalization in propionate-mediated Gαi/o signaling in heterologous and enteroendocrine cells. We have previously shown that GPCRs exhibit divergent sorting to distinct endosomal compartments between early endosomes (EEs) and very early endosomes (VEEs), and this post-endocytic organization is critical for both GPCR sorting fate and endosomal signaling (Jean-Alphonse et al., 2014Jean-Alphonse F. Bowersox S. Chen S. Beard G. Puthenveedu M.A. Hanyaloglu A.C. Spatially restricted G protein-coupled receptor activity via divergent endocytic compartments.J. Biol. Chem. 2014; 289: 3960-3977Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar; Sposini et al., 2017Sposini S. Jean-Alphonse F.G. Ayoub M.A. Oqua A. West C. Lavery S. Brosens J.J. Reiter E. Hanyaloglu A.C. Integration of GPCR signaling and sorting from very early endosomes via opposing APPL1 mechanisms.Cell Rep. 2017; 21: 2855-2867Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). As internalization of FFA2 is essential for its Gαi/o signaling, we next determined the postendo" @default.
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• W3048662837 title "Internalization-Dependent Free Fatty Acid Receptor 2 Signaling Is Essential for Propionate-Induced Anorectic Gut Hormone Release" @default.
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