GPR41/FFAR3 and GPR43/FFAR2 as Cosensors for Short-Chain Fatty Acids in Enteroendocrine Cells vs FFAR3 in Enteric Neurons and FFAR2 in Enteric Leukocytes

Biology of Incretins: GLP-1 and GIP

Background and Objectives:The gut hormones peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) acutely suppress appetite. The short chain fatty acid (SCFA) receptor, free fatty acid receptor 2 (FFA2) is present on colonic enteroendocrine L cells, and a role has been suggested for SCFAs in appetite regulation. Here, we characterise the in vitro and in vivo effects of colonic propionate on PYY and GLP-1 release in rodents, and investigate the role of FFA2 in mediating these effects using FFA2 knockout mice.Methods:We used Wistar rats, C57BL6 mice and free fatty acid receptor 2 knockout (FFA−/−) mice on a C57BL6 background to explore the impact of the SCFA propionate on PYY and GLP-1 release. Isolated colonic crypt cultures were used to assess the effects of propionate on gut hormone release in vitro. We subsequently developed an in vivo technique to assess gut hormone release into the portal vein following colonic infusion of propionate.Results:Propionate stimulated the secretion of both PYY and GLP-1 from wild-type primary murine colonic crypt cultures. This effect was significantly attenuated in cultures from FFA2−/− mice. Intra-colonic infusion of propionate elevated PYY and GLP-1 levels in jugular vein plasma in rats and in portal vein plasma in both rats and mice. However, propionate did not significantly stimulate gut hormone release in FFA2−/− mice.Conclusions:Intra-colonic administration of propionate stimulates the concurrent release of both GLP-1 and PYY in rats and mice. These data demonstrate that FFA2 deficiency impairs SCFA-induced gut hormone secretion both in vitro and in vivo.

Selective FFA2 Agonism Appears to Act via Intestinal PYY to Reduce Transit and Food Intake but Does Not Improve Glucose Tolerance in Mouse Models.

Gastric inhibitory polypeptide (GIP) is an incretin secreted from enteroendocrine K cells in response to meal ingestion. Recently free fatty acid receptor G protein-coupled receptor (GPR) 120 was identified as a lipid sensor involved in glucagon-like peptide-1 secretion. However, Gpr 120 gene expression and its role in K cells remain unclear, partly due to difficulties in separation of K cells from other intestinal epithelial cells. In this study, we purified K cells using GIP-green fluorescent protein (GFP) knock-in mice, in which K cells can be visualized by GFP fluorescence. GFP-positive cells (K cells) were observed in the small intestine but not in the stomach and colon. K cell number and GIP content in K cells were significantly higher in the upper small intestine than those in the lower small intestine. We also examined the expression levels of several free fatty acid receptors in K cells. Among free fatty acid receptors, GPR120 was highly expressed in the K cells of the upper small intestine compared with the lower small intestine. To clarify the role of GPR120 on K cells in vivo, we used GPR120-deficient mice (GPR120(-/-)). GPR120(-/-) exhibited significantly lower GIP secretion (75% reduction, P < .01) after lard oil ingestion compared with that in wild-type mice. Consistently, pharmacological inhibition of GPR120 with grifolic acid methyl ether in wild-type mice significantly attenuated lard oil-induced GIP secretion. In conclusion, GPR120 is expressed abundantly in K cells of the upper small intestine and plays a critical role in lipid-induced GIP secretion.

Glucagon-like peptide 1 (GLP-1) is a multifunctional hormone in glucose metabolism and intestinal function released by enteroendocrine L-cells. The plasma concentration of GLP-1 is increased by indigestible carbohydrates and luminal infusion of short-chain fatty acids (SCFAs). However, the triggers and modulators of the GLP-1 release remain unclear. We hypothesized that SCFAs produced by bacterial fermentation are involved in enteroendocrine cell proliferation and hormone release through free fatty acid receptor 2 (FFA2, also known as FFAR2 or GPR43) in the large intestine. Fructo-oligosaccharide (Fructo-OS), fermentable indigestible carbohydrate, was used as a source of SCFAs. Rats were fed an indigestible-carbohydrate-free diet (control) or a 5% Fructo-OS-containing diet for 28days. FFA2-, GLP-1-, and 5-hydroxytryptamine (5-HT)-positive enteroendocrine cells were quantified immunohistochemically in the colon, cecum, and terminal ileum. The same analysis was performed in surgical specimens from human lower intestine. The coexpression of FFA2 with GLP-1 was investigated both in rats and humans. Fructo-OS supplementation in rats increased the densities of FFA2-positive enteroendocrine cells in rat proximal colon, by over two-fold, relative to control, in parallel with GLP-1-containing L-cells. The segmental distributions of these cells in human were similar to rats fed the control diet. The FFA2-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HT-containing EC cells, in both human and rat colon and terminal ileum. Fermentable indigestible carbohydrate increases the number of FFA2-positive L-cells in the proximal colon. FFA2 activation by SCFAs might be an important trigger for produce and release GLP-1 by enteroendocrine L-cells in the lower intestine.

Aims/hypothesisLipids are a potent stimulus for the secretion of glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide (GIP). Traditionally, this effect was thought to involve the sensing of lipid digestion products by free fatty acid receptor 1 (FFA1) and G-protein coupled receptor 119 (GPR119) on the apical surface of enteroendocrine cells. However, recent evidence suggests that lipids may in fact be sensed basolaterally, and that fatty acid absorption and chylomicron synthesis may be a prerequisite for their stimulatory effect on gut peptide release. Therefore, we investigated the effect of chylomicrons on GLP-1 and GIP secretion in vitro.MethodsThe effect of chylomicrons on incretin secretion was investigated using GLUTag cells and duodenal cultures of both murine and human origin. The role of lipoprotein lipase (LPL) and FFA1 in GLUTag cells was assessed by pharmacological inhibition and small (short) interfering RNA (siRNA)-mediated knockdown. The effect of chylomicrons on intracellular calcium concentration ([Ca2+]i) was determined by imaging GLUTag cells loaded with Fura-2. In the primary setting, the contributions of FFA1 and GPR119 were investigated using L cell-specific Gpr119 knockout cultures treated with the FFA1 antagonist GW1100.ResultsChylomicrons stimulated GLP-1 release from GLUTag cells, and both GLP-1 and GIP secretion from human and murine duodenal cultures. Chylomicron-triggered GLP-1 secretion from GLUTag cells was largely abolished following lipase inhibition with orlistat or siRNA-mediated knockdown of Lpl. In GLUTag cells, both GW1100 and siRNA-mediated Ffar1 knockdown reduced GLP-1 secretion in response to chylomicrons, and, consistent with FFA1 Gq-coupling, chylomicrons triggered an increase in [Ca2+]i. However, LPL and FFA1 inhibition had no significant effect on chylomicron-mediated incretin secretion in murine cultures. Furthermore, the loss of GPR119 had no impact on GLP-1 secretion in response to chylomicrons, even in the presence of GW1100.Conclusions/interpretationChylomicrons stimulate incretin hormone secretion from GLUTag cells as well as from human and murine duodenal cultures. In GLUTag cells, the molecular pathway was found to involve LPL-mediated lipolysis, leading to the release of lipid species that activated FFA1 and elevated intracellular calcium.

ADVERTISEMENT RETURN TO ISSUEPREVPatent HighlightNEXTGPR40 Receptor Agonists for the Treatment of Type 2 Diabetes and Related DiseasesAhmed F. Abdel-Magid*Ahmed F. Abdel-MagidTherachem Research Medilab, LLC., 100 Jade Park, Chelsea, Alabama 35043, United States*E-mail: [email protected]More by Ahmed F. Abdel-MagidCite this: ACS Med. Chem. Lett. 2018, 9, 9, 870–871Publication Date (Web):August 13, 2018Publication History Received27 July 2018Published online13 August 2018Published inissue 13 September 2018https://pubs.acs.org/doi/10.1021/acsmedchemlett.8b00343https://doi.org/10.1021/acsmedchemlett.8b00343editorialACS PublicationsCopyright © 2018 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views1638Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (441 KB) Get e-AlertscloseSUBJECTS:Anatomy,Carbohydrates,Diabetes,Peptides and proteins,Receptors Get e-Alerts

The K cell is a specific sub-type of enteroendocrine cell located in the proximal small intestine that produces glucose-dependent insulinotropic polypeptide (GIP), xenin, and potentially other unknown hormones. Because GIP promotes weight gain and insulin resistance, reducing hormone release from K cells could lead to weight loss and increased insulin sensitivity. However, the consequences of coordinately reducing circulating levels of all K cell-derived hormones are unknown. To reduce the number of functioning K cells, regulatory elements from the rat GIP promoter/gene were used to express an attenuated diphtheria toxin A chain in transgenic mice. K cell number, GIP transcripts, and plasma GIP levels were profoundly reduced in the GIP/DT transgenic mice. Other enteroendocrine cell types were not ablated. Food intake, body weight, and blood glucose levels in response to insulin or intraperitoneal glucose were similar in control and GIP/DT mice fed standard chow. In contrast to single or double incretin receptor knock-out mice, the incretin response was absent in GIP/DT animals suggesting K cells produce GIP plus an additional incretin hormone. Following high fat feeding for 21-35 weeks, the incretin response was partially restored in GIP/DT mice. Transgenic versus wild-type mice demonstrated significantly reduced body weight (25%), plasma leptin levels (77%), and daily food intake (16%) plus enhanced energy expenditure (10%) and insulin sensitivity. Regardless of diet, long term glucose homeostasis was not grossly perturbed in the transgenic animals. In conclusion, studies using GIP/DT mice demonstrate an important role for K cells in the regulation of body weight and insulin sensitivity.

Free fatty acid receptors (FFAs) are highly enriched in enteroendocrine cells providing pathways to link dietary fats and microbially generated short-chain fatty acids (SCFAs) to the secretion of a variety of gut hormones. FFA1 and FFA4 are receptors for long-chain fatty acids that have been linked to the elevation of plasma gut hormones after fat ingestion. FFA2 and FFA3 are receptors for SCFA, which are generated at high concentrations by microbial fermentation of dietary fiber and have also been implicated in enhancement of gut hormone secretion. FFAs are candidate drug targets for increasing the secretion of intestinal hormones such as glucagon-like peptide-1 and peptide YY as potential new treatments for type 2 diabetes and obesity. This review will examine aspects of intestinal physiology and pharmacology related to the function of FFAs in enteroendocrine cells.

Diets enriched in nondigestible prebiotic fiber, which is fermented by intestinal microbes to short-chain fatty acids (SCFAs; eg, acetate, propionate, and butyrate), have beneficial effects on diet induced obesity and glucose homeostasis in a number of different models (1). Cross talk between the host and its complement of gut microbiota is a topical area of research, not least because of changes in the microbiome correlate with obesity and diabetes (2), and SCFAs could be considered part of a shared language between proand eukaryotes. Among the many likely targets for SCFAs in eukaryotic cells, two G-protein coupled sensors have been identified: the free fatty acid receptors, FFAR2 and FFAR3 (3, 4). A number of reports have implicated these receptors in the modulation by SCFAs of hormone secretion and inflammation in the gut, but it is fair to say that the jury is still out regarding their physiological roles and potential drugability. The study by Nohr et al (5) in this issue revisits the expression patterns of ffar2 and ffar3 within the intestine using transgenic mice in which expression of a fluorescent marker is driven by the respective receptor promoter. Consistent with previous reports based on receptor-specific antibodies (6) or reverse transcription PCR in purified cells (7), they found strong ffar3-dependent labeling in enteroendocrine cells (EECs) but interestingly also detected fluorescence in the enteric nervous system. Ffar2driven expression, by contrast, was not detected in enteric nerves, was restricted to a subset of EECs in which it had been detected previously (6, 7), and was abundant in leukocytes within the lamina propria, consistent with previous reports of a chemoattractant role for this receptor in neutrophils (8, 9). Partially due to the lack of specific pharmacological ligands, studies directed at characterizing SCFA sensing have so far relied heavily on the use of knockout mice with either ffar2or ffar3-specific disruption. These models do, however, present some drawbacks. Because the two genes are located in very close proximity, alteration in one locus has been found to affect expression of the other gene (10, 11), and complex interactions between different cell types expressing these receptors cannot be adequately addressed in global knockout animals. This has likely contributed to some of the apparently conflicting findings reported in the literature. For example, ffar2 / animals have been reported to have either reduced (9) or increased (8) inflammatory responses, and although bone marrow transplant experiments originally pointed toward a predominant role of FFAR2 in neutrophils (8), a recent report instead emphasizes the role of epithelial-expressed ffar2 and ffar3 for intestinal inflammatory responses (12). The new mice enabling live imaging of ffar2or ffar3-expressing cell populations in conjunction with the newly developed receptor specific agonists will undoubtedly facilitate further analysis of the physiological roles and pharmacological potential of each receptor. Nohr et al (5) focused on EECs, which are crucial in the postand interprandial control of appetite and nutrient disposal. Analysis of hormones in postprandial plasma shows that the gut releases a variety of peptides, including glucagon-like peptides (GLPs) 1 and 2, cholecystokinin, glucose-dependent insulinotropic polypeptide, secretin, and neurotensin, whose profiles reflect the composition and quantity of food in the gastrointestinal tract. In turn, these hormones promote a diverse array of physiological responses including increased insulin secretion, reduced appetite, and altered gut motility, tuned to ensure the optimal absorption and peripheral disposal of incoming nutrients. SCFA-triggered GLP-1 release had previously been

Expression of the short chain fatty acid receptor GPR41/FFAR3 in autonomic and somatic sensory ganglia

Free fatty acid receptor 4 (FFAR4), also known as G-protein coupled receptor 120 (GPR120), is a long-chain unsaturated fatty acid receptor expressed in adipocytes, endothelial cells, and macrophages. Activation of FFAR4 helps maintain metabolic homeostasis by regulating adipogenesis, insulin sensitivity, and inflammation. While FFAR4 is best known for its role its role in preventing obesity and diabetes, recent studies have demonstrated that FFAR4 may also play an important role in the development of atherosclerosis and cardiovascular disease (CVD). Given FFAR4’s importance in anti-inflammatory signaling and high expression levels in macrophages, we designed experiments to test the hypothesis that FFAR4 prevents the development of atherosclerosis by reversing macrophage foam cell formation, a hallmark of early atherogenesis. In these studies, we isolated peritoneal macrophages from wild-type C57/Bl6 mice and incubated them with 20 μg/ml oxidized low-density lipoprotein (oxLDL) to generate foam cells. We then investigated the effects of FFAR4 activation on lipid accumulation, cytokine secretion, and cholesterol efflux. We found that activation of FFAR4 with synthetic agonist GW9508 reduced lipid accumulation as observed by decreased Oil Red O staining and reduced cellular cholesterol content. Activation of FFAR4 by GW9508 also decreased macrophage secretion of pro-inflammatory cytokines interleukin 1 beta (IL-1β) by 5.3-fold, interleukin 6 (IL-6) by 2.4-fold, monocyte chemoattractant protein-1 (MCP-1) by 44.1-fold, and tumor necrosis factor alpha (TNFα) by 2.4-fold. Additionally, activation of FFAR4 by GW9508 significantly increased [ 3 H] cholesterol efflux to high-density lipoprotein (HDL) from peritoneal macrophages. Taken together, our results support an exciting and novel protective role for FFAR4 in the reversal of foam cell formation and could emerge this receptor as a new target for treating CVD by preventing accumulation of atherosclerotic plaque.

ObjectivePresent clinicians with an updated overview of empagliflozin for the treatment of type 2 diabetes mellitus (T2DM), with focus on its use in combination regimens. MethodsUsing the Medline database, keyword searches were undertaken to identify literature reporting the use of empagliflozin treatment in clinical trials with a minimum duration of 12 weeks relating to patients with T2DM.

Background: In the last few decades, many studies have shown that pesticides have a close relationship with increasing blood glucose levels and the incidence of diabetes. Some examples of pesticides include fenthion, permethrin, and carbaryl. Recently, free fatty acid receptor 2 (FFAR2) was identified as having a critical function in preventing insulin resistance. Activation of FFAR2 will reduce fat accumulation and induce glucagon-like peptide 1 (GLP-1) secretion, which plays an important role in regulating type 2 diabetes mellitus (T2DM) prevention.Objective: This study aims to determine a comparison of the binding ability between fenthion, permethrin, and carbaryl to FFAR2 protein for predicting the mechanism of pesticide toxicity to T2DM through an in silico study.Methods: This is an exploratory bioinformatic study. The protein structure was FFAR2 receptor (UniProt: O15552), while the ligand was fenthion (PubChem CID: 3346), permethrin (PubChem CID: 40326), and carbaryl (PubChem CID: 6129). This molecular docking was conducted in October 2022 using Asus X202XE with Intel® Core™ i3-3217U CPU equipped with BIOVIA Discovery Studio, AutoDockTools, and AutoDock Vina. Results: The binding affinity values generated after docking between fenthion, permethrin, and carbaryl with FFAR2 indicate that the binding affinity comparison is permethrin &lt; carbaryl &lt; fenthion. This explains that permethrin could form a stronger bond with FFAR2 protein than other pesticides. However, the visualisation results of the form of bond interactions show that permethrin does not bind to the active site of FFAR2, so it could not be called an inhibitor. This is different from fenthion and carbaryl, which could bind to several amino acid residues on the active site of FFAR2 and have the potential to become inhibitors.Conclusion: Carbaryl is a pesticide with the strongest FFAR2 inhibitor. Carbaryl could cause type 2 DM through its inhibitory pathway to FFAR2.

Free fatty acid receptor 1 (FFA1) and FFA4 belong to a family of free fatty acid (FFA) receptors. FFA1- and FFA4-mediated signaling regulates a variety of malignant properties in cancer cells. It is known that stromal cells in the tumor microenvironment promote tumor progression. In the present study, to assess the roles of FFA1 and FFA4 in cellular functions modulated by endothelial cells, highly migratory MG63-CR7(F2) cells were generated from osteosarcoma MG-63 cells, using endothelial F2 cell supernatants. Expression levels of FFAR1 and FFAR4 genes in MG63-CR7(F2) cells were significantly higher than those of MG-63 cells. In cell survival assay, cells were treated with cisplatin (CDDP) every 24 h for 2 days. The cell survival rate of MG-63 cells was significantly elevated by an FFA1 agonist TUG-770 as well as an FFA4 agonist TUG-891. Moreover, the cell survival rate of MG63-CR7(F2) cells was higher than that of MG-63 cells in the presence of TUG-770 or TUG-891, correlating with FFAR1 and FFAR4 expression levels. To validate the effects of FFA1 and FFA4 on cell survival to CDDP, FFA1 and FFA4 knockdown cell were generated from MG-63 cells. The cell survival rate of MG-63 cells was markedly inhibited by FFA1 or FFA4 knockdown. These results suggest that FFA1 and FFA4 may play an important role in the modulation of cellular functions by endothelial cells in osteosarcoma cells.