Figure 6. GLP-1 secretion is enhanced in Gpr17-deficient intestinal organoids derived from Gpr17fl/fl;Vil1Cre mice.
(A) Gpr17 mRNA expression in the gut of Gpr17fl/fl (WT) and Gpr17fl/fl;Vil1Cre (KO) mice. Gene expression was calculated as 2^-∆∆ct. β-actin was used as an internal control. Gene expression was expressed relative to duodenum (n = 5 mice for WT, n = 10 mice for KO). Unpaired t tests were performed for statistical analysis.
(B) Bright-field images of intestinal organoids derived from adult WT and constitutive Gpr17 KO mice.
(C) qRT-PCR of Gpr17 mRNA in organoids (n = 3 mice). Gene expression was normalized to β-actin. Representative gel electrophoresis image of qRT-PCR products was shown. Unpaired two-tailed Student’s t test was performed.
(D) qRT-PCR of Gcg and Gip mRNA in organoids (n = 5 mice).
(E) GLP-1 (n = 19 replicates for each group) and GIP (n = 7 replicates for each group) secretion from intestinal organoids under basal conditions (1 mmol/L glucose) for 2 h. Unpaired two-tailed Student’s t test was performed.
(F) Gpr17 deficiency enhanced fatty acid-induced GLP-1 secretion (n = 7 replicates for each group), but not GIP (n = 5 replicates for each group). Organoids were incubated with palmitoleic acid, a monounsaturated fatty acid, at 40 µmol/L for 2 h.
(G) Gpr17 deficiency enhanced bile acid receptor agonist-induced GLP-1 secretion (n = 11 replicates for each group), but not GIP (n = 5 replicates for each group). Organoids were incubated with GPBAR-A, an agonist of bile acid receptor (TGR5), at 20 µmol/L for 2 h.
(H and I) Gpr17 deficiency enhanced cAMP-elevating agents-induced GLP-1 secretion (n = 3~13 replicates) (H), but not GIP (n = 3 replicates) (I). Organoids were incubated with cAMP-elevating agents, forskolin (0, 0.3, 1, 3, and 10 µmol/L) and 10 µmol/L IBMX for 2 h. p values were calculated using two-way ANOVA test. Percentage of GLP-1 secretion was calculated by measuring GLP-1 levels in the supernatants and cell lysates and normalized to WT organoids in parallel on the same day. Data were obtained from at least three independent experiments.
*p < 0.05, **p < 0.01, ***p < 0.001. Data are displayed as means ± SEM.