Development of betulinic acid as an agonist of TGR5 receptor using a new in vitro assay

Figures & data

Figure 1 Chemical structure of betulinic acid.

Figure 2 Role of TGR5 in glucose uptake in Chinese hamster ovary (CHO-K1) cells.

Notes: Successful transfection of CHO-K1 cells with TGR5 is shown in A. Dose-dependent changes in glucose uptake by betulinic acid in TGR5-transfected CHO-K1 cells (TGR5-CHO-K1 cells) compared to cells transfected with empty vector (CHO-K1 cells) are shown in B. Effects induced by lithocholic acid are shown in C. The uptake of glucose was measured using a commercial kit. Values (mean ± SE) were obtained from eight determinations in each group. *P<0.05, **P<0.01, and ***P<0.001 compared to the vehicle-treated group (0 mol/L).
Abbreviations: CHO-K1, Chinese hamster ovary cells; 2-NBDG, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose; SE, standard error.

Figure 3 Changes in cAMP levels in CHO-K1 cells.

Notes: Dose-dependent increases in cAMP induced by betulinic acid in TGR5-transfected CHO-K1 cells (TGR5-CHO-K1 cells) compared with cells transfected with empty vector (CHO-K1 cells) are shown in A. Effects induced by lithocholic acid are shown in B. The amount of cAMP was measured using a commercial kit. Values (mean ± SE) were obtained from eight determinations in each group. *P<0.05 and **P<0.01 compared with the vehicle-treated group (0 mol/L).
Abbreviations: cAMP, cyclic adenosine monophosphate; CHO-K1, Chinese hamster ovary cells; SE, standard error.

Figure 4 Effect of protein kinase A inhibition on the changes in glucose uptake in CHO-K1 cells.

Notes: Betulinic acid-induced changes in glucose uptake in TGR5-CHO-K1 cells were reduced by protein kinase A inhibitor (PKI), as shown in A. Effects induced by lithocholic acid are shown in B. Values (mean ± SE) were obtained from eight determinations in each group. *P<0.05 and **P<0.01 compared with the vehicle-treated group. ^#P<0.05 and ^##P<0.01 compared with the basal group without treatment.
Abbreviations: CHO-K1, Chinese hamster ovary cells; 2-NBDG, 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxyglucose; SE, standard error.

Figure 5 Role of TGR5 in GLP-1 secretion from NCI-H716 cells.

Notes: Success of TGR5 silencing identified in NCI-H716 cells is shown in A where siTGR5 indicates the treatment with TGR5-specifc siRNA and scramble indicates the negative control. Dose-dependent increases in GLP-1 secretion by betulinic acid in scramble-transfected NCI-H716 cells, marked as TGR5 (+) cells, compared with cells that received siRNA targeting TGR5, marked as TGR5 (−) cells, are shown in B. Effects induced by lithocholic acid are shown in C. The amount of GLP-1 was measured using a commercial kit. Values (mean ± SE) were obtained from eight determinations in each group. *P<0.05 and **P<0.01 compared with the vehicle-treated group (0 mol/L). ^#P<0.05 compared with that in TGR5 (+) cells at the same concentration.
Abbreviations: GLP-1, glucagon-like peptide-1; SE, standard error; siRNA, small interfering RNA.

Figure 6 Role of TGR5 in calcium influx into NCI-H716 cells.

Notes: Dose-dependent increases in cellular calcium (Ca²⁺) induced by betulinic acid in scramble-transfected NCI-H716 cells, marked as TGR5 (+) cells, compared with cells that received siRNA targeting TGR5, marked as TGR5 (−) cells, are shown in A. Effects induced by lithocholic acid are shown in B. Calcium concentration was measured using fura-2 assay. Values (mean ± SE) were obtained from eight determinations in each group. *P<0.05 and **P<0.01 compared with the vehicle-treated group (0 mol/L). ^#P<0.05 compared with that in TGR5 (+) cells at the same concentration.
Abbreviations: siRNA, small interfering RNA; SE, standard error.