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. 2022 Mar 1;11:e73777. doi: 10.7554/eLife.73777

Figure 2. Both FFA2 and FFA3 are expressed in myenteric neurons and promote increased gut transit.

(A) Myenteric plexus dissected from mice expressing a β-galactosidase reporter gene, driven by the Ffar2 (upper panel) or Ffar3 (lower panel) gene promoter sequences were immunostained with anti-HuC/D to identify enteric neurons (left-hand panels, red) and with X-gal to identify receptor-expressing cells (second panels, green). Merged images (third panels) showed coexpression. Myenteric plexus immunostained with X-gal and Glial Fibrillary Acidic Protein (GFAP) to identify enteric glia did not show any coexpression (right-hand panels). Blue: staining with 4′,6-diamidino-2-phenylindole (DAPI) to identify cell nuclei. Scale bar = 20 µm. Male hFFA2-DREADD-HA (B), CRE-MINUS (C), FFA2-KO-βGAL (D), or FFA3-KO-βGAL (E) mice were acclimatized for 7 days with free access to drinking water. Individual animals were then gavaged with carmine red and total GI transit time measured. Following the initial transit studies, mice were provided with 4-methoxy-3-methyl-benzoic acid (MOMBA) (15 mM) or C3 (150 mM) in the drinking water as indicated. After a further 7 days GI transit of all mice was again measured. MOMBA was then removed and the mice were again provided with water followed by a further gavage with carmine red 7 days later. Data are for individual animals (*p < 0.05, **p < 0.01, ns = not significant). One-way analysis of variance followed by Bonferroni’s Multiple Comparison Test.

Figure 2—source data 1. GI transit measurements for Figure 2.

Figure 2.

Figure 2—figure supplement 1. 4-Methoxy-3-methyl-benzoic acid (MOMBA) promotes release of GLP-1 and peptide YY (PYY) in colonic preparations from hFFA2-DREADD-HA-expressing mice.

Figure 2—figure supplement 1.

(A) Colonic crypt-containing preparations were isolated from hFFA2-DREADD-HA-expressing mice. MOMBA-promoted release of GLP-1 in a concentration-dependent fashion (*p < 0.05, ****p < 0.0001) whilst C3 did not. At maximally effective concentrations MOMBA was as effective as sorbic acid (ns = not significantly different). Isobutylmethylxanthine (IBMX) (100 µM) provided a positive control for release of GLP-1. Data represent means ± standard error of the mean (SEM) from five different preparations, One-way analysis of variance followed by Bonferroni’s Multiple Comparison Test. (B) MOMBA (open symbols) promoted sustained release of GLP-1 release from colonic tissue of hFFA2-DREADD-HA-expressing mice and this was prevented by coexposure to CATPB (closed symbols). Data represent means ± SEM from four different preparations (***p < 0.001, unpaired t-test). (C) MOMBA did not promote release of GLP-1 from colonic tissue obtained from CRE-MINUS animals. Data represent means ± SEM from four different preparations. Following a period of flow of buffer (control) into colonic tubes from hFFA2-DREADD-HA (D) or CRE-MINUS (E) animals introduction of MOMBA (open symbols)-promoted transient PYY release from tissue of hFFA2-DREADD-HA-expressing mice (*p < 0.05, unpaired t-test) but not from CRE-MINUS animals. In tissue from hFFA2-DREADD-HA mice effect of MOMBA was prevented by coexposure to CATPB (filled symbols). Data represent means ± SEM from four (D) or five (E) different preparations. (F) Immunostaining of colonic sections from hFFA2-DREADD-HA (top panels) or wild-type C57BL/6 (bottom panels) with either anti-PYY (green, left) or anti-HA (red, middle) identified subsets of PYY-expressing cells that were also positive for the hFFA2-DREADD-HA receptor. Chevrons highlight exemplar cells that were positive for PYY and HA or PYY only. Scale bar = 20 µm.
Figure 2—figure supplement 1—source data 1. GLP-1 and peptide YY (PYY) secretion for Figure 2—figure supplement 1.