Fig. 1.

Acetoacetate is a ligand for GPR43. (A and B) cAMP levels in response to monocarboxylic acids (5 mM each) treatment (A) or acetoacetate and acetate in a dose-dependent manner (B) in Flp-in GPR43 T-REx HEK293 cells. After 24 h in culture, cells were treated with doxycycline (10 μg/mL) and cultured for another 24 h. Cells precultured with 3-isobutyl-1-methylxanthine (IBMX) for 30 min were treated in the presence of each ligand for 10 min. The intracellular cAMP levels were determined using a cAMP assay kit, and data are presented as relative to the forskolin-induced cAMP levels (n = 4 to 6). (C) Effects of acetoacetate on ERK1/2 phosphorylation in Flp-in GPR43 T-REx HEK293 cells. After 24 h in culture with doxycycline (10 μg/mL), cells were cultured for another 24 h in serum-free DMEM. The cells were treated with various concentrations (0.1 to 30 mM) of acetoacetate or 1 mM propionate for 10 min (n = 6 or 7). (D) Mobilization of [Ca²⁺]_i induced by acetate, propionate, acetoacetate, and β-hydroxybutyrate was monitored in Flp-in GPR43 T-REx HEK293 cells; data are presented as Ca²⁺ intensity. Open symbols represent values from cells without doxycycline treatment; closed symbols, values from cells with doxycycline treatment. (E) cAMP levels in response to acetoacetate in mouse embryonic fibroblast (MEF)-derived adipocytes. Adipocytes precultured with IBMX for 30 min were treated in the presence of 500 μM or 1 mM acetoacetate or 1 mM propionate for 10 min (n = 8). (F) Effects of acetoacetate on ERK1/2 phosphorylation in MEF-derived adipocytes. The adipocytes were treated with 500 μM or 1 mM acetoacetate or 1 mM propionate for 10 min (n = 8). (G) [Ca²⁺]_i induced by acetoacetate in MEF-derived adipocytes; data indicate the peak of Ca²⁺ intensity (n = 8). Hatched bars represent adipocytes from Gpr43^−/− mice (E–G). **P < 0.01; *P < 0.05, compared with (−), Dunnett’s test. All data are presented as mean ± SEM.