Title: Functional Characterization of Human Receptors for Short Chain Fatty Acids and Their Role in Polymorphonuclear Cell Activation
Abstract: Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream. Basal acetate concentrations in the blood (about 100 μm) can further increase to millimolar concentrations following alcohol intake. It was known previously that SCFAs can activate leukocytes, particularly neutrophils. In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Propionate was the most potent agonist for both GPR41 and GPR43. Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41. The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca2+ release, ERK1/2 activation, and inhibition of cAMP accumulation. They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxin-sensitive Gi/o family, whereas GPR43 displayed a dual coupling through Gi/o and Pertussis toxin-insensitive Gq protein families. The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions. In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection. The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca2+ release and chemotaxis. Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression. GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations. Short chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are produced at high concentration by bacteria in the gut and subsequently released in the bloodstream. Basal acetate concentrations in the blood (about 100 μm) can further increase to millimolar concentrations following alcohol intake. It was known previously that SCFAs can activate leukocytes, particularly neutrophils. In the present work, we have identified two previously orphan G protein-coupled receptors, GPR41 and GPR43, as receptors for SCFAs. Propionate was the most potent agonist for both GPR41 and GPR43. Acetate was more selective for GPR43, whereas butyrate and isobutyrate were more active on GPR41. The two receptors were coupled to inositol 1,4,5-trisphosphate formation, intracellular Ca2+ release, ERK1/2 activation, and inhibition of cAMP accumulation. They exhibited, however, a differential coupling to G proteins; GPR41 coupled exclusively though the Pertussis toxin-sensitive Gi/o family, whereas GPR43 displayed a dual coupling through Gi/o and Pertussis toxin-insensitive Gq protein families. The broad expression profile of GPR41 in a number of tissues does not allow us to infer clear hypotheses regarding its biological functions. In contrast, the highly selective expression of GPR43 in leukocytes, particularly polymorphonuclear cells, suggests a role in the recruitment of these cell populations toward sites of bacterial infection. The pharmacology of GPR43 matches indeed the effects of SCFAs on neutrophils, in terms of intracellular Ca2+ release and chemotaxis. Such a neutrophil-specific SCFA receptor is potentially involved in the development of a variety of diseases characterized by either excessive or inefficient neutrophil recruitment and activation, such as inflammatory bowel diseases or alcoholism-associated immune depression. GPR43 might therefore constitute a target allowing us to modulate immune responses in these pathological situations. G protein-coupled receptors (GPCRs) 1The abbreviations used are: GPCR, G protein-coupled receptor; FBS, fetal bovine serum; fMLP, formyl-Met-Leu-Phe; PBMC, peripheral blood mononuclear cells; PTX, Pertussis toxin; SCFA, short chain fatty acid; PMN, polymorphonuclear cells; RT, reverse transcription; [35S]GTPγS, 35S-labeled guanosine 5′-3-O-(thio)triphosphate; CHO, Chinese hamster ovary; ERK, extracellular signal-regulated kinase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 3-(N-morpholino)propane sulfonic acid.1The abbreviations used are: GPCR, G protein-coupled receptor; FBS, fetal bovine serum; fMLP, formyl-Met-Leu-Phe; PBMC, peripheral blood mononuclear cells; PTX, Pertussis toxin; SCFA, short chain fatty acid; PMN, polymorphonuclear cells; RT, reverse transcription; [35S]GTPγS, 35S-labeled guanosine 5′-3-O-(thio)triphosphate; CHO, Chinese hamster ovary; ERK, extracellular signal-regulated kinase; BisTris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MOPS, 3-(N-morpholino)propane sulfonic acid. constitute one of the largest gene families yet identified (1Bockaert J. Pin J.P. EMBO J. 1999; 18: 1723-1729Crossref PubMed Scopus (1218) Google Scholar). In addition to about 160 characterized receptors, around 125 human genes encode proteins obviously belonging to this family of receptors, but their ligands and functions remain to be determined. These so far uncharacterized receptors are referred to as orphan GPCRs, but they are expected to play, by analogy with characterized members of the family, important roles in the regulation of physiological processes. For some orphan receptors, sequence similarity with well known receptors allows to construct hypotheses regarding the chemical nature of their ligands or their involvement in physiological processes. However, many orphan receptors are clustered in subfamilies with low similarity to characterized receptors.Orphan receptors led in a number of cases to the discovery of molecules that were not recognized previously as functional extracellular mediators. The chemical diversity among endogenous ligands of GPCRs is unique as it includes ions, bioamines, lipids, peptides, and large proteins, as well as a large number of odorant molecules. The recent identification of prokineticins (2Lin D.C. Bullock C.M. Ehlert F.J. Chen J.L. Tian H. Zhou Q.Y. J. Biol. Chem. 2002; 277: 19276-19280Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), UDP-glucose (3Chambers J.K. Macdonald L.E. Sarau H.M. Ames R.S. Freeman K. Foley J.J. Zhu Y. McLaughlin M.M. Murdock P. McMillan L. Trill J. Swift A. Aiyar N. Taylor P. Vawter L. Naheed S. Szekeres P. Hervieu G. Scott C. Watson J.M. Murphy A.J. Duzic E. Klein C. Bergsma D.J. Wilson S. Livi G.P. J. Biol. Chem. 2000; 275: 10767-10771Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), lysophosphatidylcholine (4Kabarowski J.H. Zhu K. Le L.Q. Witte O.N. Xu Y. Science. 2001; 293: 702-705Crossref PubMed Scopus (276) Google Scholar, 5Zhu K. Baudhuin L.M. Hong G. Williams F.S. Cristina K.L. Kabarowski J.H. Witte O.N. Xu Y. J. Biol. Chem. 2001; 276: 41325-41335Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar), sphingosylphosphorylcholine (5Zhu K. Baudhuin L.M. Hong G. Williams F.S. Cristina K.L. Kabarowski J.H. Witte O.N. Xu Y. J. Biol. Chem. 2001; 276: 41325-41335Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 6Xu Y. Zhu K. Hong G. Wu W. Baudhuin L.M. Xiao Y. Damron D.S. Nat. Cell Biol. 2000; 2: 261-267Crossref PubMed Scopus (176) Google Scholar), relaxin (7Hsu S.Y. Nakabayashi K. Nishi S. Kumagai J. Kudo M. Sherwood O.D. Hsueh A.J. Science. 2002; 295: 671-674Crossref PubMed Scopus (669) Google Scholar), eicosanoid (8Hosoi T. Koguchi Y. Sugikawa E. Chikada A. Ogawa K. Tsuda N. Suto N. Tsunoda S. Taniguchi T. Ohnuki T. J. Biol. Chem. 2002; 277: 31459-31465Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), kisspeptin (9Kotani M. Detheux M. Vandenbogaerde A. Communi D. Vanderwinden J.M. Le Poul E. Brezillon S. Tyldesley R. Suarez-Huerta N. Vandeput F. Blanpain C. Schiffmann S.N. Vassart G. Parmentier M. J. Biol. Chem. 2001; 276: 34631-34636Abstract Full Text Full Text PDF PubMed Scopus (1202) Google Scholar, 10Ohtaki T. Shintani Y. Honda S. Matsumoto H. Hori A. Kanehashi K. Terao Y. Kumano S. Takatsu Y. Masuda Y. Ishibashi Y. Watanabe T. Asada M. Yamada T. Suenaga M. Kitada C. Usuki S. Kurokawa T. Onda H. Nishimura O. Fujino M. Nature. 2001; 411: 613-617Crossref PubMed Scopus (1162) Google Scholar), and psychosin (11Im D.S. Heise C.E. Nguyen T. O'Dowd B.F. Lynch K.R. J. Cell Biol. 2001; 153: 429-434Crossref PubMed Scopus (153) Google Scholar) as new ligands for GPCRs has uncovered completely new extracellular pathways regulating cellular and tissue functions.Among orphan receptors, a cluster of receptors poorly related to other subfamilies includes four members, GPR40, GPR41, GPR42, and GPR43 (Fig. 1) (12Sawzdargo M. George S.R. Nguyen T. Xu S. Kolakowski L.F. O'Dowd B.F. Biochem. Biophys. Res. Commun. 1997; 239: 543-547Crossref PubMed Scopus (169) Google Scholar). GPR41 and GPR42 have the same length and share 98% amino acid identity. The four genes encoding these receptors are intronless and are clustered onto chromosomal region 19q13.1. Although little information is available concerning these receptors, GPR41 was shown to induce apoptosis via a p53/Bax pathway in an ischemia/reperfusion paradigm (13Kimura M. Mizukami Y. Miura T. Fujimoto K. Kobayashi S. Matsuzaki M. J. Biol. Chem. 2001; 276: 26453-26460Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). In addition, GPR43 and its murine orthologue LSSIG are induced during the differentiation of leukocyte progenitor cells to monocytes or neutrophils and were found mainly in hematopoietic tissues, suggesting that this receptor could have an important function in the differentiation and/or activation of leukocytes (14Senga T. Iwamoto S. Yoshida T. Yokota T. Adachi K. Azuma E. Hamaguchi M. Iwamoto T. Blood. 2003; 101: 1185-1187Crossref PubMed Scopus (69) Google Scholar).In this report, we showed that propionate, acetate, and other SCFAs act as specific activators of GPR41 and GPR43. An extensive pharmacological study revealed differences in the rank order of potency of SCFA toward each receptor, as well as in the G protein coupling leading to intracellular cascade activation. Pharmacological data obtained on human polymorphonuclear cells involved GPR43 as the main functional SCFA receptor on these cells.EXPERIMENTAL PROCEDURESReagents—Culture media, antibiotics, fetal bovine serum (FBS), and trypsin were from Bio-Whittaker. FuGENE 6, restriction, and DNA modifying enzymes were from Roche Diagnostics. [35S]GTPγS and myo-d-[2-3H]inositol (17.7 Ci/mmol) was from Amersham Biosciences. Dowex AG1X8 (formate form) was from Bio-Rad. Short chain fatty acids (SCFAs) and Pertussis toxin were from Sigma. Forskolin and isobutylmethylxanthine were from Eurobiochem (Louvain la Neuve, Belgium). Oligonucleotide primers were from Eurogentec (Liège, Belgium). Total and messenger RNAs were from Ambion (Huntingdon, Great Britain).Cloning and Sequencing of Human GPR43 and Human GPR41— Oligonucleotide primers were synthesized on the basis of the sequence of the human GPR41 and GPR43 receptors (GenBank™ accession numbers AF024688 and AF024690, respectively). For GPR43 cloning, sense primer 5′-CCGGAATTCACCATGCTGCCGGACTGGAAG-3′ and antisense primer 5′-CTTGTCTAGACTACTCTGTAGTGAAGTC-3′ were used in a PCR amplification using human genomic DNA as template and Pfu DNA polymerase (Stratagene) under the following conditions: 1 min at 94 °C, 30 s at 52 °C, 1 min at 72 °C, 3 cycles; 1 min at 94 °C, 30 s at 63 °C, 1 min at 72 °C, 30 cycles. A fragment of 1 kb containing the entire coding sequence of human GPR43 gene was amplified, digested by EcoRI and XbaI, and cloned in the bicistronic pEFIN5 expression vector. In the bicistronic vector, designated pEFIN5, both the recombinant receptor and the neomycin phosphotransferase selection marker are transcribed from a single promoter element.For GPR41 cloning, sense primer 5′-CCGGATATCACCATGGATACAGGCCCCGAC-3′ and antisense primer 5′-CTTGTCTAGACTAGCTTTCAGCACAGGC-3′ were used in a similar strategy, except that the coding sequence was cloned using EcoRV and XbaI. All the inserts of the resulting plasmids were sequenced on both strands using the Big-Dye Terminator cycle sequencing kit (Applied Biosystems). Sequence alignment was performed using the ClustalX software, version 1.8 (15Thompson J.D. Gibson T.J. Plewniak F. Jeanmougin F. Higgins D.G. Nucleic Acids Res. 1997; 25: 4876-4882Crossref PubMed Scopus (35206) Google Scholar), and dendrograms were constructed using TreeView.Tissue Distribution of GPR41 and GPR43—Reverse transcription (RT)-PCR experiments were carried out using a panel of total RNA (peripheral blood mononuclear cells (PBMC), dendritic cells, monocytes, T lymphocytes, small intestine) and poly(A)+ RNA (thymus, spleen, lymph node, bone marrow, lung, stomach, adipose, breast). The total RNA from neutrophils was prepared from venous blood of healthy donors using TriPure (Roche Diagnostics). Approximately 50 ng of poly(A)+ RNA or 500 ng of total RNA was reverse-transcribed with Superscript II (Invitrogen) and used for PCR. Human GPR43 and GPR41 receptors transcripts were detected by PCR using the following primers: 5′-TTCTACAGCAGCATCTACTG-3′ (GPR43 forward), 5′-GAAGCACACCAGGAAATTGA-3′ (GPR43 reverse), 5′-TACGTCATAGAATTCTCAGG-3′ (GPR41 forward), and 5′-TGTTCACTGGTCTTTCTTTC-3′ (GPR41 reverse). The expected sizes of the amplified DNA bands were 439 and 508 bp for GPR43 and GPR41, respectively. PCR was performed using the Taq polymerase under the following conditions: 94 °C for 5 min; 30 cycles at 94 °C for 1 min, 53 °C for 1 min 30 s, and 72 °C for 40 s (for GPR43); or 94 °C for 5 min, 30 cycles at 94 °C for 1 min, 52 °C for 1.5 min, and 72 °C for 35 s (for GPR41). A control was performed with glyceraldehyde-3-phosphate dehydrogenase cDNA fragment (509 bp) as described previously (16Brezillon S. Lannoy V. Franssen J.D. Le Poul E. Dupriez V. Lucchetti J. Detheux M. Parmentier M. J. Biol. Chem. 2003; 278: 776-783Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Aliquots of the PCR were analyzed by 1% agarose gel electrophoresis.Cell Culture and Transfection—The recombinant pEFIN5-GPR41, pEFIN5-GPR43 plasmids, and the empty pEFIN5 vector were transfected in CHO-K1 cells (CRL-9618; ATCC, Manassas, VA), WTA11 cells (a CHO-K1 cell line coexpressing mitochondrial apoaequorin and Gα16), or human embryonic kidney 293 cells (ATCC CRL-1573), using FuGENE 6. The transfected cells were selected with 400 μg/ml G418 in Nutrient Ham's F-12 medium supplemented with 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin, from 2 days after transfection. The medium of WTA11 cells contained, in addition, 250 μg/ml zeocin. The resistant clones were selected by RT-PCR and sequencing. COS-7 and human embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium containing 10% FBS, 1 mm sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin and transfected using LipofectAMINE 2000 (Invitrogen), with the pEFIN5 plasmids encoding GPR41 or GPR43. The cells were used in functional assays 2 days after transfection.Aequorin Assay—The functional response to SCFAs was analyzed by measuring the luminescence of aequorin as described previously (17Detheux M. Standker L. Vakili J. Munch J. Forssmann U. Adermann K. Pohlmann S. Vassart G. Kirchhoff F. Parmentier M. Forssmann W.G. J. Exp. Med. 2000; 192: 1501-1508Crossref PubMed Scopus (130) Google Scholar). For all assays, data were analyzed with the PRISM software (Graph-Pad Prism Software, San Diego, CA) using nonlinear regression applied to a sigmoidal dose-response model, as for all assays used in this work. cAMP Assay—CHO-K1 cells were spread on Petri dishes (250,000 cells per 35-mm dish) and cultured overnight in Nutrient Ham's F-12 medium containing 10% FBS, 100 units/ml penicillin, 100 μg/ml streptomycin, and 400 μg/ml G418. Cells were preincubated for 30 min in Krebs-Ringer HEPES buffer composed of 25 mm HEPES, pH 7.4, 124 mm NaCl, 5 mm KCl, 1.25 mm MgSO4, 1.45 mm CaCl2, 1.25 mm KH2PO4, and 8 mm glucose and then incubated for 20 min in the same medium supplemented with 10 μm forskolin and variable concentrations of agonists. The reaction was stopped by removing the supernatant and the addition of 100 μl of lysis buffer in each well. The cAMP contents were quantified by enzyme-linked immunosorbent assay (CS200 kit; Applied Biosystems).[35S]GTPγS Binding Assay—The measurement of agonist-stimulated [35S]GTPγS binding to membranes of cells expressing human GPR41 or human GPR43 was performed as described previously (9Kotani M. Detheux M. Vandenbogaerde A. Communi D. Vanderwinden J.M. Le Poul E. Brezillon S. Tyldesley R. Suarez-Huerta N. Vandeput F. Blanpain C. Schiffmann S.N. Vassart G. Parmentier M. J. Biol. Chem. 2001; 276: 34631-34636Abstract Full Text Full Text PDF PubMed Scopus (1202) Google Scholar). Briefly, membranes (10 μg) from CHO-GPR41 or CHO-GPR43 cells were incubated for 15 min at room temperature in binding buffer (20 mm HEPES, pH 7.4, 100 mm NaCl, 3 mm MgCl2, 3 μm GDP, 10 μg/ml saponin) containing different concentrations of SCFA in 96-well microplates (Basic FlashPlates; PerkinElmer Life Sciences). After addition of 0.1 nm [35S]GTPγS, microplates were shaken for 1 min and further incubated at 30 °C for 30 min. The incubation was stopped by centrifugation of the microplate for 10 min, at 800 × g and 4 °C, and removal of the supernatant. Microplates were counted in a TopCount (PerkinElmer Life Sciences) for 1 min per well.Fluorescence-based Intracellular Ca2+ Mobilization—CHO/GPR43 and CHO/GPR41 cells were seeded into black 96-well assay plates the day before the experiment. Cells are loaded for 1 h with 4 μm Fluo-3/AM (Molecular Probes) in Hank's balanced salt buffer and washed before being transferred to a microplate reader (FDSS; Hamamatsu Photonics KK, Hamamatsu, Japan) for compounds injection and simultaneous fluorescence recording for 3 min (excitation and emission wavelengths, 340 and 510 nm, respectively). Results were expressed as a percentage of fluorescence change as compared with basal level.Phosphoinositide Accumulation—Cells expressing GPR41 or GPR43 receptors were labeled overnight with 10 μCi/ml [3H]myo-inositol in culture medium. Cells were then washed three times and incubated in Krebs buffer (10 mm HEPES, pH 7.4, 150 mm NaCl, 4.2 mm KCl, 1 mm CaCl2, 0.5 mm MgCl2, 5.6 mm d-glucose) for 15 min in medium containing1mm LiCl. SCFA agonists were then added for 30 min. The reaction was stopped by replacing the incubation medium with 1 ml of 5% HClO4. The total inositol phosphate pool was then extracted and purified on Dowex columns as described by Joly et al. (18Joly C. Gomeza J. Brabet I. Curry K. Bockaert J. Pin J.P. J. Neurosci. 1995; 15: 3970-3981Crossref PubMed Google Scholar). Results were expressed as the ratio between the radioactivity collected in the inositol phosphate fraction over the radioactivity recovered from the cellular membranes solubilized in 10% Triton and 0.1 n NaOH and used as standard. The use of this ratio allows for greater homogeneity in the data, as it reduces variations resulting from differences in cell numbers from individual wells.Mitogen-activated Protein Kinase Assay—ERK1/2 activation was assayed by Western blotting, using an anti-phospho-p42/44 monoclonal antibody. Briefly, cells serum-starved for 24 h were collected and resuspended in serum-free Dulbecco's modified Eagle's medium. After stimulation of cells at the indicated time with 10 mm propionate or acetate, in the presence or not of 100 ng/ml Pertussis toxin, cells were collected by centrifugation (12.000 rpm, 3 min) and heated to 100 °C for 5 min in lysis buffer (100 mm Tris-HCl, pH 6.8, 4 mm EDTA, 4% SDS, 20% glycerol, and 0.02% β-mercaptoethanol). For Western blot analysis, solubilized proteins corresponding to ∼6 × 106 cells were loaded onto Nupage 10% BisTris gel (Invitrogen) in a Nupage MOPS SDS running buffer. After transfer to nitrocellulose membranes, proteins were probed with mouse anti-phospho p42/p44 (1:1000) antibody (Cell Signaling Technology).Polymorphonuclear Cell Pharmacology: Intracellular Calcium Release and Chemotaxis Assays—Polymorphonuclear cells were purified from buffy coats of healthy volunteers. PMN chemotaxis was performed in Boyden microchambers (Neuro Probe, Gaithersburg, MD) with polyvinylpyrrolidone-free polycarbonate membranes (5-μm pore size; Corning Separations Division, Acton, MA) as described previously (19Struyf S. Proost P. Schols D. De Clercq E. Opdenakker G. Lenaerts J.P. Detheux M. Parmentier M. De Meester I. Scharpe S. Van Damme J. J. Immunol. 1999; 162: 4903-4909PubMed Google Scholar). For intracellular Ca2+ assays, cells were incubated in Hanks' balanced salt medium containing 0.1% bovine serum albumin and 2.8 μg/ml Fura-2/AM (Molecular Probes) at 37 °C for 45 min. Cells were washed, resuspended at 5 × 106 cells/ml, and allowed to re-equilibrate for 10 min at 37 °C. Cells were then transferred to cuvettes, and calcium transients were monitored through fluorescence measurements using an LSB 50B spectrofluorimeter (PerkinElmer Life Sciences).RESULTSIdentification of GPR43 and GPR41 as Receptors for Short Chain Fatty Acids—In the frame of a general strategy for characterizing ligands for orphan G protein-coupled receptors, a CHO-K1 cell line coexpressing GPR43, Gα16, and apoaequorin (GPR43-WTA11) was established and screened in an aequorin-based functional assay against a large collection of reference compounds comprising peptides, lipids, carbohydrates, and small chemical compounds. A biological activity specific for GPR43-expressing cells was observed for a number of peptide solutions containing acetate as the counter ion. Control tests revealed that the acetate ion itself, and not the peptides, was responsible for the agonist activity on GPR43. A pH effect could rapidly be excluded, as neutral acetate buffers displayed the same activity as acetic acid. Furthermore, different salts of acetate (Na+,NH3+, and K+) confirmed the activity of the acetate anion. We also excluded that the effect of acetate on intracellular calcium release was subsequent to the acidification of the cell cytosol, as several organic acids with a similar pK a were not active on GPR43-expressing cells. This specificity of the GPR43 response to acetate was further demonstrated by testing in the same assay a set of about 60 orphan and characterized receptors belonging to different GPCR classes and subclasses, in addition to the wild-type cell lines WTA11. None of these responded to acetate. The determination of concentration-action curves allowed us to estimate the EC50 of GPR43 for sodium acetate at 268 ± 26 μm (Fig. 2A). Additional SCFAs were tested at this stage and were found to activate GPR43, as well, in the aequorin-based assay (data not shown).Fig. 2Aequorin-based functional assay of GPR41 and GPR43. CHO-K1 cell lines coexpressing apoaequorin and Gα16 (WTA11 cells) were further transfected with a bicistronic vector expressing GPR43 (A) or GPR41 (B). The selected stable clones were used in a functional assay based on the activation of aequorin, following the release of intracellular Ca2+, in response to acetate or propionate. The data represent the mean ± S.E. for triplicate data points. The displayed curves are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)We also screened the receptors structurally related to GPR43 and found that GPR41, which shares 42% amino acid identity with GPR43, was also activated by acetate, although to a lower extent (Fig. 2B). Indeed, the EC50 of GPR41 for acetate was estimated at 1390 ± 220 μm, and the other SCFA propionate was found to display a higher potency on this receptor, with an EC50 of 11.6 ± 1.4 μm (Fig. 2B).Intracellular Coupling of GPR41 and GPR43—The natural coupling properties and the intracellular signaling pathways activated by GPR43 and GPR41, upon stimulation by acetate or propionate, were investigated in CHO-K1 cells expressing the human receptors but devoid of aequorin or additional coupling proteins such as Gα16 (CHO/GPR43 and CHO/GPR41 cells). We first demonstrated that both receptors coupled negatively to adenylyl cyclase through a Pertussis toxin-sensitive G protein (Gi/o class), while being unable to promote accumulation of cAMP in the absence of forskolin (not shown). This cAMP accumulation assay was used to characterize further the detailed pharmacology of the receptors (see Fig. 3, A and B, and see below). A [35S]GTPγS binding assay was used alternatively as a functional test to monitor Gi/o coupling of these receptors in a cell-free assay, to exclude further the possibility that cell activation would result from nonspecific actions on the cell metabolism or cytoplasmic components. The activity of acetate and propionate on GPR43 and GPR41 was confirmed in this assay (Fig. 4, A and B). Propionate was equipotent on GPR43 (EC50 = 259 ± 67 μm) and GPR41 (EC50 = 274 ± 75 μm), whereas acetate was more potent on GPR43 (EC50 = 537 ± 31 μm) than on GPR41 (EC50 = 1299 ± 65 μm). PTX treatment inhibited the response to SCFAs for both receptors. Stimulation of GPR41 and GPR43 also resulted in the release of intracellular calcium, with a similar rank order of potency (see Fig. 4, C and D and Table I). Although the EC50 values observed in both assays were similar for GPR41, they were higher in the Ca2+ assay as compared with the cAMP assay for GPR43. Furthermore, PTX abolished the response of GPR41 but not of GPR43 (Fig. 4, C and D). This suggested a unique Gi/o coupling for GPR41 and a dual coupling through the Gi/o and Gq families for GPR43.Fig. 3Pharmacology of GPR41 or GPR43 in a cAMP accumulation assay. CHO-K1 cell lines expressing GPR43 (A) or GPR41 (B) were incubated with various concentrations of SCFA and analogs, together with 5 μm forskolin. The data represent the mean ± S.E. for triplicate data points. The displayed curves are representative of at least three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Intracellular cascades activated by GPR41 and GPR43. A and B, the binding of [35S]GTPγS to membranes of CHO-K1 cells expressing GPR43 (A) or GPR41 (B) was measured following stimulation by acetate or propionate. C and D, intracellular calcium mobilization was measured in CHO-K1 cells expressing GPR43 (C) or GPR41 (D), after culturing the cells in the presence or absence of Pertussis toxin. The data represent the mean ± S.E. for triplicate data points, and the displayed curves are representative of at least three independent experiments. E and F, inositol phosphate accumulation was determined in COS-7 cells transiently expressing GPR43 (E) or cotransfected with GPR43 or GPR41 and the chimeric Gqi5 protein (F). G and H, phosphorylation of ERK1/2 mitogen-activated protein kinases following stimulation of GPR43 (G) and GPR41 (H) expressed in CHO-K1 cells by acetate or propionate for 5 min. The cellular extract (20-μg proteins) was separated by SDS-polyacrylamide gel electrophoresis, transferred to nylon membranes, and labeled with antibodies specific for the phosphorylated forms of ERK1 and ERK2.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table IpEC 50 values of SCFA and related molecules for GPR41 and GPR43GPR43GPR41CarboncAMPCa2+cAMPCa2+Formate12.61 ± 0.06 (8)1.99 ± 0.03 (8)>10 mM (10)>10 mM (10)Acetate24.46 ± 0.05 (3)3.99 ± 0.02 (2)2.99 ± 0.04 (8)2.97 ± 0.02 (8)Propionate34.85 ± 0.07 (1)4.10 ± 0.02 (1)5.21 ± 0.07 (1)4.70 ± 0.10 (1)Butyrate44.55 ± 0.1 (2)3.47 ± 0.03 (3)4.38 ± 0.10 (3)4.24 ± 0.06 (3)Isobutyrate43.84 ± 0.05 (4)3.22 ± 0.08 (4)4.52 ± 0.07 (2)4.31 ± 0.01 (2)L-OH-butyrate42.32 ± 0.02 (10)1.92 ± 0.08 (9)2.29 ± 0.10 (9)2.06 ± 0.16 (9)Pivalate52.59 ± 0.14 (9)2.34 ± 0.10 (7)3.63 ± 0.16 (7)3.19 ± 0.01 (7)Valerate52.78 ± 0.15 (7)2.72 ± 0.08 (5)4.38 ± 0.14 (4)4.11 ± 0.06 (4)Isovalerate52.67 ± 0.06 (6)2.51 ± 0.06 (6)4.24 ± 0.14 (5)3.91 ± 0.02 (5)Caproate62.86 ± 0.06 (5)>5 mM (10)3.99 ± 0.07 (6)3.87 ± 0.06 (6)Heptanoate7>10 mM (11)>5 mM (10)>100 mM (11)NDCaprylate8>10 mM (11)>5 mM (11)>100 mM (11)ND Open table in a new tab The activity of SCFAs was confirmed following expression of GPR43 and GPR41 in other cell lines (COS-7 and human embryonic kidney 293), with similar EC50 values observed for acetate and propionate. Moreover, transient expression of GPR43 in COS-7 cells led to the accumulation of inositol phosphate products in a PTX-independent mechanism with an EC50 of 325 ± 36 μm for propionate and 132 ± 40 μm for acetate (Fig. 4E). Stimulation of GPR41 expressed in COS-7 cells resulted in the accumulation of inositol phosphates, only following the cotransfection of the chimeric G protein Gqi5 (Fig. 4F). Furthermore, the cotransfection of GPR43 and Gqi5 increased significantly the basal level of inositol phosphates, as compared with control conditions, or the expression of one of the plasmids alone, suggesting that GPR43 is endowed with constitutive activity (data not shown).Stimulation of GPR43 and GPR41 express