Review Bioscience Microflora Vol. 29 (1), 31–40, 2010 Roles of Short-Chain Fatty Acids and their Receptors in Colonic Motility

Shin-ichiro KARAKI1 and Atsukazu KUWAHARA1*

1Laboratory of Physiology, Institute for Environmental Sciences, University of Shizuoka, 52–1 Yada, Suruga-Ku, Shizuoka 422-8526, Japan Received for Publication, October 29, 2009

Short chain fatty acids (SCFAs) are major anions in the large intestine. They are produced by bacterial fermentation of dietary fiber. However, the mechanism by which intraluminal SCFAs are sensed is unknown. Free fatty acids including SCFAs have recently been demonstrated to act as ligands for several G--coupled receptors (GPCRs: FFA1, FFA2, FFA3, GPR84, GPR109A and GPR120). SCFAs are ligands for FFA2 and FFA3. These receptors are proposed to play a variety of physiological and pathophysiological roles in the intestine. In rat and human colons, FFA2 and/or FFA3 are located in mucosal enteroendocrine cells containing peptide YY (PYY) and are related to energy balance. Among SCFAs, propionate and butyrate induce concentration-dependent phasic and tonic contractions in rat colonic circular muscle. These responses are not observed in mucosal free preparations. Thus, FFA2 and FFA3 are important molecular devices for monitoring the chemical composition in the colonic lumen. For the local function of SCFAs, it should be stressed that individual SCFAs have different modes of action on colonic smooth muscles. These different actions may be due to the relative contributions of FFA2 and FFA3 to the control of intestinal muscle activity. FFA2 and FFA3 may also contribute to the whole body energy balance through the release of gastrointestinal hormones related to feeding and satiety control. This review summarizes recent findings about the roles of deorphanized FFA receptors, especially, FFA2 and FFA3 and their contributions to the regulation of colonic motility. Key words: G-protein coupled receptor; short-chain fatty acid; FFA2; FFA3; human colon; colonic motility

INTRODUCTION 43), transepithelial chloride secretion (26, 67), Short chain fatty acids (SCFAs) are 2–6 carbon colonocyte proliferation (48, 51), and to prevent monocarboxylates which are a subgroup of fatty acids. colorectal cancer (13). As a remote effect, infusion of They include acetate (C2), propionate (C3) and n- SCFAs into the colon (47) or ileum (10, 11) have also butyrate (C4), all of which are predominantly found in the been reported to inhibit proximal intestinal motility large intestine (62). They are produced by bacterial which is variously known as the “colonic brake”, “ileal fermentation of undigested carbohydrates from ingested brake” or “ileocolonic brake”. With respect to intestinal dietary fiber. SCFAs are major anions, present at about motility, SCFAs-induced colonic motility is reported to 100 mM in the lumen of the non-ruminant mammalian disappear in mucosal free preparations (38). Therefore, large intestine. Molar ratios of SCFAs in human fecal the effects of SCFAs in the intestinal lumen are content are reported as 50–60 mM acetate, 15–20 mM considered to be induced via activation of specific propionate, 10–20 mM butyrate (12). Luminal SCFAs receptors and/or via absorption by epithelial cells, but the are not only absorbed as nutrients by the intestinal mechanisms by which intraluminal SCFAs are sensed are epithelia but also influence various physiological and not known. pathophysiological actions in the intestine as chemical Recently, many G protein-coupled receptors (GPCRs) stimuli. For example, SCFAs have been reported to have been deorphanized. Among them, free fatty acid stimulate smooth muscle contractions (19, 38, 39, 42, (FFA) receptors have been identified as membrane receptors and play significant roles in nutritional regulation. Individual FFA receptors are expressed *Corresponding author. Mailing address: Laboratory of Physiology, Institute for Environmental Sciences, University of Shizuoka, 52–1 differentially, and they may play different functional Yada, Suruga-ku, Shizuoka 422-8526, Japan. roles. This finding has prompted re-evaluation of the Phone: +81-54-264-5707. FAX: +81-54-264-5707. mechanisms of actions of FFAs in health and disease. E-mail: [email protected] FFA1 (originally termed GPR40) and GPR120 are The review was presented at the Function of Intestinal Microbiota and activated by medium- and long-chain FFAs, whereas Food(FIMF) Joint Forum 2008, Tokyo, 2008. FFA2 (previously designated GPR43) and FFA3

31 32 S. KARAKI and A. KUWAHARA

Table 1. Free fatty acid receptors

Nomenclatures Other Names G- Effectors Agonists Major expression cells Medium ~ Long-chain fatty acids Pancreatic  cells, FFA1 GPR40 G Ca2+  q/11 (C12 ~ C18) Thiazolidinedione Enteroendocrine K cells cAMP  SCFAs (C2 ~ C7) Immune cells, FFA2 GPR43 G G i/o q Ca2+  acetate = propionate = butyrate Enteroendocrine L cells SCFAs (C2 ~ C7) Adipocytes, FFA3 GPR41 G cAMP  i/o propionate = butyrate > acetate Enteroendocrine L cells Enteroendocrine cells GPR120 G Ca2+  Long-chain fatty acids (C14 ~) q/11 in the colon Medium-chain fatty acids GPR84 G cAMP  Immune cells i/o (C9 ~ C14) HM74A, cAMP  Adipocytes, macrophages GPR109A G SCFAs (C4 ~ C8) Nicotinate PUMA-G i/o Ca2+  Intestinal epithelial cells

(previously designated GPR41) are activated by SCFAs revealed high expression in -producing pancreatic (56). In this review, we summarize recent findings about islets (2, 27). FFA1 was found to be enriched 2- to 100- the roles of deorphanized FFA receptors, especially, fold in pancreatic islets as compared to the whole FFA2 and FFA3 and their contributions to the regulation pancreas (4, 46). These results suggest that FFA1 acts as of colonic motility. a receptor for fatty acid-induced insulin secretion. As well as -cells, FFA1 is also reported to be present in - FREE FATTY ACID RECEPTORS cells (18). Moreover, FFA1 expression has been detected FFAs are known to possess a wide range of in various pancreas-derived cell lines, including MIN6, physiological effects through cellular metabolism and -TC-3, HIT-T15 and INS-1E (4, 27, 30, 52). FFA1 cell surface receptors. expression has also been detected in human and rat islets The encoding FFA1, FFA2 and FFA3, originally (17, 49, 60). Outside of the pancreas, the presence of termed GPR40, GPR43 and GPR41, respectively, have FFA1 mRNA has been reported in the small and large been identified as tandemly encoded genes present on intestines (4, 14, 27) and in the STC-1 cells. Expression human 19q13 (50). FFA1 receptor of FFA1 was demonstrated in the enteroendocrine cells of remained classified as an orphan until 2003. In 2003, the stomach and small intestine which contain a variety of three groups independently reported that the FFA1 was an gut hormones including glucagon-like peptide-1 (GLP-1) endogenous ligand for medium- and long-chain saturated and glucose-dependent insulinotropic polypeptide (GIP), and unsaturated fatty acids (4, 27, 30). Medium- and which both increase insulin secretion(14). Instead of long-chain FFAs also activate GPR84 and GPR120 (24, incretin hormones, in the pylorus and duodenum, FFA1 60). On the other hand, SCFAs activate FFA2 (GPR43) expression colocalized with gastrin, ghrelin, and FFA3 (GPR41) (5, 32, 41). FFA1, FFA2, and FFA3 cholecystokinin (CCK), peptide YY (PYY), secretin, represent a family of receptors because they are more serotonin and substance P expression (14). Outside the closely related to each other than any other known GI tract, FFA1 has been detected in the brain, immune GPCRs. Despite this, the family exhibits somewhat cells, MCF-7 human breast cell line and MDA-MB-231 limited similarity, 43% between FFA2 and FFA3, and 33 breast cancer cells (22, 23, 35, 36, 52, 68). and 34% for FFA1 compared aganist FFA2 and FFA3, Another medium- and long-chain fatty acids receptor, respectively (6). The receptors have been given a variety GPR120, is highly expressed in the human and mouse of names in the literature and these are summarized in intestinal tract and mouse enteroendocrine STC-1 cells Table 1. (24). GPR120 is expressed in GLP-1-containing intestinal L and K cells (24, 40). GPR84 is also a Tissue distribution 1 (FFA1) medium-chain FFAs receptor. GPR84 is stimulated by All three initial reports on FFA1 (GPR40) showed high medium-chain FFA with carbon chain lengths of 9–14 levels of receptor mRNA in the pancreas (4, 27, 30). (63). GPR84 is not activated by short-chain and long- Expression analysis of FFA1 using RT-PCR, chain saturated and unsaturated FFAs, which do activate immunohistochemistry, and in situ hybridization FFA1, FFA2, FFA3 and GPR120 (63). Human GPR84 is ROLES OF SHORT-CHAIN FATTY ACIDS AND THEIR RECEPTORS IN COLONIC MOTILITY 33 expressed in the brain, heart, muscle, colon, thymus, knockout mice, release of GLP-1 and GIP was spleen, kidney, liver, intestine, placenta, lung and significantly reduced in response to a high-fat diet (14). leukocytes (64). GPR109A (also termed HM74A in These results suggest that incretin secretion might be humans and PUMA-G in mice), a nicotinic acid (niacin, impaired in FFA1 knockout mice and indicate a role for vitamin B3) receptor, has been reported to be activated by FFA1 in the secretion of these hormones. Another C4 - C8 fatty acids (57), and is expressed in adipocytes, GPCR, GPR120, is also activated by medium- to long- activated macrophages (61), and the apical membrane of chain fatty acids and induces GLP-1 secretion in mouse intestinal epithelial cells (59). enteroendocrine STC-1 cells. (24). Oral administration of FFA and direct administration to the colon also Physiological roles of FFA1 increase circulating GLP-1 and insulin levels in mice FFA1 is activated by more than 40 different medium- (24). Both FFA1 and GPR120 are activated in a smimlar and long-chain fatty acids with micromolar range manner by FFAs but FFA1 directly and GPR120 potency, and of these eicosatrienoic acid (C20:3) was the indirectly promote glucose-stimulated insulin secretion most potent with a pEC of 5.71 ± 0.11 (4). (15, 24). FFAs with a carbon chain length of 14–18, and Interestingly, the potency of the saturated fatty acids was unsaturated FFAs with a chain length of 16–22 also dependent on chain length, whereas carbon chain length stimulate GPR120. A functional analysis of GPR84 or degree of saturation did not appear to correlate with indicated that GPR84 has a role in the regulation of early potency among unsaturated fatty acid (4). Ito et al. IL - 4 expression in activated T cells (61). Microglia showed that methyl linoleate, a noncarboxyl-containing also express GPR84 during endotoxemia, suggesting a compound related to linoleic acid (C18:2), was unable to role for GPR84 in the regulation of microglia and activate the FFA1 receptor and they suggested that a neuroinflammatory processes (3). carboxyl group was important for an agonist (27). Expression of FFA1 has also been detected in breast There is a strong link between , type 2 diabetes, cancer cell lines as mentioned above (23, 68). Recent and circulating plasma levels of fatty acids. studies of the breast cancer cell line, MDA-MB-231, Administration of FFAs stimulates insulin secretion. showed that oleic acid stimulated cell proliferation but Therefore, FFAs are important to the pancreatic -cell for plamitic acid induced apoptosis (22, 23). Thus, FFA1 its normal function, its capacity to compensate for insulin may be involved in cell proliferation in the mammary resistance and its failure in type 2 diabetes. Islet tissues gland. deprived of FFA fail to produce glucose-stimulated insulin secretion and this insulin secretion is rapidly Tissue distribution of free fatty acid receptor 2 and 3 reversed by replacement with exogenous FFAs (54). (FFA2 & 3) Thus, FFAs seem to be required for basal insulin At the same time as the discovery of the FFA1, FFA2 secretion. On the other hand, chronic elevation of FFAs and FFA3 were also found to be receptors for short-chain has been shown to impair insulin secretion (45) and fatty acids (5, 32, 41). FFA2 mRNA has been detected in induce -cell apoptosis (15). a variety of tissues, but its highest expression is found in The most attractive evidence for the role of FFA1 in immune cells (5, 32, 41). Considerable levels of FFA2 normal -cell function and in the development of type 2 mRNA were also detected in bone marrow and spleen but diabetes has come from the study of FFA1 knockout these are thought to reflect the expression of the receptor mouse models. FFA1 knockout and overexpression in on immune cells (32). Nilsson et al. found expression of vivo have been shown to mediate both acute and chronic the FFA2 in skeletal muscle and the heart (41), whereas effects of long-chain fatty acids on murine insulin FFA2 has also been reported in adipose tissue (20, 25), secretion (55). Activation of FFA1 is coupled to an the breast cancer cell line, MCF-7 (68) and rat the distal 2+ 2+ increase in intracellular Ca concentration ([Ca ]i) (4, ileum and colon (28). 27, 30, 60). FFA3 has a more widespread expression pattern than Medium- and long-chain FFAs also affect the secretion FFA2. High levels of expression were observed in of several gut “satiety” hormones such as CCK, GLP-1 adipose tissues, the pancreas, spleen, lymph nodes, bone and PYY. GIP and GLP-1 are secreted from intestinal K- marrow, and peripheral blood mononuclear cells (5, 32). and L-cells, respectively, and the secretion of GIP and However, there is some debate about the expression of GLP-1 can be stimulated both by glucose and FFAs (1). FFA3 in adipocytes, Hong et al. were unable to detect In mice, plasma levels of GLP-1 and GIP increase in FFA3 expression in human adipose tissue, in cultured response to the ingestion of a high-fat diet. In FFA1 gene preadipocytes or adipocytes, or in 3T3-L1 cells, despite 34 S. KARAKI and A. KUWAHARA

in muscle plus submucosal layers both in the rat distal ileum and colon (28). In the human ascending colon, mRNA for FFA2 was also detected in extracts of whole wall, and FFA2 protein was detected in extracts of whole wall and in the separated mucosa but not in extracts of submucosa and muscle layer (29). These results indicate that FFA2 is expressed by cells in the mucosa, but not by enteric neurons or smooth muscle. Until 2009, there were no reports of FFA3 being expressed in the gut (56), but recently, we found that FFA3 protein and mRNA are expressed in human colonic mucosa (58) and that the expression levels are higher in colonic mucosa than in submucosa or muscle. To identify the cellular distribution of FFA2 in the rat and human colon, immunohistochemical staining was performed using anti-FFA2 antiserum. Immunoreactivity for FFA2 occurred at a low level in enterocytes within mucosa both in the rat and human colon. FFA2 immunoreactivity was also found in a population of enteroendocrine cells and small cells in the lamina propria in the human ascending colon. FFA2- immunreactive cells in the epithelia had the morphology of enteroendocrine cells as shown in Fig. 1A. Fig. 1. Immunohistochemistry of FFA2 and FFA3 Immunoreactivities for FFA2 in rat the ileum and colon in the human large intestine. FFA2 (GPR43) showed similar patterns to those in the human colon. (A) and FFA3 (GPR41) in the human colon FFA2 immunoreactive enteroendocrine cells in the and rectum were stained with rabbit anti- human ascending colon were open type enteroendocrine human/rat GPR43 (code: RY1505) and human GPR41 (code: RY1494) antibodies as the cells, which extended their cell body to the luminal primary antibodies, respectively, and Alexa surface. Until recently, there was no report for the tissue 594 (red)-labeled anti-rabbit IgG antibody as localization of FFA3 in the intestine, so we examined the the secondary antibody with DAPI for staining cellular distribution of FFA3 using human colonic nuclei (blue). Arrows indicate GPR43- and GPR41-immunoreactive enteroendocrine cells tissues. FFA3 immunoreactivity in human colon was in the bottom of crypts. observed as dotted staining in apical cytoplasm of enterocytes, enteroendocrine cells and several lamina propria cells as shown in Fig. 1B. FFA3-immunoreactive using the same probes as used in the original studies for enteroendocrine cells were also open type with thin cell FFA3 receptor mRNA (25). The discrepancies in FFA3 bodies extending to the luminal surface (58). However, expression reported by these studies have yet to be the number of FFA3 immunoreactive enteroendocrine resolved. cells was less than that of FFA2 cells (29, 58). As mentioned above, SCFAs affect various colonic Furthermore, double-immunostaining for FFA2 and functions including motility and ion transport and a large FFA3 revealed that they were not colocalized with each amount of SCFAs is constantly present in the large other. intestine. Therefore, it was expected that FFA2 and FFA3 Previous physiological studies reported the SCFAs- would be expressed in the large intestine and involved in induced 5-hydroxytryptamine (5-HT) and PYY release the regulation of large intestinal functions (26, 37–38, 42, (8, 19). Thus, we performed double-staining for FFA2 and 43, 66, 67). Accordingly, we started to explore the 5-HT, and FFA2 and PYY. No FFA2-immunoreactive expression of FFA2 and FFA3 using antisera for FFA2 enteroendocrine cells were positive for 5-HT, whereas and FFA3. Messenger RNA for FFA2 was detected in FFA2 immunoreactive enteroendocrine cells were extracts of whole wall and separated mucosa from the rat colocalized with PYY in both the human and rat colon (28, distal ileum and colon (28). FFA2 protein was detected 29). We also performed double-staining for FFA3 and 5- by western blotting in the mucosa and whole wall but not HT or PYY. FFA3 immunoreactive enteroendocrine cells ROLES OF SHORT-CHAIN FATTY ACIDS AND THEIR RECEPTORS IN COLONIC MOTILITY 35 in the human colon were also colocalized with PYY importance of SCFAs has been highlighted only recently. similar to those of FFA2 but not 5-HT (58). However, A possible direct influence of SCFAs on intestinal FFA2 and FFA3 were not colocalized in the same motility in monogastric animals was first suggest by enteroendocrine cells. Yajima, who recorded a tonic contraction of rat colonic muscle strips in response to propionate, butyrate or Physiological roles of FFA2 & 3 valerate in vitro (66). The concentration-dependent FFA2 is expressed at high levels in a variety of immune contractile effect occurred only when SCFAs were cells such as neutrophils, monocytes and applied on the mucosal side and disappeared when the polymorphonuclear cells (5, 32, 41). SCFAs are known mucosa was removed, suggesting the presence of sensory to mediate the activation of leukocytes by Ca2+ mechanisms near the epithelium. As indicated above, mobilization (7). Furthermore, SCFAs evoke a FFA2- and FFA3-immunoreactive intestinal cells were chemotactic response in leukocytes; leukocytes move found in both rat and human colons (28, 29, 58). toward sites of bacterial infection. (31). These results Therefore, we investigated the contribution of FFA2 and suggest that SCFAs might be involved in the activation of FFA3 to the regulation of colonic motility using in vitro leukocytes through FFA2. On the other hand, FFA3 is animal models. expressed abundantly in adipose tissues (5) and is involved in leptin production in adipose tissues by CIRCULAR MUSCLE stimulation of SCFAs (9). FFA2 mRNA has also been When recorded mechanically, in vitro and in vivo detected in adipose tissues (20, 25). Ge et al. have colonic motor activity in most species including mice and recently reported that adipocytes treated with FFA2 rats, is characterized by two distinct types of contraction: natural ligands, acetate or propionate, show a reduction in (1) rhythmic phasic contractions, and (2) spontaneous lipolytic activity and that the effect was abolished in contractions, which are also termed giant contractions adipocytes isolated from a FFA2 knockout mouse (20). (GCs) by Gonzalez and Sarna (21). We showed that They further showed that the activation of FFA2 by propionate increases the frequency and decreases the acetate results in the reduction in plasma free fatty acid mean amplitude of spontaneous GCs (39). However, levels. These results suggest that FFA2 may be involved acetate and butyrate had no such effects. The propionate- in the regulation of lipid homeostasis through the induced increase in the frequency of GCs was blocked by inhibition of lipolysis. For the signal transduction, FFA2 the muscarinic receptor antagonist, atropine. Therefore, and FFA3 are coupled to the formation of inositol 1,4,5- the propionate-induced increase in the frequency of GCs triphosphate, intracellular Ca2+ mobilization, the seems to be mediated by cholinergic motor neurons. The activation of ERK 1/2, and the inhibition of intracellular propionate-induced decrease in the mean amplitude of cAMP accumulation (34). However, FFA2 and FFA3 GCs was prevented by the cyclooxygenase (COX) exhibit differential coupling to G-proteins; FFA3 couples inhibitor piroxicam (39). GCs of the colonic circular exclusively through the pertussis toxin-sensitive Gi/o muscle layer are thought to enhance the propulsion of family, whereas FFA2 couples through the Gi/o and luminal contents because at least part of their contractions pertussis toxin-insensitive Gq family (5). propagate in the anal direction (53). Thus the stimulatory Normal blood concentrations of SCFAs are in range of effect of propionate on the frequency of GCs seems to be 100 to 150 M acetate, 4 to 5 M propionate, and 1 to 3 important for the propulsion of feces in the colon. M butyrate (12). On the other hand, a large amount of Regarding the involvement of FFAs on the frequency SCFAs are produced by commensal bacteria in the regulation of GCs induced by SCFAs, the rank order healthy large intestine through anaerobic bacterial potency of the SCFAs did not correspond to that seen for fermentation of unabsorbed carbohydrates and dietary the activation of FFA2 or FFA3 artificially expressed in fibers. In the human colon, the total concentration of HEK293 cells (5, 32). Therefore, at present, the precise SCFAs reaches between 60 and 100 mM, of which mechanisms in which induvidual FFAs contribute to the acetate is the most prevalent, with butyrate and frequency regulation of GCs induced by SCFAs remains propionate being found in lesser about approximately unknown. However, propionate did not increase the similar concentrations at a molar ratio of 60:20:20 (12). frequency of GCs in a preparation without mucosa. Thus, Therefore, SCFAs are considered to have a key role in the luminal propionate is probably sensed by FFAs in the maintenance of colonic functions including colonic mucosa triggering a release of some sensory mediators in motility and/or ion transport. the mucosa which increase the frequency of GCs. In non-ruminant mammals, the physiological SCFAs also affect basal circular muscle activity. For 36 S. KARAKI and A. KUWAHARA

mucosa causing release of 5-HT from the enterochromaffin cells containing 5-HT. Released 5-HT may act on 5-HT4 receptors located on the endings of intrinsic primary afferent neurons, that in turn would activate cholinergic motor neurons to contract the circular muscle. On the other hand, the tonic contraction was attenuated by the non-selective COX inhibitor, piroxicam and the COX-1 inhibitor, SC-560. Therefore, propionate probably induces the release of COX products to cause the tonic contractions. As atropine and TTX had no effect on the tonic contraction, it is probably triggered by the prostaglandins on the circular muscle. With respect to the involvement of FFAs in SCFAs-induced circular muscle contractions, the rank order potency of the SCFAs corresponds to that seen for the activation of FFA3. Therefore, propionate-induced circular muscle contractions might be involved in FFA3. SCFAs have been reported to enhance colonic motility via the release of 5-HT (19, 38). There are two possible source of 5-HT for the propionate-induced circular Fig. 2. Propionate-induced phasic and tonic contractions muscle contraction: enteroendocrine cells that are in circular muscle strip in the rat distal colon. Circular numerous throughout the gastrointestinal tract (16), and muscle strips (approximately 6–8 mm long and 2 mm 5-HT containing mast cells that occur in the mucosal wide) with (A) and without (B) mucosa were tied at lamina propria of the rat intestine (69). However, one end to a glass rod and at the other to an isometric force transducer and suspended in baths containing immunohistochemical studies have shown that FFA2 or Krebs-Ringer solution. The contractile responses to the FFA3 are not expressed in 5-HT containing addition of propionate to the bathing solutions were enteroendocrine cells (28, 29, 58). Therefore, FFA2- measured. Preparation with mucosa responded to both immunoreactive mast cells in the lamina propria might be propionate and acetylcholine (A). On the other hand, mucosa-free preparations responded to acetylcholine the source of 5-HT released by SCFAs. but not to propionate. SCFAs are also known to inhibit upper gastrointestinal motility, sometimes called ileal or colonic brake, via PYY release into the blood circulation (11, 33). Therefore, the example, propionate evokes phasic and tonic muscle majority of PYY-containing enteroendocrine cells contractions in the rat distal colon (38, 39, 42, 43). probably express FFA2 as SCFAs sensors, and SCFAs Propionate and butyrate concentration-dependently (10 might stimulate PYY release through FFA2. This is M–10 mM) induce rapid, large amplitude phasic supported by the observation that acetate, which is contractions followed by tonic contraction in strips of selective for FFA2, has been reported to cause an increase circular muscle from the rat distal colon. However, in the release of PYY (34). Since SCFAs are produced by acetate has no effect on basal muscle activity. The bacterial fermentation of the carbohydrates of dietary propionate-induced phasic and tonic contractions are not fiber in the large intestinal lumen as mentioned above, the observed in mucosal-free preparations as shown in Fig. 2. presence of SCFAs or individual ratios of SCFAs in the These results suggest that propionate does not directly act colonic lumen may reflect the activity of luminal bacterial on the circular muscle. We further analyzed the flora and SCFA receptors and possibly monitor the propionate-induced circular muscle contractions and activity of bacteria to maintain colonic health. Indeed, found that the propionate-induced circular muscle FFA2 and FFA3 have been reported to be highly contractions were attenuated by atropine, tetrodotoxin expressed by cells of the immune defense system and 5-HT4 receptor antagonist, SB204070. Therefore, including polymorphonuclear cells (FFA2 and FFA3), the phasic contractions seem to depend on the integrity of monocytes (FFA2) and dendritic cells (FFA3) (32). Our the mucosa, cholinergic motor neurons and 5-HT4 previous study also showed that mucosal mast cells receptors. These results also suggest that propionate acts express FFA2 (28). Polumorphonuclear cells, monocytes on SCFA receptors, FFA2 or FFA3, expressed in the (macrophages in the tissue), and dendritic cells are the ROLES OF SHORT-CHAIN FATTY ACIDS AND THEIR RECEPTORS IN COLONIC MOTILITY 37 phagocytes for non-selective antigens involved in innate studies and those of others. SCFA receptors, FFA2 and immunity. Therefore, the evidence seems to support the FFA3 are located in mucosal enteroendocrine cells hypothesis that one of the roles of FFA2 and FFA3 in the containing PYY which isrelated to energy balance. Thus, intestinal mucosa may be concerned with host defense FFA2 and FFA3 are important molecular devices for mechanisms. monitoring the chemical composition in the colonic lumen. With respect to the local functions of SCFAs and LONGITUDINAL MUSCLE FFAs, it should be stressed that individual SCFAs have In comparison to the circular muscle, the contribution different modes of action on colonic smooth muscles. of the longitudinal smooth muscle to colonic propulsion These different actions may be due to the relative has been less studied. However, the longitudinal as well contributions of FFA2 and FFA3 on the control of as the circular muscle layers are also important during intestinal muscle activity. Regarding the remote effects peristalsis because GCs are observed not only in circular of FFA2 or FFA3 on the whole body energy balance, muscle layer, but also in the longitudinal muscle layer of FFA2 or FFA3 may contribute to the release of the colon (44). Therefore, we also investigated the gastrointestinal hormones related to feeding and satiety spontaneous longitudinal muscle contractions induced by control. SCFAs. At more than 1 mM of SCFAs (mixture of acetate, propionate and butyrate) , the frequency of Acknowledgements. This work was partly supported by spontaneous longitudinal muscle contractions were a grant-in-aid from the Japan Society for the Promotion of concentration-dependently decreased and reached a Science (no.21590235), by the Smoking Research maximum at 5 mM (42). However, the SCFAs did not Foundation, by the Salt Research Foundation, and by the affect the amplitude or duration of the spontaneous Food Science Institute Foundation to A. 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