Targeting extra-oral bitter taste receptors modulates gastrointestinal motility with effects on satiation

Abstract

Bitter taste receptors (TAS2Rs) are present in extra-oral tissues, including gut endocrine cells. This study explored the presence and mechanism of action of TAS2R agonists on gut smooth muscle in vitro and investigated functional effects of intra-gastric administration of TAS2R agonists on gastric motility and satiation. TAS2Rs and taste signalling elements were expressed in smooth muscle tissue along the mouse gut and in human gastric smooth muscle cells (hGSMC). Bitter tastants induced concentration and region-dependent contractility changes in mouse intestinal muscle strips. Contractions induced by denatonium benzoate (DB) in gastric fundus were mediated via increases in intracellular Ca(2+) release and extracellular Ca(2+)-influx, partially masked by a hyperpolarizing K(+)-efflux. Intra-gastric administration of DB in mice induced a TAS2R-dependent delay in gastric emptying. In hGSMC, bitter compounds evoked Ca(2+)-rises and increased ERK-phosphorylation. Healthy volunteers showed an impaired fundic relaxation in response to nutrient infusion and a decreased nutrient volume tolerance and increased satiation during an oral nutrient challenge test after intra-gastric DB administration. These findings suggest a potential role for intestinal TAS2Rs as therapeutic targets to alter gastrointestinal motility and hence to interfere with hunger signalling.

Figure 1. Expression of bitter taste signalling elements in mouse gut muscle and contractility responses to DB in the presence and absence of Ca²⁺-activated K⁺-channel blockers in mouse fundic smooth muscle strips.

RT-PCR transcripts coding for (a) mTAS2Rs in mouse gut smooth muscle and tongue circumvallate papillae and (b) taste signalling molecules in mouse fundic smooth muscle tissue. (c) Representative tracing of the effects of increasing concentrations of DB, in absence or presence of 100 nM charybdotoxin (CbTX), on contractility in mouse fundic muscle strips, expressed as gram-force/mm² (gF/mm²). (d) Concentration-response curves to DB in the absence (n = 30) or presence of CbTX (n = 71 mice), iberiotoxin (n = 4), TRAM-34 (n = 4) or apamin (n = 3) in mouse fundic strips. ACh = acetylcholine, IP = isoprotenerol, NG = nitroglycerine.

Figure 2. Specific contractility responses of various bitter taste receptor agonists in smooth muscle strips from different regions of the mouse gut.

Representative tracing of the effects of increasing concentrations of DB, in the absence or presence of 100 nM CbTX, on contractility in mouse antral (a) and colonic (b) muscle strips. Cumulative concentration-dependent contractility changes induced by the bitter agonists DB, chloroquine, PTC and salicin, in the presence of 100 nM CbTX in mouse antral (c) and colonic (d) muscle strips. (N = 3–21 strips, n = 3–12 mice).

Figure 3. Effects of pharmacological blockers on DB-induced contractile responses in mouse fundic muscle strips and comparison of the responses to DB in fundic muscle strips of WT and α-gust^−/− or TRPM5^−/− mice.

Effect of pharmacological blockers on contractility changes induced by DB in mouse fundic smooth muscle strips. CbTX-treated strips were pre-incubated with (a) probenecid (n = 7, P < 0.05), (b) gallein (n = 6, P < 0.001), (c) U-73122 (n = 6, P < 0.05) or (d) 2-APB (n = 4, P < 0.01) before addition of DB at increasing concentrations. Comparison of DB-induced contractions in WT (N = 15–26 strips, n = 5–21 mice) and (e) α-gust^−/− (N = 16 strips, n = 9 mice, P < 0.05) or (f) TRPM5^−/− (N = 12 strips, n = 3 mice, P > 0.05) mice in the presence of CbTX.

Figure 4. Effect of the bitter agonists DB or PTC on gastric emptying in mice in the absence or presence of the TAS2R antagonist probenecid.

Effect of intra-gastric administration of bitter agonists on gastric half-excretion time (T_1/2, a,c) and lag time (T_lag, b,d). Mice (n = 12) were pretreated with vehicle or probenecid (i.p., 50 mg/kg), 15 min before oral gavage of DB (a,b) or PTC (c,d). Two-way ANOVA; *P < 0.05, ***P < 0.001 bitter vs water; #P < 0.05 probenecid vs vehicle.

Figure 5. Expression of bitter taste signalling elements in human gastric smooth muscle cells and functional responses to bitter agonists in these cells.

(a) RT-PCR transcripts coding for hTAS2Rs, α-gustducin and α-transducin in hGSMCs and human lung tissue. (b) Immunofluorescent stainings of hGSMCs for α-gustducin and α-transducin (scale bar = 50 μm). Negative controls include cells incubated with rabbit serum (α-gustducin) or with the specific antigen peptide (α-transducin). Nuclei are stained with DAPI. (c) Mean concentration-response curve of [Ca²⁺]_i-rises in response to increasing concentrations of DB (n = 8 spots, P < 0.001) and chloroquine (n = 15 spots, P < 0.05) in hGSMCs. (d,e) Representative tracing of [Ca²⁺]_i-rises in response to increasing concentrations of DB and chloroquine in a hGSMC. (f) Ratio (n = 4) of pErk over Erk, normalized to vinculin, in hGSMC in response to Hepes-buffer or 5 mM DB. *P < 0.05 vs Hepes buffer. Inlay: Representative western blot of Erk1/2 phosphorylation after stimulation.

Figure 6

(a) Intra-gastric pressure in healthy volunteers (n = 12) during intra-gastric nutrient infusion after pretreatment with 1 μmol/kg DB or placebo. (*P < 0.05; **P < 0.01). (b) Overview of the proposed pathway for the contraction induced by DB in mouse fundic smooth muscle strips.