Butyrate responses in the ascending colon are neuronally mediated, involve PYY but not GLP-1

I. R. Tough, H. Williams, H. M. Cox. IOPPN, Wolfson CARD, King\\'s College London, London, United Kingdom.

Introduction: Butyrate is a short-chain fatty acid produced by fermentation, with mouse colonic luminal concentrations of ∼10-20 mM. Free fatty acid receptors (FFA)2 and 3 are located on enteroendocrine L-cells (containing PYY and GLP-1), with FFA2 (Gq-coupled) additionally present on enteric leukocytes and FFA3 (Gi-coupled) on neurons (1). Whilst acting on both receptors, butyrate shows FFA3 preference (2). This study aimed to determine the mechanisms underpinning the functional butyrate response in the ascending colon (AC).

Method: AC mucosa, with intact submucosal innervation, was dissected from male and female wild-type (WT) or PYY-/- (KO) mice (>10 weeks, C57Bl6/129Sv). Mucosae were voltage-clamped and short-circuit current (Isc) measured prior to basolateral tetrodotoxin (TTX; 100 nM) or vehicle pre-treatment, as described previously (3). Following vasoactive intestinal polypeptide (10 nM) stimulation, 5 mM butyrate (the maximal concentration established between 1-20 mM) was added apically or basolaterally. Endogenous GLP-1 mediation of butyrate responses was determined using the GLP-1 antagonist, exendin 9-39 (1 μM), with subsequent GLP-1 agonist, exendin 4 (100 nM) added as a control. Mean+/-1sem were obtained from 5-6 different mice and statistical significance determined using unpaired Student’s t-test or one-way ANOVA.

Results: Biphasic 5 mM butyrate responses were observed in WT AC (primary increase in Isc, +2.4±1.6 μA.cm^-2, followed by a secondary decrease of -9.5±2.5 μA.cm^-2,n=5), which were significantly larger than 1 mM (+1.2±0.8 μA.cm^-2 followed by -1.4±0.4 μA.cm^-2,n=5, P<0.05) and no different to 10 or 20 mM. There were no significant differences between control apical and basolateral butyrate responses. The decreases in Isc showed TTX-sensitivity: inhibited to 24.8±15.0% of apical responses, P<0.05 and 6.5±5.2% of basolateral responses, P<0.001. In KO, the residual reductions in Isc seen in WT were abolished, whilst the increases (+12.7±3.2 μM.cm^-2,n=5) showed partial TTX-sensitivity (+5.6±0.7 μA.cm^-2,n=5). Exendin 9-39 did not inhibit butyrate-induced increases in Isc, despite inhibiting subsequent exendin 4 responses.

Conclusion: Butyrate inhibitory responses in the AC are neuronally-mediated, possibly via FFA3 inhibition of secretomotor neurones (1), with PYY playing a role presumably via FFA2-mediated signalling (4). However, the Isc increase is not GLP-1-mediated, leaving this phase of the butyrate response unresolved.

References:

(1) Nohr MK et al. (2013). Endocrinol, 154: 3552-3564.

(2) Brown AJ et al. (2003). J Biol Chem, 278: 11312-11319.

(3) Cox HM et al. (2010). Cell Metab, 11: 532-534.

(4) Forbes S et al. (2015). Diabetes, 64: 3763-3771.

KO mice were from Prof Herbert Herzog, Garvan Institute, Sydney, Australia. Funded by the BBSRC: BB/N006763/1