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Keywords:

  • GPR43−/−;
  • GPR43+/+;
  • peristalsis;
  • short-chain fatty acids

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Abstract  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) ≈ propionate (C3) ≈ butyrate (C4) > pentanoate (C5) ∼ 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.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Short-chain fatty acids (SCFAs) are produced by anaerobic bacterial fermentation of unabsorbed carbohydrates and proteins. They comprise one to seven carbons (C), with acetate (C2), propionate (C3) and butyrate (C4) accounting for 90–95% of total colonic SCFAs,1 where they occur in molar ratio of c. 57 : 21 : 22.2 In humans, luminal concentrations of SCFAs are low in the upper gut but increase to c. 2–15 mmol L−1 in the terminal ileum and c. 60–130 mmol L−1 in the proximal colon.2 SCFAs only account for 5–10% of total body energy requirements in humans3 but are an important source of energy for maintaining colonic mucosal integrity.4 Other actions include stimulation of salt and water absorption5 and regulation of colonic mucosal blood flow.6 SCFAs may also act as luminal chemical stimuli controlling gastrointestinal (GI) motility, but whether their effects are stimulatory or inhibitory is unclear, and varies according to different experimental paradigms.1,7–12

It has been postulated that abnormal colonic fermentation could be a factor in the pathogenesis of irritable bowel syndrome (IBS), and indeed, patients with small intestinal bacterial overgrowth (SIBO) show increased concentrations of SCFAs.13 Infusion of starch14 or SCFAs1 into the ileocolonic region of healthy volunteers is associated with flatus, bloating, abdominal pain, cramps and an urge to defecate. In IBS patients, bloating and abdominal pain coincide with unabsorbed carbohydrates entering the proximal colon15 and diarrhoea- and constipation-predominant IBS patients show, respectively, higher and lower SCFA concentrations in their stools than normal individuals.16

Receptors for SCFAs are thought to exist in the enteric nervous system (ENS).17 Recently, SCFAs have been shown to activate two closely related G protein-coupled receptors, GPR41 and GPR4318 and at least one of these, GPR43, has now been shown to be present in the rat intestine.19 At present, only the rank order potency of SCFAs can distinguish between these receptors; compared with C3 and C4, C2 has lower potency at the human GPR41 receptor and is equipotent at GPR43.18 This study investigates the possibility that SCFAs modulate intestinal motility in vitro via activation of these receptors. After determining the concentrations of SCFAs present in the rat intestine and digesta, similar concentrations were evaluated for their ability to modulate movements of the rat distal colon and guinea-pig terminal ileum. Rank-order potency values suggested an involvement of GPR43 receptors in both these experiments, leading to an examination of the distribution of GPR43 receptor mRNA throughout the rat gut and an evaluation of the effects of SCFAs in the wild-type (GPR43+/+) and GPR43 gene knockout (GPR43−/−) mouse isolated distal colon. Some of these data have previously been reported to the International Symposium on Neurogastroenterology and Motility.20

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Measurement of rat intestinal straight chain carboxylic acid anion concentrations

Male, Sprague–Dawley rats (250–350 g, Charles River, UK) were maintained on a 12 h light/dark cycle with ad libitum access to water and regular rodent chow. They were culled by CO2 inhalation followed by cervical dislocation. All efforts were made to minimize the number of animals used and culling was performed in accordance with the UK Animals (Scientific Procedures) Act 1986. Digesta from the ileum, terminal ileum, caecum, proximal colon plus faecal pellets and proximal colon tissue were diluted in distilled water, homogenized, centrifuged (50228 g, 10 min, 4°C) and the supernatant filtered (0.2 mm polyethersulphone). Acid anions were separated from each other and interfering species by ion exclusion chromatography and detected by suppressed conductivity using a Dionex AMMS-ICE suppressor column. Quantification was by reference to a calibration of external standards.

Tissue bath experiments

Male rats and mice were culled as above and the intestines placed immediately in Krebs’ solution (NaCl 121.5, CaCl2 2.5, KH2PO4 1.2, KCl 4.7, MgSO4 1.2, NaHCO3 25.0, glucose 5.6 mmol L−1) equilibrated with 5% CO2 in O2. Sections of distal colon (c. 15 × c. 4 mm, rat; c. 8 × c. 2 mm, mouse) were cut parallel to the circular muscle; the mucosa was kept intact except where specified.

Tissues were suspended under 10 mN tension for isometric recording of circular muscle contraction, between two parallel platinum ring electrodes in 5 ml tissue baths containing Krebs’ solution bubbled with 5% CO2/95% O2, maintained at pH 7.4 ± 0.1 and 37°C. They were equilibrated for 60 min with bath solutions changed every 15 min. For both rats and mice, electrical field stimulation (EFS) used biphasic square-wave pulses of 0.5 ms pulse width, 5 Hz frequency at maximally effective voltage (± 50 V; Scientifica Ltd, Uckfield, UK). These parameters of EFS consistently evoked nerve-mediated responses with a good signal-to-noise ratio over background spontaneous muscle activity. EFS was applied for 20 s every 40 s for 30 min periods, each period was separated by 5 min during which the bath solutions were changed. After obtaining consistent nerve-evoked contractions, the effects of SCFAs, applied non-cumulatively into the bathing solution, were assessed. The effects of SCFAs were reversible upon washout, permitting the construction of a full concentration–response curve per preparation.

Peristalsis in guinea-pig terminal ileum

Fed, male, Dunkin Hartley guinea-pigs (300–350 g) were culled as above. Segments of terminal ileum (c. 3 cm), taken c. 5 cm from the ileocaecal junction, were flushed of their contents and suspended horizontally in 25 ml baths containing Krebs’ solution (5% CO2 in O2; 37°C, pH 7.4). The oral end of the segment was connected to an inlet tube through which glucose- (0.1%) saline (0.9%), with or without drug was perfused at 0.3–0.5 mL min−1. When SCFAs were added to the glucose–saline solution the pH was adjusted to 7.4. The aboral end was connected to an outlet tube, directly connected via a two-way tap to a pressure transducer (Harvard Apparatus, Edenbridge, Germany) and a reservoir containing Krebs’ solution. The height of the reservoir was adjusted to generate an intraluminal pressure of 22–25 mmH2O which elicited regular tetrodotoxin- (0.1 μmol L−1) and atropine- (0.1 μmol L−1) sensitive aborally propagated waves of peristalsis.

GPR43 mRNA distribution in the rat gut and CNS

Relative quantitative determinations of GPR43 mRNA and the internal reference transcript cyclophilin in the rat gut and CNS were performed using real-time reverse transcriptase-polymerase chain reaction (RT-PCR).21 Male and female rats were culled as above. Tissues were removed, washed, snap-frozen and total RNA extracted using Tri-Reagent (Molecular Research Center, Cincinnati, USA). As much as 2 μg of each RNA was then converted to cDNA using random hexamers and MuLV reverse transcriptase (Applied Biosystems Inc., Foster City, CA, USA), diluted and aliquotted so that each set of 384-well PCR plates contained cDNA equivalent to 12.5 ng DNA-free total RNA for each tissue sample. 12.5 μl PCRs were prepared using reagents created for TaqMan (for GAPDH, initial primer concentrations 900 nmol L−1, probe concentration 100 nmol L−1) and SYBR Green (for GPR43 and cyclophilin; initial primer concentration 300 nmol L−1) chemistries (Roche Molecular Systems, Pleasanton, CA, USA). The sequences of the oligonucleotide primers and 6-carboxyfluorescein (FAM)- and 6-carboxytetramethylrhodamine (TAMRA)labelled probe (Biosource International, CA, USA) were GAPDH [Rat Genome Database (RGD) symbol Gapd; acc. NM_017008]: 5′-CAAGGTCATCCATGACAACTTTG-3′ (sense), 5′-GGGCCATCCACAGTCTTCTG-3′ (antisense), FAM-5′-ACCACAGTCCATGCCATC-ACTGCCAT-3′-TAMRA; GPR43 (RGD interim symbol Gpr43; acc. NM_001005877): 5′-TTCTTACTGGGCTCCCTGCC-3′ (sense), 5′-TACCAGCGGAAGTTGGATGC-3′ (antisense); cyclophilin (RGD symbol Ppia; acc. NM_017101): 5′-TATCTGCACTGCAAGACTGA-3′ (sense), 5′CCACAATGCTCATGCC-TTCTTTCA-3′ (antisense). Real-time PCR analysis was performed using the 384-well ABI PRISMTM 7900 Sequence Detection System (Applied Biosystems Inc.) at 95°C for 10 min followed by 40 cycles of 95°C for 15 s, 60°C for 1 min.

GPR43−/− mice

These were obtained from Deltagen (San Carlos, CA, USA). A section of the mouse gene encoding GPR43 was replaced by homologous recombination in embryonic stem cells with a cassette containing the neomycin resistance and β-galactosidase genes. The resulting deletion of 55 base pairs is contained within the single exon of GPR43, starting at amino acid 47 of GPR43 and ending at 65. Embryonic stem cell transfection was examined by 5′- and 3′-PCR using gene-specific primers paired with primers recognizing the neomycin resistance gene, confirmed by Southern blot analysis. Male chimeric mice were generated by injection of the targeted ES cells into C57BL/6J blastocysts. Chimeric mice were bred with C57BL/6J mice to produce F1 heterozygotes. Germline transmission was confirmed by PCR analysis; subsequent genotyping tracked transmission of the targeting construct. F1 heterozygous males and females were mated to produce F2 wild-type (GPR43+/+), heterozygous (GPR43−/−) and homozygous null (GPR43−/−) animals. Mice were backcrossed with C57BL/6J mice and all phenotypic analysis was performed in a hybrid C57BL/6J/129 background (75%/25%, respectively). The GPR43 wildtype and knockout mouse genotypes were confirmed by digestion and extraction of DNA from tail snips, PCR (primers were GGG CCA GCT CAT TCC TCC CAC TCA T, GCG GAA GTT GGA TGC TGC TTC CAC G and GCA CAG TTC CTT GAT CCT CAC GGC C) and analysis through agarose gel electrophoresis and image capture using UV light. In addition, PCR analysis using RNA extracted from colon, subcutaneous fat and other tissues from wildtype and knockout mice (n = 2 each) confirmed the success of the GPR43 gene knockout (data not shown).

Chemicals

Drugs were freshly prepared before use. Sodium formate (C1), acetate (C2), propionate (C3), butyrate (C4), pentanoate (C5), carbachol, atropine sulphate, sodium nitroprusside (Sigma, Gillingham, Kent, UK) and tetrodotoxin (Tocris, Bristol, UK), were dissolved in distilled water.

Data analysis

Data acquisition and analysis for tissue bath and peristalsis experiments were performed using MP100 hardware and AcqKnowledge® software (Biopac Systems, Inc., Goleta, CA, USA). For EFS studies, data were expressed as the percentage change in the amplitude of the nerve-evoked post-EFS contractions, compared with the mean of three pre-drug post-EFS contractions. In peristalsis studies, the peristaltic pressure threshold, the peak amplitude pressure and the intervals between peak contractions were measured. The effects of SCFAs and the kinetics of the response were assessed over three separate time periods (c. 200 s each) spanning 10 min. All data are expressed as means ± standard error of the mean; n indicates number of animals. Differences between the means were determined using a Student's t-test for paired or unpaired data or a two-way anova followed by post-hoc Fisher's LSD test; P < 0.05 is statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Measurement of rat intestinal straight chain carboxylic acid anion concentrations

Digesta pooled from the ileum-caecum-colon had a mean pH of 7.5 ± 0.1 and osmolality of 298 ± 14 mosmol l−1 (n = 4). The concentrations of C1, C2, C3 and C4 anions in digesta from the ileum, terminal ileum, caecum, proximal colon, as well as in faeces and proximal colon tissue are shown in Fig. 1. The highest concentrations of SCFAs were in the caecum (c. 115 mmol L−1) with lower amounts in faeces (c. 57 mmol L−1) and proximal colon (c. 50 mmol L−1); concentrations <10 mmol L−1 were detected in proximal colon tissue, terminal ileum and ileum. In all samples, C2 concentrations were greatest with the highest amounts in the caecum (c. 58 mmol L−1). The molar ratios of C2 : C3 : C4 in the caecum and proximal colon were 50 : 7 : 43 and 59 : 7 : 34, respectively.

image

Figure 1.  Straight chain carboxylic acid anion concentrations in the rat GI tract. Data are expressed as means ± SEM; n = 4. Acid anions were separated by ion-exclusion chromatography and detected by suppressed conductivity.

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Effects of SCFAs on EFS-evoked responses in rat distal colon

Electrical field stimulation evoked a small-amplitude relaxation followed by a large-amplitude, ‘post-EFS’ contraction on termination of EFS; each of these responses could be prevented by tetrodotoxin 1 μmol L−1.22 C1, C2, C3, C4 and C5 (0.1–100 mmol L−1, 15 min contact, n = 4–16, each concentration) had no effect on the nerve-mediated relaxation during EFS, but concentration-dependently reduced the post-EFS contraction (Fig. 2). Threshold concentrations were between 0.3 and 1 mmol L−1 for C2, C3, C4 and C5, and >1 mmol L−1 for C1; the rank order of potency was C2 ≈ C3 ≈ C4 > C5 > C1. At 100 mmol L−1 there was a c. 85% reduction in the post-EFS contraction for C2, C3, C4 and C5 and a 63% reduction for C1. EC50 values were not determined, as the inhibitory effects did not achieve maxima, even at the highest concentrations of SCFAs permitted in this assay. There were no differences between the effects of C2, C3 and C4 at each of the concentrations (P > 0.05). In separate studies, there were no significant differences in the ability of C3 (0.1–100 mmol L−1) to decrease the amplitude of post-EFS contractions in colon preparations with or without mucosa (P > 0.05; Fig. 3).

image

Figure 2.  Formate (C1), acetate (C2), propionate (C3), butyrate (C4) and pentanoate (C5) concentration-dependently reduced neuronally mediated contractions of rat isolated distal colon circular muscle preparations. The results are expressed as the mean ± SEM reduction in the amplitudes of contractions evoked after termination of EFS (post-EFS contractions); 15 min contact, n = 4–16 at each concentration, *P < 0.05, compared with C2. The traces illustrate the inhibitory activity of each SCFA 10 mmol L−1 in single preparations of rat colon, with each contraction representing the post-EFS response. EFS was applied for 20 s every 40 s using biphasic square-wave pulses of 0.5 ms pulse width, 5 Hz frequency at maximally effective voltage.

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image

Figure 3.  Propionate (C3) concentration-dependently reduced post-EFS contractions in rat isolated distal colon preparations, with or without mucosa (15 min contact, n = 4). The results are expressed as the mean ± SEM reduction in the amplitudes of contractions and there was no significant difference at each of the concentrations. EFS was applied for 20 s every 40 s using biphasic square-wave pulses of 0.5 ms pulse width, 5 Hz frequency at maximally effective voltage.

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At the highest concentration used, 100 mmol L−1, SCFAs did not affect the pH of the Kreb's solution. The osmolarity at this concentration was c. 400 mosmol L−1 and control studies using saline adjusted to 400 mosmol L−1 had no effect on the amplitude of post-EFS contractions, 9 ± 6% change in amplitude cf. vehicle 4 ± 4%, n = 4, P = 0.2. C2, 30 mmol L−1, had no effect on the amplitude of contractions induced by a submaximally effective concentration of carbachol (10 μmol L−1, 60 s contact time), suggesting that C2 acts at a pre-junctional neuronal site; 7 ± 5% change in amplitude cf. vehicle 2 ± 5%, n = 4, P = 0.2.

Effects of SCFAs on peristalsis in guinea-pig ileum

C2, C3 and C4 at 1 mmol L−1 (n = 4–6) had no significant effect on any parameter of peristalsis measured (data not shown). However, C2, C3 and C4 at 100 mmol L−1 (n = 5–8) significantly decreased the peristaltic pressure threshold, the interval between peristaltic waves and the peristaltic peak amplitude (Fig. 4). At 300 mmol L−1 (n = 4), C2, C3 and C4 disrupted peristalsis (data not shown). A mixture of C2 : C3 : C4 (59 : 7 : 34 mmol L−1, n = 6), at concentrations and a ratio shown to occur in the lumen of the rat proximal colon, exerted highly significant effects on the different parameters of peristalsis when compared with individual SCFAs (Fig. 4). These were most significant during the first time period. Saline osmolality controls (data not shown), formate and pentanoate had no significant effect on any of the parameters of peristalsis (Fig. 4). The rank order potency of SCFAs was C2 ≈ C3 ≈ C4 > C5 ≈ C1.

image

Figure 4.  Acetate (C2), propionate (C3) and butyrate (C4), but not formate (C1) or pentanoate (C5), modulate peristaltic motility in the guinea-pig isolated terminal ileum. Changes in peristaltic movements were assessed over three separate time periods (c. 200 s each) spanning 10 min. C2, C3 and C4 at 100 mmol L−1 (n = 5–8) and a mixture of C2 : C3 : C4 (59 : 7 : 34 mmol L−1, n = 6), at concentrations and a ratio shown to occur in the lumen of the rat proximal colon, significantly decreased the interval between peristaltic waves, reduced the peristaltic peak amplitude and lowered the peristaltic pressure threshold. *P < 0.05; **P < 0.01; ***P < 0.001.

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GPR43 mRNA distribution

Significant levels of GPR43 mRNA and the housekeeping gene cyclophilin (data not shown) were detected throughout the rat gut (n = 6–8); lowest levels were observed in the oesophagus and stomach and the highest in the colon (Fig. 5). Little to no GPR43 mRNA was detected in the CNS (n = 3–4).

image

Figure 5.  mRNA distributions of GPR43 throughout the full-thickness rat GI tract and selected CNS regions. Relative expression levels for each transcript were measured using real-time PCR where equivalent amounts of cDNAs were compared. Results are expressed in relative arbitrary units (RAU) as mean ± SEM (n = 6–8 for GI regions and n = 3–4 for CNS regions).

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Effects of SCFAs on EFS-evoked responses in GPR43+/+ and GPR43−/− mouse colon

Similar to the rat, the majority of distal colon preparations from GPR43+/+ mice, evoked biphasic responses. During EFS a small-amplitude relaxation was observed in 80% of preparations, while in the remaining 20%, a small contraction occurred [−8 ± 1% (n = 3) and 46 ± 28% (n = 2) of the amplitude of post-EFS contractions, respectively]. Termination of EFS consistently evoked a large amplitude ‘post-EFS’ contraction. All responses to EFS were prevented by 1 μmol L−1 tetrodotoxin (n = 4, 20 min contact). Addition of atropine (1 μmol L−1, 20 min contact) prevented any contraction during EFS and increased the amplitude of the relaxation response by 156 ± 44% (n = 4) whilst reducing the amplitude of the post-EFS contraction by 83 ± 16% (n = 5).

C1, C2, C3 and C4 (0.1–100 mmol L−1, 15 min contact, n = 3–6) had no effects on nerve-mediated relaxations during EFS but concentration-dependently reduced post-EFS contractions in both the GPR43+/+ and GPR43−/− mouse colons (Fig. 6a,b); the effects on any small contractions occurring during EFS were not assessed due to the inconsistency of the response. Threshold concentrations were between 0.3 and 1 mmol L−1 for all of the SCFAs tested; EC50 values could not be determined. For both GPR43+/+ and GPR43−/−, the rank order of potency was C2 ≈ C3 ≈ C4 > C1 and at the lower concentrations, this rank-order was similarly reflected in terms of efficacy.

image

Figure 6.  Formate (C1), acetate (C2), propionate (C3) and butyrate (C4) concentration-dependently reduced neuronally mediated contractions of (a) GPR43+/+ and (b) GPR43−/− mouse isolated colon circular muscle preparations. The results are expressed as the mean ± SEM reduction in the amplitudes of contractions evoked after termination of EFS (post-EFS contractions); 15 min contact, n = 3–6 at each concentration. There were no signifcant differences in the effects of C1, C2, C3 or C4, at each of the concentrations, between GPR43+/+ and GPR43−/− mice colons. EFS was applied for 20 s every 40 s using biphasic square-wave pulses of 0.5 ms pulse width, 5 Hz frequency at maximally effective voltage.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Physiologically relevant concentrations of SCFAs may exert regionally specific effects on intestinal motility, as suggested by this study with rodent distal colon and guinea-pig terminal ileum. Although our studies do not exclude the possibility of a species-dependent difference in excitatory or inhibitory functions of SCFAs, our results are consistent with the findings of others who have recognized the different roles of SCFAs in regulating the motility of these distinct regions of the gut.

In the present study, SCFAs decreased electrically stimulated, nerve-mediated, post-EFS contractions in mouse and rat isolated distal colon circular preparations; correlations between the abilities of drugs which modulate this type of activity and their effects on GI motility, suggests that in vivo, SCFAs will tend to inhibit colonic motility.23 The same activity was also seen in preparations of rat colon with the mucosa removed, indicating that in these experiments, the actions of the SCFAs were unlikely to be mediated via the release of 5-HT or other substances from the neuroendocrine and mast cells located within the mucosa; such an activity has previously been suggested by Mitsui et al.24 Furthermore, the inability of C2 to affect the amplitude of contractions induced by a sub-maximally effective concentration of carbachol suggests that SCFAs exert their effects via a prejunctional, predominantly cholinergic site of action, given the sensitivity of the electrically evoked contractions to atropine. Inhibitory activity has also been observed in guinea-pig isolated colon, when measuring the transit times of faecal pellets (unpublished observation), and in rat isolated colon where SCFAs inhibit spontaneous contractions10,25 and fluid output.10 By contrast, others have shown that SCFAs may induce biphasic, tetrodotoxin-sensitive contractions in rat isolated colon.24,26 In the present experiments, this type of response was not clearly observed, possibly because the SCFAs were applied to tissues undergoing regular periods of EFS rather than at rest. Stimulatory effects of intraluminal SCFAs on rat colonic transit have also been observed in vivo; these were prevented by local anaesthetics,11,12 5-HT3 receptor antagonists and vagotomy12 indicating the involvement of a local neural mechanism. There is no evidence that SCFAs influence colonic motility in man.27,28 The reasons for these different observations in the colon are currently not clear, but regional, dietary, species and even methodological differences need to be explored. Interestingly, more consistent observations have been made using the terminal ileum. Thus, in several species including humans, the presence of SCFAs in the terminal ileum, following intraluminal infusion or colo-ileal reflux, stimulates peristalsis and increases tonic activity.7,8,29,30 The present experiments with guinea-pig isolated terminal ileum are consistent with these observations, in that a reduced threshold and amplitude was matched with an increased frequency of peristaltic contractions when C2, C3 and C4 were administered singly or more especially in combination at concentrations and molar ratios known to exist in the rat proximal colon.

The amounts of SCFAs can vary considerably with diet, rates of absorption, utilization for bacterial synthesis and the net transport of water.14 Nevertheless, the concentrations of SCFAs used in the present study were considered physiological, as they were similar to the concentrations of SCFA anions in rat intestinal luminal samples and similar to those observed in other species, including humans.2,31,32 Topping & Clifton,31 for example, report concentrations of C2, C3 and C4 within human colon in the range of 20–43, 6–13 and 6–15 mmol L−1. SCFA-induced motility effects, in isolated preparations, have also been reported over similar concentration ranges (0.01–100 mmol L−1)10,25,26,33,34 with threshold concentrations ranging between 0.01 and 1 mmol L−1.

The rank order potency of SCFAs at inhibiting post-EFS contractions in the rodent colon and stimulating peristaltic motility in the guinea-pig terminal ileum (C2 ≈ C3 ≈ C4 > C5 ≈ C1) was consistent with that found using GPR43 receptors transiently transfected in HEK293 cells, where the order of potency was C3 ≈ C2 ≈ C4 > C5 ≈ C1 for GPR43 and C3 ≈ C4 ≈ C5 > C2 > C1 for GPR41.18 However, the same rank-order of inhibitory effects of SCFAs on post-EFS contractions was observed in the GPR43−/− mouse colon. This was initially a surprising finding and it remains a possibility that the function of the GPR43 receptor has been conserved within the knock-out mouse by upregulation of another receptor, such as GPR41. We did not look for this possibility, but the similar rank-order of inhibitory activity shown by the SCFAs in both the wild-type and knock-out mice tends to argue against this idea. Recently, Karaki et al.19 demonstrated the presence of the GPR43 receptor in the PYY-containing enterochromaffin cells and 5-HT-expressing mast cells of the rat intestine, but not in the muscle or ENS of the same tissue. These findings argue against a role for the GPR43 receptor within the ENS and as such, find consistency with the maintained ability of SCFAs to modulate neuronal activity in the GPR43 KO mice.

In summary, we have shown that SCFAs exert effects on intestinal motility at concentrations which exist in the lumen and tissue of rat intestines. SCFAs were inhibitory in the rodent distal colon, an action which may retard substrates for salvageable energy, and stimulatory in guinea-pig terminal ileum. It is possible that the latter serves as a protective mechanism against inappropriate colo-ileal reflux or if present in humans, could be involved in the generation of symptoms associated with SIBO, where there is likely to be an elevation in the levels of SCFAs. Surprisingly, inhibition of post-EFS contractions in rodent colons was not mediated via GPR43, despite the similar rank-order of SCFA potency in both the colon and at the recombinant GPR43 receptor. If not acting via the GPR43 receptor, then what alternative mechanisms are possible? As shown in the present experiments and by others, the effects of the SCFAs are independent of pH or osmolality changes10–12,33 but it remains a possibility that following infusion of the SCFAs into the ENS, neuronal function is affected by local acidification. Additionally, a direct ability of butyrate to hyperpolarise rat cultured myenteric neurones has been reported35,36 and although the mechanism of action has not been determined, several other studies have described an ability of certain SCFAs to modulate enteric nerve activity.37,38 Whatever the mechanism of action, the similar rank-order of SCFA potency in both the colon and the recombinant GPR43 receptor, the role of the GPR43 receptor in intestinal function and the mechanisms by which SCFAs modulate intestinal motility, are each areas which remain to be clarified.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Judy Latcham (LAS, GSK, UK) for breeding and husbandry of GPR43 gene knockouts, David Hurp (DPG, GSK, UK) for providing data to help genotype the wildtype and knockout mice, Andrew J. Brown (ADCP, GSK, UK) for expert advice on SCFA receptor pharmacology and Simon Bate for statistical analysis.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
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