GPBAR1, FFA1, and GPR119 agonists induce motilin secretion. (A) Bulk RNA sequencing data showing transcripts per million (TPM) of fat and bile sensing receptors in MLN-Venus and negative cells, (BeD) Motilin secretion in response to indicated agonists (expressed as fold change versus basal condition measured in parallel). *p < 0.05, **p < 0.01, ***p < 0.001 by Welch's t-test, or Browne-Forsyth and Welch ANOVA with Dunnett's multiple comparisons (n ¼ 6e14 wells from 3 to 7 independent experiments), (E) Fura2 (340/380 nm) ratio in one representative MLN-Venus cell during perfusion of FFA1 agonist AM1638 (10 mM, pink), (F) Calcium signal across several cells, measured as fold change in maximal fura2 ratio, as in (E). ***p < 0.001 by one-sample test (n ¼ 77 cells from 7 independent experiments), (G) Action potentials evoked by incremental (þ2 pA) 500 ms current steps from 12 to 18 pA in the presence (blue) or absence (black) of GPBAR-A (3 mM). The dotted line at 0 mV represents the threshold used to count action potential spikes. The same axis scale is applied across all traces, and the current injection protocol applied is shown below voltage traces, (H) The number of action potentials recorded as in (G). *p < 0.05, **p < 0.01 by two-way ANOVA with Sidak's multiple comparisons (n ¼ 5 cells). Mean AE SEM presented.

Contexts in source publication

Context 1

... fat ingestion and gallbladder emptying stimulate motilin release in humans [16e18], we investigated the molecular mechanisms underlying M-cell sensing of bile and products of lipid digestion. Our RNA sequencing data demonstrated enriched expression of the G-protein bile acid receptor 1 (GPBAR1), long-chain fatty acid receptors, namely FFAR1 and FFAR4 (also known as FFA1/GPR40 and FFA4/GPR120, respectively), and the monoacylglycerol receptor GPR119 ( Figure 4A). As the endogenous ligands for these receptors are non-specific, we used selective synthetic ligands to study their function. ...

Context 2

... the endogenous ligands for these receptors are non-specific, we used selective synthetic ligands to study their function. Motilin secretion was significantly stimulated by the GPBAR1-agonist GPBAR-A (3 mM, 3.4-fold), the GPR119 agonist AR231453 (100 nM, 1.5-fold), and the FFA1 agonist AM1638 (10 mM, 2.4-fold) (Figure 4BeD). Even though there was a trend towards stimulated motilin secretion with the FFA4 agonist TUG891 at the higher concentration (30 mM) tested (1.3- fold; p ¼ 0.06), at this concentration it is known to also agonise FFA1 [34]. ...

Context 3

... though there was a trend towards stimulated motilin secretion with the FFA4 agonist TUG891 at the higher concentration (30 mM) tested (1.3- fold; p ¼ 0.06), at this concentration it is known to also agonise FFA1 [34]. Neither a lower concentration of TUG891 (1 mM), which should not activate FFA1, nor the FFA4-selective compound A [35] stimulated motilin secretion ( Figure 4D). This supports long-chain fatty acid signalling via FFA1 as a potent stimulus for motilin release. ...

Context 4

... gain further mechanistic insight into FFA1-and GPBAR1-dependent stimulation of motilin release, we measured intracellular calcium levels (after loading cells with fura2) or electrical activity of MLN-Venus cells in response to agonist application. Activation of the G q -coupled receptor FFA1 with AM1638 increased the calcium-dependent fura2 fluorescence ratio by >15% in 19/77 MLN-Venus cells (Figure 4EeF). Stimulation of the G s -coupled receptor GPBAR1 significantly increased the evoked action potential firing rate in MLN-Venus cells (Figure 4Ge H). ...

Context 5

... of the G q -coupled receptor FFA1 with AM1638 increased the calcium-dependent fura2 fluorescence ratio by >15% in 19/77 MLN-Venus cells (Figure 4EeF). Stimulation of the G s -coupled receptor GPBAR1 significantly increased the evoked action potential firing rate in MLN-Venus cells (Figure 4Ge H). A similar effect on evoked action potential firing rate was described for human ileal L-cells [20], which suggests a common mechanism of action of GPBAR1 on these two enteroendocrine cell types. ...

Context 6

... examined our RNA sequencing dataset to identify potential pHsensing mechanisms [38]. We found highly enriched expression in MLN-Venus cells of the acid-sensitive transient receptor potential channel TRPC4 [39] but inhibition of TRPC4 with ML204 did not alter motilin secretion ( Figure S4AeB). The two-pore potassium channels TALK1/KCNK16 and TASK1/KCNK3 were also enriched in M-cells ( Figure S4C), but would not account for the observed pH-dependent inward current, while known proton-sensing GPCRs were largely undetectable ( Figure S4D). ...

Context 7

... found highly enriched expression in MLN-Venus cells of the acid-sensitive transient receptor potential channel TRPC4 [39] but inhibition of TRPC4 with ML204 did not alter motilin secretion ( Figure S4AeB). The two-pore potassium channels TALK1/KCNK16 and TASK1/KCNK3 were also enriched in M-cells ( Figure S4C), but would not account for the observed pH-dependent inward current, while known proton-sensing GPCRs were largely undetectable ( Figure S4D). Several acid-sensing ion channels (ASIC1-4) were expressed in MLNVenus cells ( Figure 6A). ...

Context 8

... found highly enriched expression in MLN-Venus cells of the acid-sensitive transient receptor potential channel TRPC4 [39] but inhibition of TRPC4 with ML204 did not alter motilin secretion ( Figure S4AeB). The two-pore potassium channels TALK1/KCNK16 and TASK1/KCNK3 were also enriched in M-cells ( Figure S4C), but would not account for the observed pH-dependent inward current, while known proton-sensing GPCRs were largely undetectable ( Figure S4D). Several acid-sensing ion channels (ASIC1-4) were expressed in MLNVenus cells ( Figure 6A). ...