Identification and characterization of the bovine G protein-coupled receptor GPR41 and GPR43 genes. A Wang;Z Gu;B Heid;R M Akers;H Jiang. 2009. J Dairy Sci. 92. PMID: 19448003

Volatile fatty acids (VFA), including acetate, propionate, and butyrate, are not only a primary source of energy, but also regulate rumen development, insulin and glucagon secretion, and other physiological processes in cattle and sheep. The mechanism underlying the regulatory effects of VFA is unknown. Recent "reverse pharmacology" studies identified human G protein-coupled receptors GPR41 and GPR43 as receptors for short-chain fatty acids. It is possible that proteins similar to human GPR41 and GPR43 mediate the regulatory effects of VFA in cattle. In this study, we determined first, whether the bovine genome contains genes similar to the human GPR41 and GPR43 genes; second, whether and where these genes are expressed in cattle; and third, if the proteins encoded by these genes can be activated by acetate, propionate, and butyrate. A search of GenBank revealed bovine genomic sequences and expressed sequence tags highly similar to the human GPR41 and GPR43 DNA and cDNA sequences. The protein-coding and 5' untranslated regions of the bovine GPR41 and GPR43 mRNA were cloned and sequenced from spleen tissue. Based on these sequences, the bovine GPR41 gene contains 3 exons and its transcription is initiated at 2 leader exons, generating 2 GPR41 mRNA variants differing in the 5' untranslated region. The bovine GPR43 gene contains 2 exons and transcription of this gene is initiated from a single start site. The amino acid sequences deduced from the bovine GPR41 and GPR43 mRNA sequences are more than 75% identical to those of the human GPR41 and GPR43 and are predicted to encode 7 transmembrane domains, typical of G protein-coupled receptors. Both bovine GPR41 and GPR43 mRNA were detected in a variety of tissues including rumen and pancreas. In a cell system, interaction of the overexpressed bovine GPR41 or GPR43 protein with acetate, propionate, or butyrate inhibited luciferase reporter expression from a cyclic AMP-responsive promoter, suggesting that the bovine GPR41 and GPR43 proteins couple to Galpha(i/11). In total, these results demonstrate that the bovine genome encodes functional GPR41 and GPR43 genes and suggest that GPR41 and GPR43 may play a role in the regulatory effects of VFA in cattle.
Propionate induces the release of granules from bovine neutrophils. M D Carretta;I Conejeros;M A Hidalgo;R A Burgos. 2013. J Dairy Sci. 96. PMID: 23403200

Short-chain fatty acids (SCFA) are produced by bacterial fermentation in the rumen of cattle and are the primary energy source in ruminants. Propionate is one of the main SCFA and it can exert multiple effects on the inflammatory process and neutrophil function via calcium (Ca(2+)) release, reactive oxygen species, and intracellular pH changes. However, currently no evidence has shown whether propionate can induce granule release from bovine neutrophils. The purpose of this study was to analyze the effect of propionate on granule release and to evaluate the expression of two G-protein coupled receptors-GPR41 and GPR43-that are activated by propionate. Neutrophil degranulation was assessed by quantifying the release of the neutrophil enzymes myeloperoxidase (MPO), lactoferrin, and matrix metalloprotease-9 (MMP-9) as markers of azurophil, specific granules, and gelatinase granules, respectively. Isolated bovine neutrophils were treated with millimolar concentrations of propionate (0.3, 3 and 30mM), and the cell-free supernatants were recovered. The stimulation of neutrophils with 0.3mM propionate induced the release of lactoferrin and MMP-9 as revealed by ELISA and gelatin zymography, respectively. Propionate at 30mM induced the release of MPO as demonstrated using an enzymatic assay. The role of intracellular Ca(2+) influx and the signaling pathways that may regulate the propionate effect on granules release were also determined. Reverse transcription (RT)-PCR and real-time PCR were performed to analyze the expression of GPR41 and GPR43 mRNA in bovine neutrophils. Both mRNA were detected, whereas the expression of GPR43 was higher than that of GPR41, and the synthetic agonists for this receptor, phenylacetamides 1 and 2, caused an increase in intracellular Ca(2+), lactoferrin, and MMP-9 release. These results support that propionate-induced granule release is mediated by intracellular Ca(2+) influx and activation of extracellular signal-regulated kinase ERK 1/2. We also propose a potential role of GPR43 in propionate-induced granule release from bovine neutrophils that may be involved in regulatory effects of propionate in the innate immune response in cattle.
The Short-Chain Fatty Acid Propionate Inhibits Adipogenic Differentiation of Human Chorion-Derived Mesenchymal Stem Cells Through the Free Fatty Acid Receptor 2. Judit Iván;Evelin Major;Adrienn Sipos;Katalin Kovács;Dániel Horváth;István Tamás;Péter Bay;Viktor Dombrádi;Beáta Lontay. 2017. Stem Cells Dev. 26. PMID: 28992793

Free fatty acid receptor 2 (FFAR2, also known as GPR43) is a G-protein-coupled receptor activated by short-chain fatty acids that are produced by gut microbiota through fermentation of nondigestible carbohydrates. FFAR2 functions as a metabolic sensor and is expressed in metabolically active tissues, such as adipose tissue. Earlier studies proved the connection between FFAR2 and adipocyte differentiation in mice. The aim of this study was to investigate the implication of FFAR2 receptor in adipogenesis in human chorion-derived mesenchymal stem cells (cMSCs). The short-chain fatty acid, propionate, and phenylacetamide a selective FFAR2 agonist resulted in a marked suppression of lipid droplet accumulation during the adipogenic differentiation of cMSCs. Western blot studies revealed that FFAR2 was detectable at any time point of the differentiation period. The direct involvement of FFAR2 in the differentiation into adipocytes was proven by the downregulation of its gene expression in cMSCs by lentiviral messenger RNA (mRNA) silencing transduction particles. Our results showed that a significant suppression in lipid accumulation upon FFAR2 agonist treatments was elicited by FFAR2-silencing. Based on these results we suggest that propionate inhibits the formation of adipocytes from MSCs and acts on adipogenesis predominantly via FFAR2.
Propionate suppresses hepatic gluconeogenesis via GPR43/AMPK signaling pathway. Hiroki Yoshida;Megumi Ishii;Mitsugu Akagawa. 2019. Arch Biochem Biophys. 672. PMID: 31356781

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are generated by gut microbial fermentation of dietary fiber. SCFAs may exert multiple beneficial effects on human lipid and glucose metabolism. However, their actions and underlying mechanisms are not fully elucidated. In this study, we examined the direct effects of propionate on hepatic glucose and lipid metabolism using human HepG2 hepatocytes. Here, we demonstrate that propionate at a physiologically-relevant concentration effectively suppresses palmitate-enhanced glucose production in HepG2 cells but does not affect intracellular neutral lipid levels. Our results indicated that propionate can decline in gluconeogenesis by down-regulation of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) through activation of AMP-activated protein kinase (AMPK), which is a major regulator of the hepatic glucose metabolism. Mechanistic studies also revealed that propionate-stimulated AMPK phosphorylation can be ascribed to Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) activation in response to an increase in intracellular Ca2+ concentration. Moreover, siRNA-mediated knockdown of the propionate receptor GPR43 prevented propionate-inducible activation of AMPK and abrogates the gluconeogenesis-inhibitory action. Thus, our data indicate that the binding of propionate to hepatic GPR43 elicits CaMKKβ-dependent activation of AMPK through intracellular Ca2+ increase, leading to suppression of gluconeogenesis. The present study suggests the potential efficacy of propionate in preventive and therapeutic management of diabetes.
Activation of G protein-coupled receptor 43 in adipocytes leads to inhibition of lipolysis and suppression of plasma free fatty acids. Hongfei Ge;Xiaofan Li;Jennifer Weiszmann;Ping Wang;Helene Baribault;Jin-Long Chen;Hui Tian;Yang Li. 2008. Endocrinology. 149. PMID: 18499755

G protein-coupled receptor 43 (GPR43) has been identified as a receptor for short-chain fatty acids that include acetate and propionate. A potential involvement of GPR43 in immune and inflammatory response has been previously suggested because its expression is highly enriched in immune cells. GPR43 is also expressed in a number of other tissues including adipocytes; however, the functional consequences of GPR43 activation in these other tissues are not clear. In this report, we focus on the potential functions of GPR43 in adipocytes. We show that adipocytes treated with GPR43 natural ligands, acetate and propionate, exhibit a reduction in lipolytic activity. This inhibition of lipolysis is the result of GPR43 activation, because this effect is abolished in adipocytes isolated from GPR43 knockout animals. In a mouse in vivo model, we show that the activation of GPR43 by acetate results in the reduction in plasma free fatty acid levels without inducing the flushing side effect that has been observed by the activation of nicotinic acid receptor, GPR109A. These results suggest a potential role for GPR43 in regulating plasma lipid profiles and perhaps aspects of metabolic syndrome.
Short-chain fatty acid receptor, GPR43, is expressed by enteroendocrine cells and mucosal mast cells in rat intestine. Shin-ichiro Karaki;Retsu Mitsui;Hisayoshi Hayashi;Ikuo Kato;Hiroshi Sugiya;Toshihiko Iwanaga;John B Furness;Atsukazu Kuwahara. 2006. Cell Tissue Res. 324. PMID: 16453106

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are the major anions in the large intestinal lumen. They are produced from dietary fiber by bacterial fermentation and are known to have a variety of physiological and pathophysiological effects on the intestine. In the present study, we investigated the expression of the SCFA receptor, GPR43, in the rat distal ileum and colon. Expression of GPR43 was detected by reverse transcriptase/polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. mRNA for GPR43 was detected, by RT-PCR, in extracts of the whole wall and separated mucosa from the ileum and colon and from muscle plus submucosa from the ileum, but not from muscle plus submucosa preparations from the colon. We raised a rabbit antiserum against a synthesized fragment of rat GPR43; this was specific for rat GPR43. GPR43 protein was detected by Western blot analysis in extracts of whole wall and separated mucosa, but not in muscle plus submucosa extracts. By immunohistochemistry, GPR43 immunoreactivity was localized to enteroendocrine cells expressing peptide YY (PYY), whereas 5-hydroxytryptamine (5-HT)-immunoreactive (IR) enteroendocrine cells were not immunoreactive for GPR43. Mast cells of the lamina propria expressing 5-HT were also GPR43-IR. The results of the present study suggest that the PYY-containing enteroendocrine cells and 5-HT-containing mucosal mast cells sense SCFAs via the GPR43 receptor. This is consistent with physiological data showing that SCFAs stimulate the release of PYY and 5-HT from the ileum and colon.
The diet-derived short chain fatty acid propionate improves beta-cell function in humans and stimulates insulin secretion from human islets in vitro. Attilio Pingitore;Edward S Chambers;Thomas Hill;Inmaculada Ruz Maldonado;Bo Liu;Gavin Bewick;Douglas J Morrison;Tom Preston;Gareth A Wallis;Catriona Tedford;Ramón Castañera González;Guo C Huang;Pratik Choudhary;Gary Frost;Shanta J Persaud. 2016. Diabetes Obes Metab. 19. PMID: 27761989

AIMS: Diet-derived short chain fatty acids (SCFAs) improve glucose homeostasis in vivo, but the role of individual SCFAs and their mechanisms of action have not been defined. This study evaluated the effects of increasing colonic delivery of the SCFA propionate on β-cell function in humans and the direct effects of propionate on isolated human islets in vitro. MATERIALS AND METHODS: For 24 weeks human subjects ingested an inulin-propionate ester that delivers propionate to the colon. Acute insulin, GLP-1 and non-esterified fatty acid (NEFA) levels were quantified pre- and post-supplementation in response to a mixed meal test. Expression of the SCFA receptor FFAR2 in human islets was determined by western blotting and immunohistochemistry. Dynamic insulin secretion from perifused human islets was quantified by radioimmunoassay and islet apoptosis was determined by quantification of caspase 3/7 activities. RESULTS: Colonic propionate delivery in vivo was associated with improved β-cell function with increased insulin secretion that was independent of changes in GLP-1 levels. Human islet β-cells expressed FFAR2 and propionate potentiated dynamic glucose-stimulated insulin secretion in vitro, an effect that was dependent on signalling via protein kinase C. Propionate also protected human islets from apoptosis induced by the NEFA sodium palmitate and inflammatory cytokines. CONCLUSIONS: Our results indicate that propionate has beneficial effects on β-cell function in vivo, and in vitro analyses demonstrated that it has direct effects to potentiate glucose-stimulated insulin release and maintain β-cell mass through inhibition of apoptosis. These observations support ingestion of propiogenic dietary fibres to maintain healthy glucose homeostasis.
G-protein-coupled receptor for short-chain fatty acids suppresses colon cancer. Yong Tang;Yakun Chen;Hongmei Jiang;Gregory T Robbins;Daotai Nie. 2010. Int J Cancer. 128. PMID: 20979106

GPR43 is a G-protein-coupled receptor for short-chain fatty acids (SCFAs). Expression of GPR43 is detected in hematopoietic tissues and the large intestine. SCFAs are derived from bacterial fermentation and metabolism of undigested dietary fibers and have been recognized for their cancer prevention activities in the colon. The role of SCFAs, particularly butyrate, in colon cancer therapy has been extensively studied, and its tumor suppressive functions are believed to be due to their intracellular actions, notably inhibition of histone deacetylase. In our study, we show that SCFAs also exert their antitumor effects via receptor GPR43 and that GPR43 is frequently lost in colon cancer cells. Immunohistostaining revealed that GPR43 immunoreactivity was high in normal colon tissues (N = 31) but was markedly reduced or completely lost in most colorectal adenocarcinoma tissues (N = 70) and their corresponding lymph node metastatic adenocarcinomas (N = 38). RT-PCR analysis detected the presence of full length GPR43 mRNA in only one (HT-29) of nine established human colon cancer cell lines. Restoration of GPR43 expression in HCT8 human colonic adenocarcinoma cells induced G0/G1 cell cycle arrest and activated caspases, leading to increased apoptotic cell death after propionate/butyrate treatment. Restored GPR43 expression, coupled with propionate treatment, induced an upregulation of p21 and a decrease in the levels of cyclin D3 and cyclin-dependent kinases (CDKs) 1 and 2, while the CDK4 and CDK6 levels remained unchanged. Our results suggest that GPR43 functions as a tumor suppressor by mediating SCFA-induced cell proliferation inhibition and apoptotic cell death in colon cancer.
A novel antiinflammatory role for the short-chain fatty acids in human labor. Chiara Voltolini;Sharon Battersby;Sophie L Etherington;Felice Petraglia;Jane E Norman;Henry N Jabbour. 2011. Endocrinology. 153. PMID: 22186417

Human parturition is an inflammatory process that can be activated prematurely by pathological stimuli. This study investigated the expression of G protein-coupled receptors GPR43 and GPR41 receptors in human uteroplacental tissues and the role of short-chain fatty acids (SCFA) in modulating inflammatory pathways in fetal membranes. Expression of GPR43 and GPR41 was investigated in uteroplacental tissues collected from women delivering at term or preterm after ethical approval and patient informed consent. The effect of SCFA on expression of inflammatory genes was assessed in amnion explants after culture with a mimetic of infection (lipopolysaccharide, LPS). Sodium propionate effect on LPS-induced neutrophil chemotaxis was evaluated by transwell assay. GPR43 and GPR41 mRNA expression was higher in myometrium and fetal membranes collected from women after the onset of labor. GPR43 protein expression localized to immune cells and vascular endothelium in the myometrium and epithelium of fetal membranes. Treatment with LPS significantly increased mRNA expression of GPR43 and inflammatory genes. Cotreatment with LPS and sodium propionate decreased LPS-induced expression of inflammatory genes including IL-6, IL-8, cyclooxygenase-2, IL-1α, intercellular adhesion molecule-1, and platelet endothelial cell adhesion molecule-1 but not IL-1β or lymphocyte function-associated antigen-1. Sodium propionate reduced LPS-induced neutrophil chemotaxis and protein secretion of the neutrophil chemoattractant IL-8. Finally, fetal membrane expression of GPR43 was significantly higher in women delivering preterm with evidence of infection. GPR43-SCFA interactions may represent novel pathways that regulate inflammatory processes involved in human labor. Suppression of inflammatory pathways by SCFA may be therapeutically beneficial for pregnant women at risk of pathogen-induced preterm delivery.
Butyrate and propionate protect against diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent mechanisms. Hua V Lin;Andrea Frassetto;Edward J Kowalik;Andrea R Nawrocki;Mofei M Lu;Jennifer R Kosinski;James A Hubert;Daphne Szeto;Xiaorui Yao;Gail Forrest;Donald J Marsh. 2012. PLoS One. 7. PMID: 22506074

Short-chain fatty acids (SCFAs), primarily acetate, propionate, and butyrate, are metabolites formed by gut microbiota from complex dietary carbohydrates. Butyrate and acetate were reported to protect against diet-induced obesity without causing hypophagia, while propionate was shown to reduce food intake. However, the underlying mechanisms for these effects are unclear. It was suggested that SCFAs may regulate gut hormones via their endogenous receptors Free fatty acid receptors 2 (FFAR2) and 3 (FFAR3), but direct evidence is lacking. We examined the effects of SCFA administration in mice, and show that butyrate, propionate, and acetate all protected against diet-induced obesity and insulin resistance. Butyrate and propionate, but not acetate, induce gut hormones and reduce food intake. As FFAR3 is the common receptor activated by butyrate and propionate, we examined these effects in FFAR3-deficient mice. The effects of butyrate and propionate on body weight and food intake are independent of FFAR3. In addition, FFAR3 plays a minor role in butyrate stimulation of Glucagon-like peptide-1, and is not required for butyrate- and propionate-dependent induction of Glucose-dependent insulinotropic peptide. Finally, FFAR3-deficient mice show normal body weight and glucose homeostasis. Stimulation of gut hormones and food intake inhibition by butyrate and propionate may represent a novel mechanism by which gut microbiota regulates host metabolism. These effects are largely intact in FFAR3-deficient mice, indicating additional mediators are required for these beneficial effects.
An essential role of Ffar2 (Gpr43) in dietary fibre-mediated promotion of healthy composition of gut microbiota and suppression of intestinal carcinogenesis. S Sivaprakasam;A Gurav;A V Paschall;G L Coe;K Chaudhary;Y Cai;R Kolhe;P Martin;D Browning;L Huang;H Shi;H Sifuentes;M Vijay-Kumar;S A Thompson;D H Munn;A Mellor;T L McGaha;P Shiao;C W Cutler;K Liu;V Ganapathy;H Li;N Singh. 2016. Oncogenesis. 5. PMID: 27348268

Composition of the gut microbiota has profound effects on intestinal carcinogenesis. Diet and host genetics play critical roles in shaping the composition of gut microbiota. Whether diet and host genes interact with each other to bring specific changes in gut microbiota that affect intestinal carcinogenesis is unknown. Ability of dietary fibre to specifically increase beneficial gut microbiota at the expense of pathogenic bacteria in vivo via unknown mechanism is an important process that suppresses intestinal inflammation and carcinogenesis. Free fatty acid receptor 2 (FFAR2 or GPR43) is a receptor for short-chain fatty acids (acetate, propionate and butyrate), metabolites of dietary fibre fermentation by gut microbiota. Here, we show FFAR2 is down modulated in human colon cancers than matched adjacent healthy tissue. Consistent with this, Ffar2(-/-) mice are hypersusceptible to development of intestinal carcinogenesis. Dietary fibre suppressed colon carcinogenesis in an Ffar2-dependent manner. Ffar2 played an essential role in dietary fibre-mediated promotion of beneficial gut microbiota, Bifidobacterium species (spp) and suppression of Helicobacter hepaticus and Prevotellaceae. Moreover, numbers of Bifidobacterium is reduced, whereas those of Prevotellaceae are increased in human colon cancers than matched adjacent normal tissue. Administration of Bifidobacterium mitigated intestinal inflammation and carcinogenesis in Ffar2(-/-) mice. Taken together, these findings suggest that interplay between dietary fibre and Ffar2 play a key role in promoting healthy composition of gut microbiota that stimulates intestinal health.
The Orphan G protein-coupled receptors GPR41 and GPR43 are activated by propionate and other short chain carboxylic acids. Andrew J Brown;Susan M Goldsworthy;Ashley A Barnes;Michelle M Eilert;Lili Tcheang;Dion Daniels;Alison I Muir;Mark J Wigglesworth;Ian Kinghorn;Neil J Fraser;Nicholas B Pike;Jay C Strum;Klaudia M Steplewski;Paul R Murdock;Julie C Holder;Fiona H Marshall;Philip G Szekeres;Shelagh Wilson;Diane M Ignar;Steve M Foord;Alan Wise;Simon J Dowell. 2002. J Biol Chem. 278. PMID: 12496283

GPR41 and GPR43 are related members of a homologous family of orphan G protein-coupled receptors that are tandemly encoded at a single chromosomal locus in both humans and mice. We identified the acetate anion as an agonist of human GPR43 during routine ligand bank screening in yeast. This activity was confirmed after transient transfection of GPR43 into mammalian cells using Ca(2+) mobilization and [(35)S]guanosine 5'-O-(3-thiotriphosphate) binding assays and by coexpression with GIRK G protein-regulated potassium channels in Xenopus laevis oocytes. Other short chain carboxylic acid anions such as formate, propionate, butyrate, and pentanoate also had agonist activity. GPR41 is related to GPR43 (52% similarity; 43% identity) and was activated by similar ligands but with differing specificity for carbon chain length, with pentanoate being the most potent agonist. A third family member, GPR42, is most likely a recent gene duplication of GPR41 and may be a pseudogene. GPR41 was expressed primarily in adipose tissue, whereas the highest levels of GPR43 were found in immune cells. The identity of the cognate physiological ligands for these receptors is not clear, although propionate is known to occur in vivo at high concentrations under certain pathophysiological conditions.
Expression of the short-chain fatty acid receptor, GPR43, in the human colon. Shin-Ichiro Karaki;Hideaki Tazoe;Hisayoshi Hayashi;Hidefumi Kashiwabara;Kazunari Tooyama;Yuichi Suzuki;Atsukazu Kuwahara. 2007. J Mol Histol. 39. PMID: 17899402

Short-chain fatty acids (SCFAs), 2-4 carbon monocarboxylates including acetate, propionate and butyrate, are known to have a variety of physiological and pathophysiological effects on the intestine. Previously, we reported that the SCFA receptor, G-protein coupled receptor 43 (GPR43), is expressed by enteroendocrine and mucosal mast cells in the rat intestine. In the present study, expression and localization of GPR43 were investigated in the human large intestine. Gene and protein expression of GPR43 in the human ascending colon was analyzed by reverse transcriptase/polymerase chain reaction and Western blotting, respectively. In addition, localization of GPR43 was investigated by immunohistochemistry. In RT-PCR analysis, GPR43 mRNA was detected in whole wall mRNA samples. Western blotting analysis revealed the expression of GPR43 protein in whole wall and scraped mucosa protein samples, but not in muscle or submucosa. GPR43 immunoreactivity was observed in the intracellularly in enterocytes and in the peptide YY-immunoreactive enteroendocrine cells. These results indicate that the short chain fatty acid receptor, GPR43 is expressed by enteroendocrine L cells containing peptide YY in the human large intestine.
Short-chain fatty acid propionate alleviates Akt2 knockout-induced myocardial contractile dysfunction. Linlin Li;Yinan Hua;Jun Ren. 2011. Exp Diabetes Res. 2012. PMID: 21960994

BACKGROUND AND AIMS. Dysregulation of Akt has been implicated in diseases such as cancer and diabetes, although little is known about the role of Akt deficiency on cardiomyocyte contractile function. This study was designed to examine the effect of Akt2 knockout-induced cardiomyocyte contractile response and the effect of dietary supplementation of short-chain fatty acid propionate on Akt2 knockout-induced cardiac dysfunction, if any. METHODS AND RESULTS. Adult male wild-type (WT) and Akt2 knockout mice were treated with propionate (0.3 g/kg, p.o.) or vehicle for 7 days. Oral glucose tolerance test (OGTT) was performed. Cardiomyocyte contractile function and mitochondrial membrane potential were assessed. Expression of insulin-signaling molecules Akt, PTEN, GSK3β, and eNOS receptors for short-chain fatty acids GPR41, and GPR43 as well as protein phosphatase PP2AA, PP2AB, PP2C were evaluated using Western blot analysis. Our results revealed that Akt2 knockout led to overt glucose intolerance, compromised cardiomyocyte contractile function (reduced peak shortening and maximal velocity of shortening/relengthening as well as prolonged relengthening), loss of mitochondrial membrane potential, decreased GPR41 and elevated GPR43 expression, all of which, with the exception of glucose intolerance and elevated GPR43 level, were significantly attenuated by propionate. Neither Akt2 knockout nor propionate affected the expression of protein phosphatases, eNOS, pan, and phosphorylated PTEN and GSK3β. CONCLUSIONS. Taken together, these data depicted that Akt2 knockout may elicit cardiomyocyte contractile and mitochondrial defects and a beneficial role of propionate or short-chain fatty acids against Akt2 deficiency-induced cardiac anomalies.
Short-chain fatty acids induce tissue plasminogen activator in airway epithelial cells via GPR41&43. Y Imoto;A Kato;T Takabayashi;M Sakashita;J E Norton;L A Suh;R G Carter;A R Weibman;K E Hulse;W Stevens;K E Harris;A T Peters;L C Grammer;B K Tan;K Welch;D B Conley;R C Kern;S Fujieda;R P Schleimer. 2018. Clin Exp Allergy. 48. PMID: 29431874

BACKGROUND: Chronic rhinosinusitis (CRS) is a heterogeneous chronic inflammatory disease generally divided based on the presence or absence of nasal polyps (NPs). One of the features of NPs is excessive fibrin deposition, which is associated with down-regulation of tissue plasminogen activator (t-PA) in NPs. As t-PA is expressed in epithelial cells, and epithelium is readily accessible to topical therapies, identifying compounds that can mediate the induction of t-PA would be a potential new strategy for the treatment of NPs. OBJECTIVE: The objective of this study was to determine whether short-chain fatty acids (SCFAs) can induce t-PA in airway epithelial cells via their known receptors GPR41 and GPR43. METHODS: We performed immunohistochemistry (IHC) to determine whether receptors for SCFAs, known as G protein-coupled receptor 41/free fatty acid receptor 3 (GPR41/FFAR3) and GPR43/FFAR2, are expressed in nasal tissue. Primary normal human bronchial epithelial (NHBE) cells were stimulated with different concentrations of SCFAs to test induction of t-PA, which was analysed by expression of mRNA and protein. Mediation of responses by SCFA receptors was evaluated by specific receptor gene silencing with siRNA. RESULTS: Immunohistochemistry study revealed that airway epithelial cells expressed GPR41 and GPR43. Acetic acid, propionic acid, butyric acid and valeric acid significantly induced t-PA expression from two- to tenfolds. The strongest inducer of t-PA from NHBE cells was propionic acid; cells stimulated with propionic acid released t-PA into the supernatant in its active form. Gene silencing of GPR41 and GPR43 revealed that induction of t-PA by SCFAs was dependent upon both GPR41 and GPR43. CONCLUSIONS AND CLINICAL RELEVANCE: Short-chain fatty acids were shown to induce airway epithelial cell expression of t-PA via GPR41 and GPR43. Topical delivery of potent compounds that activate these receptors may have value by reducing fibrin deposition and shrinking nasal polyp growth.
The impact of short-chain fatty acids on GLP-1 and PYY secretion from the isolated perfused rat colon. Charlotte Bayer Christiansen;Maria Buur Nordskov Gabe;Berit Svendsen;Lars Ove Dragsted;Mette Marie Rosenkilde;Jens Juul Holst. 2018. Am J Physiol Gastrointest Liver Physiol. 315. PMID: 29494208

The colonic epithelium harbors a large number of endocrine cells, but little is known about the endocrine functions of the colon. However, the high density of glucagon like peptide-1 (GLP-1)- and peptide-YY (PYY)-secreting L cells is of great interest because of the potential antidiabetic and antiobesity effects of GLP-1 and PYY. Short-chain fatty acids (SCFAs) produced by local bacterial fermentation are suggested to activate the colonic free fatty acid receptors FFAR2 (GPR43) and FFAR3 (GPR41), stimulating the colonic L cells. We used the isolated perfused rat colon as a model of colonic endocrine secretion and studied the effects of the predominant SCFAs formed: acetate, propionate, and butyrate. We show that luminal and especially vascular infusion of acetate and butyrate significantly increases colonic GLP-1 secretion, and to a minor extent also PYY secretion, but only after enhancement of intracellular cAMP. Propionate neither affected GLP-1 nor PYY secretion whether administered luminally or vascularly. A FFAR2- and FFAR3-specific agonist [( S)-2-(4-chlorophenyl)-3,3-dimethyl- N-(5-phenylthiazol-2-yl)butamide (CFMB)/ AR420626 ] had no effect on colonic GLP-1 output, and a FFAR3 antagonist ( AR399519 ) did not decrease the SCFA-induced GLP-1 response. However, the voltage-gated Ca2+-channel blocker nifedipine, the KATP-channel opener diazoxide, and the ATP synthesis inhibitor 2,4-dinitrophenol completely abolished the responses. FFAR2 receptor studies confirmed low-potent partial agonism of acetate, propionate, and butyrate, compared with CFMB, which is a full agonist with ~750-fold higher potency than the SCFAs. In conclusion, SCFAs may increase colonic GLP-1/PYY secretion, but FFAR2/FFAR3 do not seem to be involved. Rather, SCFAs are metabolized and appear to function as a colonocyte energy source. NEW & NOTEWORTHY By the use of in situ isolated perfused rat colon we show that short-chain fatty acids (SCFAs) primarily are used as a colonocyte energy source in the rat, subsequently triggering glucagon like peptide-1 (GLP-1) secretion independent of the free fatty acid receptors FFAR2 and FFAR3. Opposite many previous studies on SCFAs and FFAR2/FFAR3 and GLP-1 secretion, this experimental model allows investigation of the physiological interactions between luminal nutrients and secretion from cells whose function depend critically on their blood supply as well as nerve and paracrine interactions.
A short-chain fatty acid, propionate, enhances the cytotoxic effect of cisplatin by modulating GPR41 signaling pathways in HepG2 cells. Mamiko Kobayashi;Daisuke Mikami;Junsuke Uwada;Takashi Yazawa;Kazuko Kamiyama;Hideki Kimura;Takanobu Taniguchi;Masayuki Iwano. 2018. Oncotarget. 9. PMID: 30140374

Short-chain fatty acids (SCFAs) such as acetate, propionate, and butyrate are generated by microbial fermentation of indigestible fiber by gut flora. SCFAs are ligands of two orphan G protein-coupled receptors, GPR41 and GPR43, that modulate cell proliferation and induce apoptosis. However, it is unclear if SCFAs enhance the effects of chemotherapy in a GPR41- or GPR43-dependent manner. The aim of this study was to investigate whether SCFAs, and particularly propionate, activate GPR41 or GPR43, and thereby enhance the antitumor effects of cisplatin in HepG2 human hepatocellular carcinoma (HCC) cells. The inhibitory effects of propionate and cisplatin on proliferation of HCC cells were determined by MTS assay. Changes in apoptosis rate were analyzed by flow cytometry. The effects of combined propionate and cisplatin on these properties in HCC cells were significantly higher than those of cisplatin alone. With combined treatment, the levels of cleaved caspase-3, active caspase-3 forms, and acetylated histone H3 were enhanced in a GPR41-dependent manner; expression of histone deacetylases (HDAC) 3, 4, 5, 6, 8 proteins was significantly reduced; and induction of TNF-α expression was significantly enhanced. These results suggest that propionate and cisplatin synergistically and significantly induce apoptosis of HepG2 cells by increasing expression of autocrine TNF-α via reduction of HDACs through GPR41 signaling. From clinical and translational perspectives, our data suggest that a combination of propionate with cisplatin may have better therapeutic effects on HCC compared with conventional treatment, and that a selective GPR41 agonist may be a candidate as an adjuvant therapeutic agent for HCC.
Functional selective ATP receptor signaling controlled by the free fatty acid receptor 2 through a novel allosteric modulation mechanism. Simon Lind;André Holdfeldt;Jonas Mårtensson;Martina Sundqvist;Lena Björkman;Huamei Forsman;Claes Dahlgren. 2019. FASEB J. 33. PMID: 30808243

A nonactivating allosteric modulator of free fatty acid receptor 2 (FFA2R, also called GPCR 43) turns both propionate (an orthosteric FFA2R agonist) and ATP (an agonist for the purinergic P2Y2 receptor), into potent activating ligands that trigger an assembly of the superoxide-generating neutrophil NADPH oxidase. The ATP-induced activation requires the participation of FFA2R, and the signaling is biased toward oxidase activation, leaving the ATP-induced rise in intracellular Ca2+ unaffected. No NADPH oxidase activity was induced by ATP when propionate replaced the allosteric modulator. Signaling downstream of propionate-activated FFA2Rs was insensitive to Gαq inhibition, but the crosstalk activation involving both FFA2R and P2Y2R relied on Gαq signaling. The receptor crosstalk, by which allosterically modulated FFA2Rs communicate with P2Y2Rs and generate NADPH oxidase activating signals downstream of Gαq, represent a novel mechanism by which GPCR activities can be regulated from inside the plasma membrane. Further, the finding that an allosteric FFA2R modulator sensitizes not only the response induced by orthosteric FFA2R agonists, but also the response induced by ATP (P2Y2R-specific agonist) and formyl peptide receptor-specific agonists, violates the receptor restriction characteristics normally defining the selectivity of allosteric GPCR modulators.-Lind, S., Holdfeldt, A., Mårtensson, J., Sundqvist, M., Björkman, L., Forsman, H., Dahlgren, C. Functional selective ATP receptor signaling controlled by the free fatty acid receptor 2 through a novel allosteric modulation mechanism.
Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation. Emmanuel Le Poul;Cecile Loison;Sofie Struyf;Jean-Yves Springael;Vincent Lannoy;Marie-Eve Decobecq;Stephane Brezillon;Vincent Dupriez;Gilbert Vassart;Jo Van Damme;Marc Parmentier;Michel Detheux. 2003. J Biol Chem. 278. PMID: 12711604

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 microm) 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.
Roles of short-chain fatty acids receptors, GPR41 and GPR43 on colonic functions. H Tazoe;Y Otomo;I Kaji;R Tanaka;S-I Karaki;A Kuwahara. 2008. J Physiol Pharmacol. 59 Suppl 2. PMID: 18812643

Short chain fatty acids (SCFAs) are the major anions in the large intestine. They are produced by a bacterial fermentation of dietary fiber. SCFAs are known to have a variety of physiological and pathphysiological effects on intestine. However, the mechanisms by which intraluminal SCFAs are sensed are not known. In 2003, two orphan G protein coupled receptors (GPRs), GPR41 and GPR43, have been cloned and demonstrated to be receptors for SCFAs. Thus, we had attempted to make antibodies raised against GPR43 and GPR41 to elucidate the roles of SCFAs on colonic functions. We have also evaluated the effects of SCFAs on colonic motility to define the physiological roles on luminal SCFAs. In rat and human colon, GPR43 protein was detected by Western blot analysis in extracts of whole wall and separated mucosa, but not in muscle plus submucosa extract. By immunohistochemistry, GPR43 immunoreactivity was localized with enteroendocrine cells expressing peptide YY, whereas 5-HT immunoreactive enteroendocrine cells were not immunoreactive for GPR43. GPR41 immunoreactivity was also found in human colon. In functional studies, propionate and butyrate concentration-dependently (10 microM - 10 mM) induced phasic and tonic contractions in rat colonic circular muscle. The propionate-induced phasic contraction was attenuated by atropine, tetrodotoxin and the 5-HT(4) receptor antagonists SB204070. However, acetate did not induce phasic or tonic contractions. Propionate-induced responses were not observed in mucosal free preparations. The present results suggest that the SCFA-induced physiological effects on colonic functions might be attributable to the activation of SCFA receptors on epithelial cells in the colon.
SCFAs induce mouse neutrophil chemotaxis through the GPR43 receptor. Marco A R Vinolo;G John Ferguson;Suhasini Kulkarni;George Damoulakis;Karen Anderson;Mohammad Bohlooly-Y;Len Stephens;Phillip T Hawkins;Rui Curi. 2011. PLoS One. 6. PMID: 21698257

Short chain fatty acids (SCFAs) have recently attracted attention as potential mediators of the effects of gut microbiota on intestinal inflammation. Some of these effects have been suggested to occur through the direct actions of SCFAs on the GPR43 receptor in neutrophils, though the precise role of this receptor in neutrophil activation is still unclear. We show that mouse bone marrow derived neutrophils (BMNs) can chemotax effectively through polycarbonate filters towards a source of acetate, propionate or butyrate. Moreover, we show that BMNs move with good speed and directionality towards a source of propionate in an EZ-Taxiscan chamber coated with fibrinogen. These effects of SCFAs were mimicked by low concentrations of the synthetic GPR43 agonist phenylacetamide-1 and were abolished in GPR43(-/-) BMNs. SCFAs and phenylacetamide-1 also elicited GPR43-dependent activation of PKB, p38 and ERK and these responses were sensitive to pertussis toxin, indicating a role for Gi proteins. Phenylacetamide-1 also elicited rapid and transient activation of Rac1/2 GTPases and phosphorylation of ribosomal protein S6. Genetic and pharmacological intervention identified important roles for PI3Kγ, Rac2, p38 and ERK, but not mTOR, in GPR43-dependent chemotaxis. These results identify GPR43 as a bona fide chemotactic receptor for neutrophils in vitro and start to define important elements in its signal transduction pathways.
Short-chain fatty acids enhance adipocyte differentiation in the stromal vascular fraction of porcine adipose tissue. Genlai Li;Wen Yao;Honglin Jiang. 2014. J Nutr. 144. PMID: 25320182

BACKGROUND: Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are the main products of microbial fermentation in the gut and might mediate some of the effects of gut microbiota and nutrition on development, metabolism, and pathogenesis of obesity and other diseases. OBJECTIVE: The objective of this study was to determine the effects of SCFAs on adipocyte differentiation and the underlying mechanism. METHODS: The stromal vascular fraction (SVF) of the porcine subcutaneous fat was used as the preadipocyte model. Adipocyte differentiation was assessed by Oil Red O staining and gene expression analysis of adipocyte markers. Chromatin immunoprecipitation was used to assess the histone acetylation amounts at the peroxisome proliferator-activated receptor γ (PPARG) and CCAAT/enhancer binding protein α (CEBPA) promoters. RESULTS: Compared with control, propionate and butyrate enhanced the formation of adipocytes by 10-20% and mRNA expression of adipocyte markers by 20-200% in porcine SVF undergoing adipocyte differentiation. Compared with control, short-term treatment of propionate and butyrate enhanced PPARG and CEBPA mRNA expression in porcine SVF by 50-100%. Neither free fatty acid receptor (FFAR) 2 nor FFAR3 mRNA was detectable in porcine SVF before or during differentiation. Neither a cAMP analogue nor an activator of AMP-activated protein kinase (AMPK) affected propionate- or butyrate-enhanced expression of PPARG or CEBPA mRNA. Trichostatin A, a specific inhibitor of histone deacetylases (HDACs), enhanced the formation of adipocytes in porcine SVF by nearly 100% and the expression of PPARG and CEBPA mRNAs by 150% and 50%, respectively. Butyrate increased whereas propionate had no significant effect on histone H3 acetylation at the CEBPA promoter in porcine SVF. CONCLUSIONS: Propionate and butyrate enhance adipocyte differentiation in porcine SVF. These effects are unlikely mediated through FFAR2, FFAR3, cAMP, or AMPK. The effect of butyrate may be partially mediated by its HDAC inhibitory activity, whereas that of propionate is independent of its HDAC inhibitory activity.
Ffar2 expression regulates leukaemic cell growth in vivo. Laure B Bindels;Paolo E Porporato;Sarah Ducastel;Martina Sboarina;Audrey M Neyrinck;Evelyne M Dewulf;Olivier Feron;Sophie Lestavel;Patrice D Cani;Bart Staels;Pierre Sonveaux;Nathalie M Delzenne. 2017. Br J Cancer. 117. PMID: 28873082

BACKGROUND: Activation of free fatty acid receptor 2 (FFAR2) by microbiota-derived metabolites (e.g., propionate) reduces leukaemic cell proliferation in vitro. This study aims to test whether Ffar2 expression per se also influences leukaemia cell growth in vivo. METHODS: Bcr-Abl-expressing BaF cells were used as a leukaemia model and the role of Ffar2 was evaluated in Balb/c mice after lentiviral shRNA transduction. RESULTS: Our data formally establish that reduced leukaemic cell proliferation is associated with increased Ffar2 expression in vivo and in vitro. Going beyond association, we point out that decreasing Ffar2 expression fosters cancer cell growth in vitro and in vivo. CONCLUSIONS: Our data demonstrate the role of Ffar2 in the control of leukaemic cell proliferation in vivo and indicate that a modulation of Ffar2 expression through nutritional tools or pharmacological agents may constitute an attractive therapeutic approach to tackle leukaemia progression in humans.
Signaling of free fatty acid receptors 2 and 3 differs in colonic mucosa following selective agonism or coagonism by luminal propionate. Iain R Tough;Sarah Forbes;Helen M Cox. 2018. Neurogastroenterol Motil. 30. PMID: 30136343

BACKGROUND: Propionate exhibits affinity for free fatty acid receptor 2 (FFA2, formerly GPR43) and FFA3 (GPR41). These two G protein-coupled receptors (GPCRs) are expressed by enteroendocrine L cells that contain anorectic peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), while FFA3 is also expressed by enteric neurons. Few studies have investigated the individual roles of FFA2 and FFA3 in propionate's gastrointestinal (GI) effects. Here, we compared FFA2, FFA3, and propionate mucosal responses utilizing selective ligands including an FFA3 antagonist, in mouse and human colonic mucosa. METHODS: Vectorial ion transport was measured in native colonic preparations from normal mouse and human colon with intact submucosal innervation. Endogenous fecal pellet propulsion was monitored in colons isolated from wild-type (WT) and PYY-/- mice. KEY RESULTS: FFA2 and FFA3 signaling differed significantly. FFA2 agonism involved endogenous L cell-derived PYY and was glucose dependent, while FFA3 agonism was independent of PYY and glucose, but required submucosal enteric neurons for activity. Tonic FFA3 activity was observed in mouse and human colon mucosa. Apical propionate responses were a combination of FFA2-PYY mediation and FFA3 neuronal GLP-1- and CGRP-dependent signaling in mouse ascending colon mucosa. Propionate also slowed WT and PYY-/- colonic transit, and this effect was blocked by a GLP-1 receptor antagonist. CONCLUSIONS & INFERENCES: We conclude that luminal propionate costimulates FFA2 and FFA3 pathways, reducing anion secretion and slowing colonic motility; FFA2 via PYY mediation and FFA3 signaling by activation of enteric sensory neurons.
Butyrate and propionate induced activated or non-activated neutrophil apoptosis via HDAC inhibitor activity but without activating GPR-41/GPR-43 pathways. Michiko Aoyama;Joji Kotani;Makoto Usami. 2009. Nutrition. 26. PMID: 20004081

OBJECTIVE: Decreased neutrophil apoptosis is implicated in persistent inflammation resulting in systemic inflammatory response syndrome and multiple organ dysfunctions syndromes. Short-chain fatty acids (SCFAs) may be a candidate to control neutrophil apoptosis because SCFAs are normally produced in the gut and related products have been approved for human use. We investigated the effects of SCFAs on apoptosis of activated and non-activated neutrophils and their mechanisms. METHODS: Purified neutrophils obtained from healthy volunteers were preincubated for 1 h with or without the G-protein receptor (GPR) inhibitor pertussis toxin (100 ng/mL) or U-73122 (50 ng/mL), extracellular signal-related protein kinase inhibitor PD98059 (10 microM), mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 (25 microM), Jun kinase inhibitor-I (2 microM), caspase-3 and -7 inhibitor Z-VAD-FMK (100 microM), caspase-8 inhibitor Z-IETD-FMK (50 microM), or caspase-9 inhibitor Z-LEHD-FMK (50 microM). The cells were then cultured with or without SCFAs or trichostatin A, a typical histone deacetylase inhibitor, in the presence or absence of lipopolysaccharide (1 microg/mL) or tumor necrosis factor-alpha (100 ng/mL). Neutrophil apoptosis was assessed by annexin V staining using flow cytometry. The GPR-41 and -43 and apoptosis-related proteins (bax, mcl-1, a1) mRNA were measured by quantitative real-time polymerase chain reaction and the expression of acetylated histone H3 was determined by western blot. RESULTS: The caspase inhibitors inhibited butyrate- and propionate-induced neutrophil apoptosis treated or untreated with lipopolysaccharide or tumor necrosis factor-alpha, whereas GPR and MAPK inhibitors had no effect. The mRNA expressions of GPR-43 and a1 protein were reduced by butyrate and propionate. The expressions of acetylated histone H3 were induced by butyrate and propionate. CONCLUSION: These results suggest that butyrate and propionate increase apoptosis of neutrophils irrespective of their activation state, by factors other than GPRs and MAPKs, and their mechanisms likely relate to their histone deacetylase inhibition activity, which may control a1 mRNA expression.
Propionate-induced epithelial K(+) and Cl(-)/HCO3(-) secretion and free fatty acid receptor 2 (FFA2, GPR43) expression in the guinea pig distal colon. Shin-ichiro Karaki;Atsukazu Kuwahara. 2010. Pflugers Arch. 461. PMID: 20945073

Propionate, a fermented product in the lumen of the large intestine, is a short-chain fatty acid (SCFA) known to have a variety of localized physiological and pathophysiological functions (e.g., luminal fluid secretion and anti-inflammatory response). In the present study, we investigated propionate-induced transepithelial ion transport and the expression of SCFA receptor, free fatty acid receptor 2 (FFA2, otherwise known as GPR43) in the guinea pig distal colon utilizing the Ussing chamber technique and immunohistochemistry. The addition of propionate to the luminal bathing solution concentration-dependently induced transient K(+) and Cl(-) and/or bicarbonate secretion within approximately 30 s and long-lasting Cl(-) secretion for approximately 60 min was first identified in the present study. The transient anion secretion was tetrodotoxin (TTX)-sensitive and mediated through the cholinergic (both nicotinic and muscarinic) neural pathway, but the transient K(+) and long-lasting Cl(-) secretion were due to TTX-insensitive mechanism. Immunohistochemistry studies showed that some chromogranin A-immunoreactive enteroendocrine cells were also immunoreactive for FFA2 but not colocalized with 5-hydroxytryptamine. In conclusion, the propionate-induced secretion consisted of the neural and non-neural three-phase secretory manner possibly mediated by the stimulation of FFA2 expressed by enteroendocrine cells.
The G-protein on cholesterol-rich membrane microdomains mediates mucosal sensing of short- chain fatty acid and secretory response in rat colon. T Yajima;R Inoue;M Yajima;T Tsuruta;S Karaki;T Hira;A Kuwahara. 2011. Acta Physiol (Oxf). 203. PMID: 21649864

AIM: Short-chain fatty acids (SCFA) stimulate colonic contraction and secretion, which are mediated by an enteric reflex via a mucosal sensing and cholinergic mechanisms. The involvement of G-protein signal transduction was examined in the secretory response to luminal propionate sensing in rat distal colon. METHODS: Mucosa-submucosa and mucosa preparations were used to measure short-circuit current (I(sc)) and acetylcholine (ACh) release respectively. Cholesterol-rich membrane microdomains, lipid rafts/caveolae, were fractionated using a sucrose gradient ultra-centrifugation after detergent-free extraction of the isolated colonic crypt. RESULTS: Luminal addition of methyl-β-cyclodextrin (10 mm) and mastoparan (30 μm), lipid rafts/caveolae disruptors, significantly inhibited luminal propionate-induced (0.5 mm) increases in I(sc) , but did not affect increases in I(sc) induced by serosal ACh (0.05 mm) or electrical field stimulation (EFS). Luminal addition of YM-254890 (10 μm), a Gα(q/11) -selective inhibitor, markedly inhibited propionate-induced increase in I(sc) , but did not affect I(sc) responses to ACh and EFS. Both methyl-β-cyclodextrin and YM-254890 significantly inhibited luminal propionate-induced non-neuronal release of ACh from colonocytes. Real-time PCR demonstrated that in mRNA expression of SCFA receptors, GPR 43 was far higher than that of GPR41 in the colon. Western blotting analysis revealed that the cholesterol-rich membrane microdomains that fractionated from colonic crypt cells were associated with caveolin-1, flotillin-1 and Gα(q/11) , but not GPR43. Uncoupling of Gα(q/11) from flotillin-1 in lipid rafts occurred under desensitization of the I(sc) response to propionate. CONCLUSIONS: These data demonstrate that the secretory response to luminal propionate in rat colon is mediated by G-protein on cholesterol-rich membrane microdomains, provably via Gα(q/11) .
Effect of short chain fatty acids on the expression of free fatty acid receptor 2 (Ffar2), Ffar3 and early-stage adipogenesis. G Frost;Z Cai;M Raven;D T Otway;R Mushtaq;J D Johnston. 2014. Nutr Diabetes. 4. PMID: 25089883

Adipose tissue has a major influence on insulin sensitivity. Stimulation of free fatty acid receptor 2 (FFAR2) has been proposed to influence adipocyte differentiation. We hypothesised that exposing preadipocytes to short chain fatty acids would induce earlier expression of nuclear receptors that co-ordinate adipogenesis, triglyceride accumulation and leptin secretion. 3T3-L1 preadipocytes were differentiated in the presence of 1 μM acetate, 0.1-10 μM propionate or vehicle control. In experiment 1, expression of Ffar2 and nuclear receptor mRNA was measured by quantitative PCR over 48 h following onset of differentiation. In experiment 2, extracellular leptin concentration and intracellular triglyceride content were measured at days 0, 2, 4, 6, 8 and 10 following the onset of differentiation. Control cells exhibited similar temporal dynamics of gene expression, triglyceride accumulation and leptin secretion as reported previously. We were unable to detect expression of Ffar3 mRNA at any stage of differentiation. Consistent with a lack of Ffar2 expression in the first 24 h of differentiation, acetate and propionate had no significant effect on nuclear receptor expression. Furthermore, acetate or propionate treatment did not alter leptin concentration or triglyceride content. In conclusion, we observed no significant effect of propionate or acetate on adipogenesis in 3T3-L1 cells using validated quantitative techniques.
Short-chain fatty acids, GPR41 and GPR43 ligands, inhibit TNF-α-induced MCP-1 expression by modulating p38 and JNK signaling pathways in human renal cortical epithelial cells. Mamiko Kobayashi;Daisuke Mikami;Hideki Kimura;Kazuko Kamiyama;Yukie Morikawa;Seiji Yokoi;Kenji Kasuno;Naoki Takahashi;Takanobu Taniguchi;Masayuki Iwano. 2017. Biochem Biophys Res Commun. 486. PMID: 28322790

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced predominantly by gut microbiota fermentation of dietary fiber. SCFAs are newly identified as endogenous ligands of two orphan G protein-coupled receptors, GPR41 and GPR43, which have the potential to modulate inflammation. Therefore, GPR41 and GPR43 may mediate the link between the gut microbiome status and various disease conditions including renal inflammation. This study aimed at investigating whether SCFAs activate GPR41 and GPR43, and thereby exert anti-inflammatory effects in human renal cortical epithelial cells (HRCEs) as a main component of kidney tissue. Immunohistochemical analyses of human renal biopsy specimens revealed the expression of GPR41 and GPR43 protein in the distal renal tubules and collecting tubules. TNF-α increased the expression of monocyte chemoattractant protein-1 (MCP-1), a potential fibrotic inducer, at least partly via enhancing phosphorylation of p38 and JNK in HRCEs. SCFAs, especially propionate, attenuated TNF-α- stimulated MCP-1 expression by inhibiting the phosphorylation of p38 and JNK. This inhibitory effect was considerably attenuated by an inactivator of the Gi/o-type G protein and a Gβγ (i/o) blocker, but not by a Gα (i/o) blocker. Furthermore, SCFA-mediated inhibition of MCP-1 expression was significantly blocked by siRNA-induced gene silencing of GPR41 and GPR43. In conclusion, SCFAs lowered TNF-α-induced MCP-1 expression by reducing phosphorylation of p38 and JNK in a GPR41/43-dependent manner in HRCEs, suggesting that SCFA modification may be a new therapeutic tool for preventing progression of renal inflammation and fibrosis.
Calcium propionate supplementation improves development of rumen epithelium in calves via stimulating G protein-coupled receptors. X Z Zhang;W B Chen;X Wu;Y W Zhang;Y M Jiang;Q X Meng;Z M Zhou. 2018. Animal. 12. PMID: 29477151

In the present study, calcium propionate (CaP) was used as feed additive in the diet of calves to investigate their effects on rumen fermentation and the development of rumen epithelium in calves. To elucidate the mechanism in which CaP improves development of calf rumen epithelium via stimulating the messenger RNA (mRNA) expression of G protein-coupled receptors, a total of 54 male Jersey calves (age=7±1 days, BW=23.1±1.2 kg) were randomly divided into three treatment groups: control without CaP supplementation (Con), 5% CaP supplementation (5% CaP) and 10% CaP supplementation (10% CaP). The experiment lasted 160 days and was divided into three feeding stages: Stage 1 (days 0 to 30), Stage 2 (days 31 to 90) and Stage 3 (days 91 to 160). Calcium propionate supplementation percentages were calculated on a dry matter basis. In total, six calves from each group were randomly selected and slaughtered on days 30, 90 and 160 at the conclusion of each experimental feeding stage. Rumen fermentation was improved with increasing concentration of CaP supplementation in calves through the first 30 days (Stage 1). No effects of CaP supplementation were observed on rumen fermentation in calves during Stage 2 (days 31 to 90). Supplementation with 5% CaP increased propionate concentration, but not acetate and butyrate in calves during Stage 3 (days 91 to 160). The rumen papillae length of calves in the 5% CaP supplementation group was greater than that of Con groups in calves after 160 days feeding. The mRNA expression of G protein-coupled receptor 41 (GPR41) and GPR43 supplemented with 5% CaP were greater than the control group and 10% CaP group in feeding 160 days calves. 5% CaP supplementation increased the mRNA expression of cyclin D1, whereas did not increase the mRNA expression of cyclin-dependent kinase 4 compared with the control group in feeding 160-day calves. These results indicate that propionate may act as a signaling molecule to improve rumen epithelium development through stimulating mRNA expression of GPR41 and GPR43.
The Anti-inflammatory Effects of Short Chain Fatty Acids on Lipopolysaccharide- or Tumor Necrosis Factor α-Stimulated Endothelial Cells via Activation of GPR41/43 and Inhibition of HDACs. Meng Li;Betty C A M van Esch;Paul A J Henricks;Gert Folkerts;Johan Garssen. 2018. Front Pharmacol. 9. PMID: 29875665

Background and Aim: Previously, we found that short chain fatty acids (SCFA) inhibit LPS or TNFα-induced endothelial inflammatory responses and excessive vascular cell adhesion molecule-1 (VCAM-1) expression, two important steps in the development of atherosclerosis. However, the mechanisms involved are still unclear. We hypothesized that the effects of SCFA are associated with activation of G-protein coupled receptor 41/43 (GPR41/43) and/or inhibition of histone deacetylases (HDACs). Methods: The expression and location of GPR41/43 and HDAC3 in human umbilical vein endothelial cells (HUVEC) were confirmed. HUVEC were pre-incubated with acetate, butyrate or propionate alone or in combination with GLPG0974 (GLPG, antagonist of GPR43) or β-hydroxybutyrate (SHB, antagonist of GPR41) and then exposed to LPS or TNFα. Interleukin (IL)-6 and IL-8 levels and VCAM-1 expression were measured. HDAC activity was measured after treatment with butyrate, propionate and trichostatin A (TSA, HDAC inhibitor). The peripheral blood mononuclear cell (PBMC) adhesive level was also determined after TSA treatment. Results: GPR41/43 were expressed on the membrane of HUVEC and HDAC3 was located in cytoplasm and nucleus. The GLPG and/or SHB treatments restored the inhibitory effects of acetate on IL-6 and IL-8 production and the inhibitory effects of butyrate or propionate on IL-6 production, but not on IL-8. In contrast, GLPG and/or SHB treatments did not affect the inhibitory effects of butyrate or propionate on TNFα-induced VCAM-1 expression. TSA showed similar effects on IL-8 production and VCAM-1 expression as butyrate and propionate. In addition, TSA significantly inhibited the adhesion of PBMC to an endothelial monolayer. Conclusion: Activation of GPR41/43 mediates the effects of acetate on IL-6 and IL-8 production and the effects of butyrate and propionate on IL-6 production. Furthermore, inhibition of HDACs mediates the effects of butyrate and propionate on IL-8 production, VCAM-1 expression, and PBMC adhesion to an endothelial monolayer. These data indicate the beneficial roles of SCFA in preventing vascular inflammation and relevant diseases by activation of GPR41/43 and inhibition of HDACs.
Short chain fatty acids stimulate insulin secretion and reduce apoptosis in mouse and human islets in vitro: Role of free fatty acid receptor 2. Attilio Pingitore;Noemi Gonzalez-Abuin;Inmaculada Ruz-Maldonado;Guo C Huang;Gary Frost;Shanta J Persaud. 2018. Diabetes Obes Metab. 21. PMID: 30203438

AIMS: To evaluate the role of free fatty acid receptor 2 (FFAR2)/G-protein coupled receptor 43 in mediating the effects of the short chain fatty acids (SCFAs) sodium acetate (SA) and sodium propionate (SP) on islet function in vitro, and to identify the intracellular signalling pathways used in SCFA-induced potentiation of glucose-induced insulin secretion. MATERIALS AND METHODS: Islets of Langerhans were isolated from wild-type and FFAR2-/- mice and from human donors without diabetes. The effects of SA and SP on dynamic insulin secretion from perifused islets were quantified by radioimmunoassay, signalling downstream of SCFAs was profiled by single-cell calcium microfluorimetry, and measurement of cAMP was performed using a fluorescence assay. Islet apoptosis was induced by exposure to cytokines or sodium palmitate, and the effects of SA and SP in regulating islet apoptosis were assessed by quantification of caspase 3/7 activities. RESULTS: Deletion of FFAR2 did not affect islet morphology or insulin content. SA and SP reversibly potentiated insulin secretion from mouse islets in a FFAR2-dependent manner. SCFA-induced potentiation of insulin secretion was coupled to Gq activation of phospholipase C and protein kinase C, with no evidence of Gi-mediated signalling. SA and SP protected human and mouse islets from apoptosis, and these pro-survival properties were dependent on islet expression of FFAR2. CONCLUSION: Our results indicate that FFAR2 directly mediates both the stimulatory effects of SA and SP on insulin secretion and their protection against islet apoptosis. We have also shown that SCFA coupling in islets occurs via Gq-coupled intracellular signalling.
Interdependent allosteric free fatty acid receptor 2 modulators synergistically induce functional selective activation and desensitization in neutrophils. Simon Lind;André Holdfeldt;Jonas Mårtensson;Martina Sundqvist;Terry P Kenakin;Lena Björkman;Huamei Forsman;Claes Dahlgren. 2020. Biochim Biophys Acta Mol Cell Res. 1867. PMID: 32092308

The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2Y2R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFAR2 when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free calcium ions (Ca2+). Furthermore, following AZ1729/Cmp58 activation, the signaling by the desensitized FFAR2s was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca2+. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFAR2 modulators represent a new regulatory mechanism that controls receptor signaling.
Gut microbiota-derived propionate reduces cancer cell proliferation in the liver. L B Bindels;P Porporato;E M Dewulf;J Verrax;A M Neyrinck;J C Martin;K P Scott;P Buc Calderon;O Feron;G G Muccioli;P Sonveaux;P D Cani;N M Delzenne. 2012. Br J Cancer. 107. PMID: 22976799

BACKGROUND: Metabolites released by the gut microbiota may influence host metabolism and immunity. We have tested the hypothesis that inulin-type fructans (ITF), by promoting microbial production of short-chain fatty acids (SCFA), influence cancer cell proliferation outside the gut. METHODS: Mice transplanted with Bcr-Abl-transfected BaF3 cells, received ITF in their drinking water. Gut microbiota was analysed by 16S rDNA polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) and qPCR. Serum Short-chain fatty acids were quantified by UHPLC-MS. Cell proliferation was evaluated in vivo, by molecular biology and histology, and in vitro. RESULTS: Inulin-type fructans treatment reduces hepatic BaF3 cell infiltration, lessens inflammation and increases portal propionate concentration. In vitro, propionate reduces BaF3 cell growth through a cAMP level-dependent pathway. Furthermore, the activation of free fatty acid receptor 2 (FFA2), a Gi/Gq-protein-coupled receptor also known as GPR43 and that binds propionate, lessens the proliferation of BaF3 and other human cancer cell lines. CONCLUSION: We show for the first time that the fermentation of nutrients such as ITF into propionate can counteract malignant cell proliferation in the liver tissue. Our results support the interest of FFA2 activation as a new strategy for cancer therapeutics. This study highlights the importance of research focusing on gut microbes-host interactions for managing systemic and severe diseases such as leukaemia.
The SCFA Receptor GPR43 and Energy Metabolism. Ikuo Kimura;Daisuke Inoue;Kanako Hirano;Gozoh Tsujimoto. 2014. Front Endocrinol (Lausanne). 5. PMID: 24926285

Free fatty acids (FFAs) are essential nutrients and act as signaling molecules in various cellular processes via binding with FFA receptors. Of these receptors, GPR43 is activated by short-chain fatty acids (SCFAs; e.g., acetate, propionate, and butyrate). During feeding, SCFAs are produced by microbial fermentation of dietary fiber in the gut, and these SCFAs become important energy sources for the host. The gut microbiota affects nutrient acquisition and energy regulation of the host and can influence the development of obesity, insulin resistance, and diabetes. Recently, GPR43 has been reported to regulate host energy homeostasis in the gastrointestinal tract and adipose tissues. Hence, GPR43 is also thought to be a potential drug target for metabolic disorders, such as obesity and diabetes. In this review, we summarize the identification, structure, and activities of GPR43, with a focus on host energy regulation, and present an essential overview of our current understanding of its physiological roles in host energy regulation that is mediated by gut microbiota. We also discuss the potential for GPR43 as a therapeutic target.
Role of Free Fatty Acid Receptor 2 (FFAR2) in the Regulation of Metabolic Homeostasis. Sameer Mohammad. 2015. Curr Drug Targets. 16. PMID: 25850624

Besides being an important source of fuel and structural components of biological membranes, free fatty acids (FFAs) are known to display a wide variety of roles that include modulation of receptor signaling and regulation of gene expression among many. FFAs play a significant role in maintaining metabolic homeostasis by activating specific G-Protein Coupled Receptors (GPCRs) in pancreatic β cells, immune cells, white adipose tissue, intestine and several other tissues. Free Fatty acid receptor 2 (FFAR2) also known as GPR43 belongs to this group of GPCRs and has been shown to participate in a number of important biological activities. FFAR2 is activated by short-chain fatty acids (SCFAs) such as acetate, propionate and butyrate. SCFAs are formed in the distal gut by bacterial fermentation of macro-fibrous material that escapes digestion in the upper gastrointestinal tract and enters the colon and have been shown to play vital role in the immune regulation and metabolic homeostasis. FFAR2 and other free fatty acid receptors are considered key components of the body's nutrient sensing mechanism and targeting these receptors is assumed to offer novel therapies for the management of diabetes and other metabolic disorders. This review aims to summarize the current state of our understanding of FFAR2 biology with a particular focus on its role in metabolic homeostasis.
Function and clinical implications of short-chain fatty acids in patients with mixed refractory constipation. Y Shi;Q Chen;Y Huang;L Ni;J Liu;J Jiang;N Li. 2016. Colorectal Dis. 18. PMID: 26921846

AIM: The present study was designed to investigate the function and clinical implications of stool short-chain fatty acids (SCFAs) in patients with mixed refractory constipation. METHOD: Ascending colon specimens obtained from 30 patients with ascending colon cancer were regarded as the control group. Ascending colon specimens obtained from patients with mixed refractory constipation were regarded as the experimental group and were divided into three subgroups, according to Wexner scores [A constipation scoring system to simplify evaluation and management of constipated patients. Dis Colon Rectum 1996; 39: 681-5] of 16-20, 21-25 and 26-30, with 30 patients in each group. The stool SCFAs were extracted and quantitatively analysed using gas chromatography-mass spectrometry (GC-MS). The expression of G protein-coupled receptor 43 (GPR43) and of choline acetyltransferase (ChAT) were detected by immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR) and western blotting of colon samples. RESULTS: The levels of acetate, propionate and butyrate were significantly lower in the experimental group than in the control group (P < 0.05). Compared with the control group, the densitometric quantification and mean density of GPR43 and ChAT proteins, and expression of GPR43 and CHAT genes, were significantly decreased in the patients with mixed refractory constipation (P < 0.05). CONCLUSION: In the patients with mixed refractory constipation, the levels of stool SCFAs, including acetate, propionate and butyrate, as well as the levels of GPR43 and ChAT expressed in the colon, which were all negatively correlated with the Wexner score, were decreased and may be associated with the pathogenesis of mixed refractory constipation.
Gut microbiota derived propionate regulates the expression of Reg3 mucosal lectins and ameliorates experimental colitis in mice. Danica Bajic;Adrian Niemann;Anna-Katharina Hillmer;Raquel Mejias-Luque;Sena Bluemel;Melissa Docampo;Maja C Funk;Elena Tonin;Michael Boutros;Bernd Schnabl;Dirk H Busch;Tsuyoshi Miki;Roland M Schmid;Marcel R M van den Brink;Markus Gerhard;Christoph K Stein-Thoeringer. None. J Crohns Colitis. . PMID: 32227170

BACKGROUND AND AIMS: Reg3 lectins are antimicrobial peptides at mucosal surfaces of the gut, whose expression is regulated by pathogenic gut microbes via IL-22- or TLR signaling. In addition to antimicrobial effects, tissue protection is hypothesized, but poorly investigated in the gut. METHODS: We applied antibiotic-induced microbiota perturbations, gnotobiotic approaches and a dextran-sodium sulfate (DSS) colitis model to assess microbial Reg3 regulation in the intestines and its role in colitis. We also used an intestinal organoid model to investigate this axis in vitro. RESULTS: First, we studied whether gut commensals are involved in Reg3 expression in mice, and found that antibiotic-mediated reduction of Clostridia downregulated intestinal Reg3B. A loss in Clostridia was accompanied by a significant reduction of short chain fatty acids (SCFA), and knock-out (KO) mice for SCFA receptors GPR43 and GPR109 expressed less intestinal Reg3B/-G. Propionate was found to induce Reg3 in intestinal organoids and in gnotobiotic mice colonized with a defined, SCFA producing microbiota. Investigating the role of Reg3B as a protective factor in colitis, we found that Reg3B-KO mice display increased inflammation and less crypt proliferation in the DSS colitis model. Propionate decreased colitis and increased proliferation. Treatment of organoids exposed to DSS with Reg3B or propionate reversed the chemical injury with a loss of expression of the stem-cell marker Lgr5 and Olfm4. CONCLUSIONS: Our results suggest that Clostridia can regulate Reg3-associated epithelial homeostasis through propionate signaling. We also provide evidence that the Reg3-propionate axis may be an important mediator of gut epithelial regeneration in colitis.
Protective Effects of Short-Chain Fatty Acids on Endothelial Dysfunction Induced by Angiotensin II. Iñaki Robles-Vera;Marta Toral;Néstor de la Visitación;Nazaret Aguilera-Sánchez;Juan Miguel Redondo;Juan Duarte. 2020. Front Physiol. 11. PMID: 32372967

Short-chain fatty acids (SCFAs) are among the main classes of bacterial metabolic products and are mainly synthesized in the colon through bacterial fermentation. Short-chain fatty acids, such as acetate, butyrate, and propionate, reduce endothelial activation induced by proinflammatory mediators, at least in part, by activation of G protein-coupled receptors (GPRs): GPR41 and GPR43. The objective of the study was to analyze the possible protective effects of SCFAs on endothelial dysfunction induced by angiotensin II (AngII). Rat aortic endothelial cells (RAECs) and rat aortas were incubated with AngII (1 μM) for 6 h in the presence or absence of SCFAs (5-10 mM). In RAECs, we found that AngII reduces the production of nitric oxide (NO) stimulated by calcium ionophore A23187; increases the production of reactive oxygen species (ROS), both from the nicotinamide adenine dinucleotide phosphate oxidase system and the mitochondria; diminishes vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser239; reduces GPR41 and GPR43 mRNA level; and reduces the endothelium-dependent relaxant response to acetylcholine in aorta. Coincubation with butyrate and acetate, but not with propionate, increases both NO production and pSer239-VASP, reduces the concentration of intracellular ROS, and improves relaxation to acetylcholine. The beneficial effects of butyrate were inhibited by the GPR41 receptor antagonist, β-hydroxybutyrate, and by the GPR43 receptor antagonist, GLPG0794. Butyrate inhibited the down-regulation of GPR41 and GPR43 induced by AngII, being without effect acetate and propionate. Neither β-hydroxybutyrate nor GLPG0794 affects the protective effect of acetate in endothelial dysfunction. In conclusion, acetate and butyrate improve endothelial dysfunction induced by AngII by increasing the bioavailability of NO. The effect of butyrate seems to be related to GPR41/43 activation, whereas acetate effects were independent of GPR41/43.
Molecular link between dietary fibre, gut microbiota and health. Jitendra Kumar;Kavita Rani;Chander Datt. 2020. Mol Biol Rep. . PMID: 32623619

Natural polysaccharides cellulose, hemicelluloses, inulin etc., galactooligosaccharides (GOS), and fructooligosaccharides (FOS) play a significant role in the improvement of gut microbiota balance and human health. These polysaccharides prevent pathogen adhesion that stimulates the immune system and gut barrier function by servicing as fermentable substrates for the gut microbiota. The gut microbiota plays a key role in the fermentation of non-digestible carbohydrates (NDCs) fibres. Moreover, the gut microbiota is responsible for the production of short-chain fatty acids (SCFAs) like acetate, propionate and butyrate. Acetate is the most abundant and it is used by many gut commensals to produce propionate and butyrate in a growth-promoting cross-feeding process. The dietary fibres affect the gut microbiome and play vital roles in signaling pathways. The SCFAs, acetate, butyrate, and propionate have been reported to affect on metabolic activities at the molecular level. Acetate affects the metabolic pathway through the G protein-coupled receptor (GPCR) and free fatty acid receptor 2 (FFAR2/GPR43) while butyrate and propionate transactivate the peroxisome proliferator-activated receptorsγ (PPARγ/NR1C3) and regulate the PPARγ target gene Angptl4 in colonic cells of the gut. The FFAR2 signaling pathway regulates the insulin-stimulated lipid accumulation in adipocytes and inflammation, however peptide tyrosine-tyrosine (PPY) and glucagon-like peptide 1 regulates appetite. The NDCs via gut microbiota dependent pathway regulate glucose homeostasis, gut integrity and hormone by GPCR, NF-kB, and AMPK-dependent processes.
Roles of GPR41 and GPR43 in leptin secretory responses of murine adipocytes to short chain fatty acids. Mohamed S Zaibi;Claire J Stocker;Jacqueline O'Dowd;Alison Davies;Mohamed Bellahcene;Michael A Cawthorne;Alastair J H Brown;David M Smith;Jonathan R S Arch. 2010. FEBS Lett. 584. PMID: 20399779

GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Galpha(i). Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Galpha(i) signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Galpha(i) signalling mediated by GPR43 in SCFA-stimulated leptin secretion.
Microbial metabolite sensor GPR43 controls severity of experimental GVHD. Hideaki Fujiwara;Melissa D Docampo;Mary Riwes;Daniel Peltier;Tomomi Toubai;Israel Henig;S Julia Wu;Stephanie Kim;Austin Taylor;Stuart Brabbs;Chen Liu;Cynthia Zajac;Katherine Oravecz-Wilson;Yaping Sun;Gabriel Núñez;John E Levine;Marcel R M van den Brink;James L M Ferrara;Pavan Reddy. 2018. Nat Commun. 9. PMID: 30201970

Microbiome-derived metabolites influence intestinal homeostasis and regulate graft-versus-host disease (GVHD), but the molecular mechanisms remain unknown. Here we show the metabolite sensor G-protein-coupled receptor 43 (GPR43) is important for attenuation of gastrointestinal GVHD in multiple clinically relevant murine models. GPR43 is critical for the protective effects of short-chain fatty acids (SCFAs), butyrate and propionate. Increased severity of GVHD in the absence of GPR43 is not due to baseline differences in the endogenous microbiota of the hosts. We confirm the ability of microbiome-derived metabolites to reduce GVHD by several methods, including co-housing, antibiotic treatment, and administration of exogenous SCFAs. The GVHD protective effect of SCFAs requires GPR43-mediated ERK phosphorylation and activation of the NLRP3 inflammasome in non-hematopoietic target tissues of the host. These data provide insight into mechanisms of microbial metabolite-mediated protection of target tissues from the damage caused allogeneic T cells.
Short-chain fatty acids and regulation of pancreatic endocrine secretion in mice. Anne Ørgaard;Sara Lind Jepsen;Jens Juul Holst. 2019. Islets. 11. PMID: 31469342

The intestinal microbiota has been demonstrated to influence host metabolism, and has been proposed to affect the development of obesity and type 2 diabetes (T2D), possibly through short-chain fatty acids (SCFAs) produced by fermentation of dietary fiber. There are some indications that SCFAs inhibit glucose-stimulated insulin secretion (GSIS) in rodents, but research on this subject is sparse. However, it has been reported that receptors for SCFAs, free fatty acid receptor 2 (FFAR2) and FFAR3 are expressed not only on gut endocrine cells secreting GLP-1 and PYY, but also on pancreatic islet cells. We hypothesized that SCFAs might influence the endocrine secretion from pancreatic islets similar to their effects on the enteroendocrine cells. We studied this using isolated perfused mouse pancreas which responded adequately to changes in glucose and to infusions of arginine. None of the SCFAs, acetate, propionate and butyrate, influenced glucagon secretion, whereas they had weak inhibitory effects on somatostatin and insulin secretion. Infusions of two specific agonists of FFAR2 and FFAR3, CFMB and Compound 4, respectively, did not influence the pancreatic secretion of insulin and glucagon, whereas both induced strong increases in the secretion of somatostatin. In conclusion, the small effects of acetate, propionate and butyrate we observed here may not be physiologically relevant, but the effects of CFMB and Compound 4 on somatostatin secretion suggest that it may be possible to manipulate pancreatic secretion pharmacologically with agonists of the FFAR2 and 3 receptors, a finding which deserves further investigation.
Short-chain fatty acids induce acute phosphorylation of the p38 mitogen-activated protein kinase/heat shock protein 27 pathway via GPR43 in the MCF-7 human breast cancer cell line. Tomo Yonezawa;Yosuke Kobayashi;Yoshiaki Obara. 2006. Cell Signal. 19. PMID: 16887331

The expression of GPR41 and 43, which have recently been identified as G-protein-coupled cell-surface receptors for short-chain fatty acids (SCFAs), was detected in a human breast cancer cell line (MCF-7) by RT-PCR. Acetate, propionate and butyrate induced an increase in intracellular Ca2+ in these cells that was not blocked by treatment with pertussis toxin (PTX). SCFAs significantly reduced forskolin-induced cAMP levels in these cells. The phosphorylation of mitogen-activated protein kinase (MAPK) p38 was selectively increased by SCFAs. The downstream substrate heat shock protein 27 (HSP27) was also phosphorylated by SCFAs at Ser-78 and-82, but not-15. Propionate induced elevations in intracellular Ca2+ and the phosphorylation of p38 were inhibited by the silencing of GPR43 using a specific siRNA. These results suggest that GPR41 and 43 mediate SCFA signaling in mammary epithelial cells and thereby play an important role in their stress management.
The relationship between the effects of short-chain fatty acids on intestinal motility in vitro and GPR43 receptor activation. N B Dass;A K John;A K Bassil;C W Crumbley;W R Shehee;F P Maurio;G B T Moore;C M Taylor;G J Sanger. 2006. Neurogastroenterol Motil. 19. PMID: 17187590

The G protein-coupled receptors, GPR41 and GPR43, are activated by short-chain fatty acids (SCFAs), with distinct rank order potencies. This study investigated the possibility that SCFAs modulate intestinal motility via these receptors. Luminal SCFA concentrations within the rat intestine were greatest in the caecum (c. 115 mmol L(-1)) and proximal colon. Using similar concentrations (0.1-100 mmol L(-1)), SCFAs were found to inhibit electrically evoked, neuronally mediated contractions of rat distal colon, possibly via a prejunctional site of action; this activity was independent of the presence or absence of the mucosa. By contrast, SCFAs reduced the amplitude but also reduced the threshold and increased the frequency of peristaltic contractions in guinea-pig terminal ileum. In each model, the rank-order of activity was acetate (C2) approximately propionate (C3) approximately butyrate (C4) > pentanoate (C5) approximately formate (C1), consistent with activity at the GPR43 receptor. GPR43 mRNA was expressed throughout the rat gut, with highest levels in the colon. However, the ability of SCFAs to inhibit neuronally mediated contractions of the colon was similar in tissues from wild-type and GPR43 gene knockout mice, with identical rank-orders of potency. In conclusion, SCFAs can modulate intestinal motility, but these effects can be independent of the GPR43 receptor.
[Effect of dietary fiber in the quantitative expression of butyrate receptor GPR43 in rats colon]. L Y Corte Osorio;H E Martínez Flores;R Ortiz Alvarado. 2011. Nutr Hosp. 26. PMID: 22072352

INTRODUCTION: Short chain fatty acids (SCFA) acetate, propionate and butyrate are the major anions produced by the bacterial fermentation of dietary fiber (DF) in colon. Recently, butyrate has been recently studied because is important to maintain colonic functions and because it has been related with a protective effect in colorectal cancer, which is mainly, explained by its potential to regulate gene expression by inhibiting enzyme histonedeacetylase (HDAC). Several investigationsshown that SCFAreceptor GPR43 is involved insignal transduction mechanisms once they bind to ligands such as butyrate to generate different physiological effects in colonocytes. OBJECTIVE: Determine if dietary fiber consumption from nopal (Opuntia ficus I.) containing a ratio of soluble-insoluble fiber 40/60, has a direct influence on the quantitative expression of butyrate-specific receptor GPR43. METHODS: Wistar rats were fed with four different diets formulated at different concentrations of dietary fiber of 0, 5, 15 and 25% of dietary fiber from opuntia, respectively. RESULTS AND DISCUSSION: The results shown an increase in the expression of GPR43 (93.1%) when rats was fed with a 5% fiber diet, using β-actin as a reference gene. The results of this investigation will contribute to determinate the relation of diet with intestinal health for the purpose of expanding the knowledge of butyric acid on colonic functions.
Differential modulation by Akkermansia muciniphila and Faecalibacterium prausnitzii of host peripheral lipid metabolism and histone acetylation in mouse gut organoids. Sabina Lukovac;Clara Belzer;Linette Pellis;Bart J Keijser;Willem M de Vos;Roy C Montijn;Guus Roeselers. 2014. mBio. 5. PMID: 25118238

UNLABELLED: The gut microbiota is essential for numerous aspects of human health. However, the underlying mechanisms of many host-microbiota interactions remain unclear. The aim of this study was to characterize effects of the microbiota on host epithelium using a novel ex vivo model based on mouse ileal organoids. We have explored the transcriptional response of organoids upon exposure to short-chain fatty acids (SCFAs) and products generated by two abundant microbiota constituents, Akkermansia muciniphila and Faecalibacterium prausnitzii. We observed that A. muciniphila metabolites affect various transcription factors and genes involved in cellular lipid metabolism and growth, supporting previous in vivo findings. Contrastingly, F. prausnitzii products exerted only weak effects on host transcription. Additionally, A. muciniphila and its metabolite propionate modulated expression of Fiaf, Gpr43, histone deacetylases (HDACs), and peroxisome proliferator-activated receptor gamma (Pparγ), important regulators of transcription factor regulation, cell cycle control, lipolysis, and satiety. This work illustrates that specific bacteria and their metabolites differentially modulate epithelial transcription in mouse organoids. We demonstrate that intestinal organoids provide a novel and powerful ex vivo model for host-microbiome interaction studies. IMPORTANCE: We investigated the influence of the gut microbiota and microbially produced short-chain fatty acids (SCFAs) on gut functioning. Many commensal bacteria in the gut produce SCFAs, particularly butyrate, acetate, and propionate, which have been demonstrated to reduce the risk of gastrointestinal disorders. Organoids-small crypt-villus structures grown from ileal intestinal stem cells-were exposed to SCFAs and two specific gut bacteria. Akkermansia muciniphila, found in the intestinal mucus, was recently shown to have a favorable effect on the disrupted metabolism associated with obesity. Faecalibacterium prausnitzii is a commensal gut bacterium, the absence of which may be associated with Crohn's disease. We showed that in our model, A. muciniphila induces stronger effects on the host than F. prausnitzii. We observed that A. muciniphila and propionate affect the expression of genes involved in host lipid metabolism and epigenetic activation or silencing of gene expression. We demonstrated that organoids provide a powerful tool for host-microbe interaction studies.
[Host energy regulation via SCFAs receptors, as dietary nutrition sensors, by gut microbiota]. Ikuo Kimura. 2014. Yakugaku Zasshi. 134. PMID: 25274213

Food intake regulates energy balance, and its dysregulation leads to metabolic disorders such as obesity and diabetes. Free fatty acids are not only essential nutrients but also act as signaling molecules in various cellular processes. Recent studies have shown that the receptors GPR40, GPR41, GPR43, and GPR120 are new drug targets for treating metabolic disorders because they are activated by free fatty acids. Two of these receptors, GPR41 and GPR43, are activated by short-chain fatty acids (SCFAs: acetate, propionate, and butyrate), which are important energy sources for the host. During feeding, SCFAs are produced by the microbial fermentation of dietary fiber in the gut. The gut microbiota affect nutrient acquisition and energy regulation of the host, and can influence the development of obesity, insulin resistance, and diabetes. Hence, GPR41 and GPR43 are also a focus of research into energy regulation via SCFAs. We report that these SCFA receptors are involved in energy homeostasis: GPR41 regulates sympathetic activity, and GPR43 regulates adipose-insulin signaling by sensing SCFAs produced by gut microbiota. We believe that these results will provide valuable insight into therapeutic targets for treating metabolic disorders and diabetes, as well as in the use of probiotics to control gut microbiota.
Expression of metabolic sensing receptors in adipose tissues of periparturient dairy cows with differing extent of negative energy balance. P Friedrichs;H Sauerwein;K Huber;L F Locher;J Rehage;U Meyer;S Dänicke;B Kuhla;M Mielenz. 2015. Animal. 10. PMID: 26556304

We recently showed that the mRNA expression of genes encoding for specific nutrient sensing receptors, namely the free fatty acid receptors (FFAR) 1, 2, 3, and the hydroxycarboxylic acid receptor (HCAR) 2, undergo characteristic changes during the transition from late pregnancy to lactation in certain adipose tissues (AT) of dairy cows. We hypothesised that divergent energy intake achieved by feeding diets with either high or low portions of concentrate (60% v. 30% concentrate on a dry matter basis) will alter the mRNA expression of FFAR 1, 2, 3, as well as HCAR2 in subcutaneous (SCAT) and retroperitoneal AT (RPAT) of dairy cows in the first 3 weeks postpartum (p.p.). For this purpose, 20 multiparous German Holstein cows were allocated to either the high concentrate ration (HC, n=10) or the low concentrate ration (LC, n=10) from day 1 to 21 p.p. Serum samples and biopsies of SCAT (tail head) and RPAT (above the peritoneum) were obtained at day -21, 1 and 21 relative to parturition. The mRNA abundances were measured by quantitative PCR. The concentrations of short-chain fatty acid (SCFA) in serum were measured by gas chromatography-flame ionisation detector. The FFAR1 and FFAR2 mRNA abundance in RPAT was higher at day -21 compared to day 1. At day 21 p.p. the FFAR2 mRNA abundance was 2.5-fold higher in RPAT of the LC animals compared to the HC cows. The FFAR3 mRNA abundance tended to lower values in SCAT of the LC group at day 21. The HCAR2 mRNA abundance was neither affected by time nor by feeding in both AT. On day 21 p.p. the HC group had 1.7-fold greater serum concentrations of propionic acid and lower concentrations of acetic acid (trend: 1.2-fold lower) compared with the LC group. Positive correlations between the mRNA abundance of HCAR2 and peroxisome proliferator-activated receptor γ-2 (PPARG2) indicate a link between HCAR2 and PPARG2 in both AT. We observed an inverse regulation of FFAR2 and FFAR3 expression over time and both receptors also showed an inverse mRNA abundance as induced by different portions of concentrate. Thus, indicating divergent nutrient sensing of both receptors in AT during the transition period. We propose that the different manifestation of negative EB in both groups at day 21 after parturition affect at least FFAR2 expression in RPAT.
Expression of short-chain fatty acid receptor GPR41 in the human colon. Hideaki Tazoe;Yasuko Otomo;Shin-Ichiro Karaki;Ikuo Kato;Yasuyuki Fukami;Masaki Terasaki;Atsukazu Kuwahara. 2009. Biomed Res. 30. PMID: 19574715

Short-chain fatty acids (SCFAs), including acetate, propionate and butyrate, are the most commonly found anions found in the monogastric mammalian large intestine, and are known to have a variety of physiological and pathophysiological effects on the gastrointestinal tract. We investigated the protein and mRNA expression levels of GPR41, a possible G protein coupled receptor for SCFA, using Western blot analysis and reverse transcriptase-polymerase chain reaction. We found that GPR41 protein and mRNA are expressed in human colonic mucosa. Immunohistochemistry for GPR41 showed that mucosal GPR41 protein is localized in cytoplasm of enterocytes and enteroendocrine cells. Moreover, GPR41-immunoreactive endocrine cells contained peptide YY but not serotonin or GPR43. The cellular population of GPR41 (0.01 +/- 0.01 cells/crypt) was much smaller than that of GPR43 (0.33 +/- 0.01 cells/crypt) in the human colon. However, the potency order of SCFA-induced phasic contraction of colonic smooth muscle that we previously reported is consistent with GPR41 (propionate >or= butyrate > acetate) but not GPR43 (propionate = butyrate = acetate). Therefore, the present study suggests that GPR41 expressed in human colonic mucosa may function as a sensor for luminal SCFAs.