Internalization-Dependent Free Fatty Acid Receptor 2 Signaling Is Essential for Propionate-Induced Anorectic Gut Hormone Release

Abstract

The ability of propionate, a short-chain fatty acid produced from the fermentation of non-digestible carbohydrates in the colon, to stimulate the release of anorectic gut hormones, such as glucagon like peptide-1 (GLP-1), is an attractive approach to enhance appetite regulation, weight management, and glycemic control. Propionate induces GLP-1 release via its G protein-coupled receptor (GPCR), free fatty acid receptor 2 (FFA2), a GPCR that activates Gαi and Gαq/11. However, how pleiotropic GPCR signaling mechanisms in the gut regulates appetite is poorly understood. Here, we identify propionate-mediated G protein signaling is spatially directed within the cell whereby FFA2 is targeted to very early endosomes. Furthermore, propionate activates a Gαi/p38 signaling pathway, which requires receptor internalization and is essential for propionate-induced GLP-1 release in enteroendocrine cells and colonic crypts. Our study reveals that intestinal metabolites engage membrane trafficking pathways and that receptor internalization could orchestrate complex GPCR pathways within the gut.

Keywords: Cell Biology; Functional Aspects of Cell Biology.

Graphical abstract

Figure 1

Propionate Stimulates GLP-1 Secretion and Activates Gαi/o but Not Gαq/11 via FFA2 (A and B) Intracellular cAMP levels measured in STC-1 cells (A) or colonic crypts (B) pre-treated with Pertussis toxin (Ptx; 200 ng/mL, 20 h) prior to pre-treatment with IBMX (0.5 mM, 5 min) and then stimulated with forskolin (FSK, 3 μM) or a combination of FSK with either NaCl or sodium propionate (Pro) (1 mM, 5 min). Data are expressed as percent change of FSK-treated cells. n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. (C and D) Intracellular calcium mobilization measured in STC-1 cells (C) or colonic crypts (D). Cultures were incubated with calcium indicator Fluo4-AM for 1 h and imaged live via confocal microscopy for 1 min before the addition of NaCl (1 mM), sodium propionate (Pro), DMSO, orthosteric FFA2 agonist Cmp1 (10 μM), or allosteric FFA2 agonist 4-CMTB (10 μM). Average maximal intensities of n = 20 cells in duplicate per six independent experiments. Two-sided Mann-Whitney U test, ∗p < 0.05. (E and F) Intracellular accumulation of IP₁ in STC-1 cells (E) or colonic crypts (F). Cultures were treated with NaCl, sodium propionate (Pro) (1 mM), DMSO, Cmp1 (10 μM), or 4-CMTB (10 μM) for 2 h. STC-1 cells or crypts, n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. Data represent mean ± SEM. (G and H) (G) STC-1 cells and (H) colonic crypts were treated with either NaCl or sodium propionate (Pro) (1 mM, 2 h STC-1, 1 h crypts), and total GLP-1 levels secreted were measured via RIA. Data are expressed as fold change of total GLP-1 and normalized to basal (NaCl) secretion within the same experiment. For STC-1 cells, n = 4 independent experiments. For crypts, n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗p < 0.05, ∗∗p < 0.01. (I) Intracellular cAMP levels measured in colonic crypts from wild-type (WT) or FFA2 knockout mice (FFA2 ^−/−) pre-treated with IBMX (0.5 mM, 5 min) and then stimulated as in (C) and (D). Data are expressed as percent change of FSK-treated cells. n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. (J) Expression levels of FFA3 in WT and FFA2 ^−/− colonic crypts. mRNA isolated from colonic crypts of WT and FFA2 ^−/− mice were used in qPCR studies with specific mouse FFA3 primers. Data are presented as ΔΔCt. Two-sided Mann-Whitney U test. Data represent mean ± SEM. See also Figures S1–S4 and Videos S1, S2, and S3.

Figure 2

Propionate-Dependent Gαi/o Signaling Requires Receptor Internalization (A) Representative confocal microscopy images of HEK 293 cells expressing FLAG-FFA2 were pre-treated with either DMSO (vehicle) or Dyngo-4a (50 μM, 45 min), fed with M1 anti-FLAG antibody prior to stimulation with either NaCl or sodium propionate (Pro) (1 mM, 20 min). Fixed cells were imaged via confocal microscopy. (B and C) Intracellular cAMP levels (B) or calcium mobilization (C) measured in HEK 293 cells expressing FLAG-FFA2 pre-treated with either DMSO (vehicle) or Dyngo-4a (50 μM, 45 min). For (B), cells were pre-treated with IBMX (0.5 mM, 5 min) and then stimulated with forskolin (FSK, 3 μM) or a combination of FSK with either NaCl or sodium propionate (Pro) (1 mM, 5 min). n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. For (C), cells were incubated with calcium indicator Fluo4-AM for 1 h and imaged live via confocal microscopy for 1 min before the addition of either NaCl or sodium propionate (Pro) (1 mM). Average maximal intensities of n = 20 cells in duplicate per four independent experiments. (D) Representative confocal microscopy images of WT or β-ARR KO HEK 293 cells expressing FLAG-FFA2. Cells were treated with FLAG antibody and ligands and imaged as in (A). (E and F) Intracellular cAMP levels (E) or calcium mobilization (F) measured in WT or β-ARR KO HEK 293 cells transiently expressing FLAG-FFA2. Samples were treated and assayed as in (B) and (C). n = 3 independent experiments for either WT or β-ARR KO HEK 293 cells transiently expressing FLAG-FFA2. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. (G) Representative confocal images of STC-1 cells transiently expressing FLAG-FFA2 pre-treated with either DMSO (vehicle) or Dyngo-4a (50 μM, 45 min) then stimulated as in (A). (H) Intracellular cAMP levels of STC-1 pre-treated with either DMSO (vehicle) or Dyngo-4a (50 μM, 45 min). Scale bar, 5 μm; scale bar in inset, 1 μm. n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. For confocal images, representative images are shown of ~10 cells/experiment. Data represent mean ± SEM. See also Figure S5.

Figure 3

FFA2 Internalizes to Endosomes Exhibiting Properties of VEEs (A) Representative confocal microscopy images of HEK 293 cells expressing FLAG-FFA2 or FLAG- β2AR or STC-1 cells expressing FLAG-FFA2 or FLAG-β2AR imaged live with confocal microscopy before and after ligand treatment. FFA2 was stimulated with sodium propionate (Pro) (1 mM), and β2AR with isoproterenol (Iso, 10 μM) for 20 min. Scale bars, 5 μm; scale bar in inset, 1 μm. (B) Bar graph showing diameter of FFA2 or β2AR in HEK 293 or STC-1 cells containing endosomes. Endosome diameter was assessed by measuring the diameter of 20 endosomes, n = 10 cells per condition, collected across three independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. (C) Representative confocal microscopy images of fixed HEK 293 cells stably expressing FLAG-FFA2 or β2AR or STC-1 cells transiently expressing FLAG-FFA2 treated with ligand for 20 min prior to “stripping” by PBS/EDTA (to remove surface bound FLAG antibody), fixation and stained with anti-EEA1 antibody. Scale bars, 5 μm; scale bar in inset, 1 μm. (D) Numbers of FFA2 or β2AR-containing endosomes positive for EEA1 quantified from (C); 200 endosomes per condition, 10 cells quantified per condition. Data represent mean ± SEM, n = 10 cells per condition, collected across three independent experiments. Two-sided Mann-Whitney U test, ∗∗p < 0.01, ##p < 0.01. (E, F) FFA2 colocalizes with APPL1. (E) Representative confocal microscopy images of fixed HEK 293 cells stably expressing FLAG-FFA2 or LHR or STC-1 cells transiently expressing FLAG-FFA2 treated with ligand (LH for LHR). Cells were treated as in (C) except that cells were stained with anti-APPL1 antibody. Scale bars, 5 μm; scale bar in inset, 1 μm. (F) Numbers of FFA2 or LHR-containing endosomes positive for APPL1 quantified from (E); 200 endosomes per condition, 10 cells quantified per condition. n = 10 cells per condition, collected across three independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. For confocal images, representative images are shown of ~10 cells/experiment. Data represent mean ± SEM. See also Figure S6.

Figure 4

FFA2 Trafficking and G Protein Signaling Is Regulated by APPL1 (A) Representative western blot of total cellular levels of APPL1 from cells transfected with either scramble or APPL1 siRNA. GAPDH was used as a loading control. (B) Representative confocal microscopy images of propionate-induced internalization and recycling following APPL1 siRNA-mediated knockdown. HEK 293 cells stably expressing FLAG-FFA2 were labeled with anti-FLAG antibody and then treated with NaCl (1 mM) or propionate (Pro) (1 mM) for 20 min, then “stripped” and incubated with ligand-free medium for 1 h to allow receptor recycling. Scale bars, 5 μm. (C) Recycling of HEK 293 cells stably expressing SEP-FFA2 was measured in real time, via TIRFM; cells were transfected either with scramble or APPL1 siRNA and stimulated with NaCl (1 mM) or sodium propionate (Pro) (1 mM) for 5 min n = 20 cells per condition, collected across four independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. (D and E) APPL1 negatively regulates propionate-mediated Gαi signaling. HEK 293 cells stably expressing FLAG-FFA2 (D) or STC-1 cells (E) transfected with either scramble of APPL1 siRNA prior to pre-treatment of IBMX (0.5 mM, 5 min) and then stimulated with forskolin (FSK, 3 μM) or a combination of FSK and NaCl or stated SCFAs (1 mM, 5 min). Data are expressed as percent change of FSK and NaCl treatment. n = 4 independent experiments. Two-sided Mann-Whitney U test, ∗p < 0.05; ∗∗p < 0.01. (F and G) FFA2 colocalizes with Gα_i within APPL1 endosomes. Representative TIRFM images of HEK 293 cells stably expressing FLAG-FFA2 (magenta), Gαi (green), APPL1 (blue) in cells stimulated either with NaCl (1 mM) or sodium propionate (Pro) (1 mM) for 5 min (F). Dotted line marks cell boundary. The lower panel of each treatment, highlighted in red, shows higher magnification image of the region of colocalization indicated by the red box in the corresponding upper-panel image. Arrows indicate FFA2 endosomes positive for Gαi only; circle indicates FFA2 endosomes positive for Gαi and APPL1; squares indicate FFA2 endosome positive for APPL1 only. Scale bars of upper-panel images, 10 μm; scale bar of lower-panel images 1 μm. (G) Quantification of FFA2 endosomes positive for Gαi, APPL1, or Gαi and APPL1; n = 12 cells per condition from (F) were quantified across three independent experiments. Two-way ANOVA, Bonferroni multiple comparisons test, ∗∗∗p < 0.001. Data represent mean ± SEM. See also Figures S7 and S8.

Figure 5

Endosomal Gαi/o Signaling Regulates Propionate-Mediated GLP-1 Release Stimulation of GLP-1 release from STC-1 cells (A) or colonic crypts (B) in the presence of Ptx. STC-1 cells or colonic crypts were pre-treated with either vehicle or Ptx (200 ng/mL, 20 h) prior to stimulation with either NaCl (1 mM) or sodium propionate (Pro) (1 mM) for 2 and 1 h for colonic crypts. For STC-1 cells, n = 4 independent experiments. For crypts, n = 8 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. Stimulation of GLP-1 release from STC-1 cells (C) or colonic crypts (D) in the presence of Gαq/11 inhibitor, YM-254890. STC-1 cells and colonic crypts were pre-treated with either DMSO or YM-254890 (YM, 10 μM, 5 min) and then treated as in (A) and (B). For STC-1 cells, n = 4 independent experiments. For crypts, n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗p < 0.05, ∗∗p < 0.01. Stimulation of GLP-1 release from STC-1 cells (E) or colonic crypts (F) in the presence of Dygno-4a. STC-1 cells or colonic crypts were pre-treated with either DMSO or Dyngo-4a (50 μM, 45 min for STC-1 cells and 100 μM, 45 min for colonic crypts), following pre-treatment, Dyngo-4a was co-incubated with ligands for an additional 5 min and then removed. Cells and crypts were treated as in (A) and (B). For STC-1 cells, n = 5 independent experiments. For crypts, n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗p < 0.01, ∗∗∗p < 0.001. Insets show propionate-induced GLP-1 release normalized to NaCl GLP-1 release. ∗∗p < 0.01, ∗∗∗p < 0.001. GLP-1 secretion of media and cells was detected via RIA and was expressed as fold change of total GLP-1 and normalized to NaCl secretion within the same experiment. Data represent mean ± SEM. See also Figures S9 and S10.

Figure 6

Endosomal Signaling of FFA2 Regulates GLP-1 Release via Activation of p38 (A) STC-1 cells were pre-treated with DMSO (vehicle) or Dyngo-4a (50 μM, 45 min) prior to stimulation with NaCl (1 mM) or propionate (Pro) (1 mM) for 5 or 30 min. Lysates were incubated with membranes spotted for 43 different phosphokinases (R&D systems). (Ai) Membranes highlighting location of phospho-kinase antibodies spotted onto the array. Signals of relevant kinases effected by Dyngo-4a are indicated by numbers. (Aii) Fold changes over NaCl in levels of phosphorylation that decreased in presence of Dyngo-4a. Data represent mean ± SEM of fold change values. (B–E) Representative western blots demonstrating phosphorylated p38 (P-p38) and total p38 (T-p38) of lysates from STC-1 cells pre-treated with either Ptx (B) or p38 inhibitor, SB 203580 (C). STC-1 cells were pre-treated with control or Ptx (200 ng/mL, 20 h) or SB 203580 (5 μM, 10 min) prior to stimulation of NaCl (1 mM) or propionate (Pro) (1 mM) at the indicated time points. Cell lysates were then collected for western blot analysis and probed for P-p38. Membranes were then stripped and re-probed with T-p38, which was used as a loading control (i). Densitometry and fold change analysis of P-p38 normalized to T-p38 of lysates pre-treated with Ptx, or SB 203580. Fold change of densitometry analysis of P-p38 levels normalized to basal of control or inhibitor at each time point stimulation with T-p38 (ii). Stimulation of GLP-1 release from STC-1 cells (D) or colonic crypts (E) in the presence of SB 203580. Both were pre-treated either with DMSO or SB 203580 (5 μM, 10 min), prior to stimulation with either NaCl (1 mM) or sodium propionate (Pro) (1 mM) for 2 h for STC-1 cells and 1 h for colonic crypts. For STC-1 cells and crypts, n = 3 independent experiments. Two-sided Mann-Whitney U test, ∗∗∗p < 0.001. Insets show propionate-induced GLP-1 release normalized to NaCl-induced GLP-1 release. ∗∗p < 0.01, ∗∗∗p < 0.001. GLP-1 secretion of media and cells detected via RIA and was expressed as fold change of total GLP-1 and normalized to NaCl secretion within the same experiment. Data represent mean ± SEM.