Short Chain Fatty Acids Enhance Expression and Activity of the Umami Taste Receptor in Enteroendocrine Cells via a Gαi/o Pathway

Abstract

The short chain fatty acids (SCFAs) acetate, butyrate and propionate, are produced by fermentation of non-digestible carbohydrates by the gut microbiota and regulate appetite, adiposity, metabolism, glycemic control, and immunity. SCFAs act at two distinct G protein coupled receptors (GPCRs), FFAR2 and FFAR3 and are expressed in intestinal enteroendocrine cells (EECs), where they mediate anorectic gut hormone release. EECs also express other GPCRs that act as nutrient sensors, thus SCFAs may elicit some of their health-promoting effects by altering GPCR expression in EECs and enhance gut sensitivity to dietary molecules. Here, we identify that exposure of the murine EEC STC-1 cell line or intestinal organoids to physiological concentrations of SCFAs enhances mRNA levels of the umami taste receptors TASR1 and TASR3, without altering levels of the SCFA GPCRs, FFAR2 and FFAR3. Treatment of EECs with propionate or butyrate, but not acetate, increased levels of umami receptor transcripts, while propionate also reduced CCK expression. This was reversed by inhibiting Gαi/o signaling with pertussis toxin, suggesting that SCFAs act through FFAR2/3 to alter gene expression. Surprisingly, neither a FFAR3 nor a FFAR2 selective ligand could increase TASR1/TASR3 mRNA levels. We assessed the functional impact of increased TASR1/TASR3 expression using unique pharmacological properties of the umami taste receptor; namely, the potentiation of signaling by inosine monophosphate. Activation of umami taste receptor induced inositol-1-phosphate and calcium signaling, and butyrate pretreatment significantly enhanced such signaling. Our study reveals that SCFAs may contribute to EEC adaptation and alter EEC sensitivity to bioactive nutrients.

Keywords: GPCR (G protein coupled receptor); enteroendocrine cell; heterotrimeric G protein; short chain fatty acid (SCFA); umami taste receptor.

Figure 1

Exposure to SCFAs significantly changes the expression profile of taste receptors in STC-1 cells. (A) RNA was extracted from STC-1 cells for qPCR analysis of taste receptors TAS1R1, TAS1R3, TAS1R2, TAS2 (108), and TAS2 (138); free fatty acids receptors FFAR2 and FFAR3; and taste-specific G-protein α-gustducin, and normalized to the levels of housekeeping gene β-actin. (B–G) STC-1 cells were treated with NaCl (control; white bars), 5 mM SCFAs (gray bars), or 10 mM SCFA (black bars) for 2 h for (B only) and between 1 and 5 h as indicated, after which RNA was extracted, purified and quantified with qPCR. (G,H) Ileal intesinal organoids were treated with 10 mM NaCl or SCFAs for 5 h and RNA extracted and quantified via qPCR. Results are expressed as a fold change in expression over the untreated control and all data represent the average ± SEM, n = 3. The red line indicates a fold change of 1. (A–F) Two-way ANOVA, with Bonferroni post hoc, $$$p < 0.001 SCFA (10 mM) vs. NaCl control, **p < 0.01, ***p < 0.001 SCFA (5 mM) vs. NaCl control. (G,H) Unpaired t-test, *p < 0.05, ***p < 0.001.

Figure 2

SCFAs and synthetic ligands differ in their ability to upregulate the umami taste receptor. (A,B) STC-1 cells were incubated with NaCl or SCFAs (10 mM) for 5 h, after which, RNA was extracted and purified. Expression of taste receptors TAS1R1 (A), TAS1R3 (B) was quantified using qPCR analysis and normalized to the levels of housekeeping gene β-actin. Data are expressed as mean ± SEM fold-change in expression over the untreated control (n = 3). T-tests vs. control; ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001. (C,D) STC-1 cells were incubated with either 4-CMTB or AZ420626 (10 μM) for 5 h, after which, RNA was extracted and purified. Expression of taste receptors TAS1R1 (C), TAS1R3 (D) was quantified using qPCR analysis and normalized to the levels of housekeeping gene β-actin. Data are expressed as mean ± SEM fold change in expression over the untreated control (n = 3). Unpaired t-tests vs. control; ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3

Inhibtion of Gα_i/o signaling impacts SCFA-mediated changes in taste receptor gene expression. (A,B) STC-1 cells were pretreated with Gα_i/o inhibitor pertussis toxin (PTX) (200 ng/μL, 18 h; black bars) or no pretreatment (white bars), followed by stimulation with either NaCl, propionate or butyrate (all 5 mM) for 5 hr. RNA was extracted and purified. Expression of taste receptors TAS1R1 (A), TAS1R3 (B) was quantified using qPCR analysis and normalized to the levels of housekeeping gene β-actin. Data are expressed as mean ± SEM fold change in expression over the NaCl control either with or without PTX exposure (n = 3). Two-way ANOVA, Bonferroni post hoc of no pretreatment vs. PTX treatment for each ligand; ns, non-significant; *p < 0.05; **p < 0.01.

Figure 4

Umami receptor signaling cascades are potentiated by butyrate pretreatment. (A) Intracellular Ca²⁺ levels measured in STC-1 cells, incubated with fluorescence Ca²⁺ indicator dye Fluo4-AM following addition of IMP (2 mM, black bars) or NaCl control (white bars; 2 mM) with L-Ala (20 mM) or L-MSG (20 mM). Data is expressed as mean ± SEM maximal fluorescence intensities over the control and is taken from 20 cells per sample, in duplicate (n = 3). T-test vs. control; *p < 0.05; **p < 0.01; ***p < 0.001. (B) Intracellular IP₁ accumulation measured in STC-1 cells on the addition of IMP (black bars) with either NaCl control (white bars; 2 mM), L-Ala (20 mM), or L-MSG (20 mM). Data is expressed as mean ± SEM across three distinct experiments; t-test *p < 0.05; **p < 0.01; ***p < 0.001 vs. control. (C) Intracellular Ca²⁺ levels measured in butyrate-naive (white) or butyrate-pretreated (gray) STC-1 cells, incubated with fluorescence Ca²⁺ indicator dye Fluo4-AM, followed by stimulation with IMP (2 mM) with L-Ala (20 mM) or L-MSG (20 mM). Data is expressed as mean ± SEM maximal fluorescence intensities over the control and are taken from 20 cells per sample, in duplicate (n = 3). T-test vs. control, *p < 0.05; ***p < 0.001. (D) Intracellular IP₁ accumulation measured in butyrate-naive (white) or butyrate-pretreated (gray) STC-1 cells after incubation with IMP (2 mM) and either L-Ala (20 mM) or L-MSG (20 mM). Data is expressed as mean ± SEM (n = 3); t-test vs. control, *p < 0.05; ***p < 0.001. (E,F) Representative fluorescence intensity plots following IMP (2 mM) and L-Ala (E; 20 mM) or L-MSG (F; 20 mM) stimulation in butyrate pretreated (red lines) and butyrate naive (black lines) STC-1 cells, expressed in arbitrary units (AU).