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. 2020 Aug 12;23(9):101449. doi: 10.1016/j.isci.2020.101449

Figure 6.

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.