Bile alcohols function as the ligands of membrane-type bile acid-activated G protein-coupled receptor

Abstract

TGR5 is a G protein-coupled receptor that is activated by bile acids, resulting in an increase in cAMP levels and the subsequent modulation of energy expenditure in brown adipose tissue and muscle. Therefore, the development of a TGR5-specific agonist could lead to the prevention and treatment of various metabolic disorders related to obesity. In the present study, we evaluated the ability of bile alcohols, which are structurally and physiologically similar to bile acids and are produced as the end products of cholesterol catabolism in evolutionarily primitive vertebrates, to act as TGR5 agonists. In a cell-based reporter assay and a cAMP production assay performed in vitro, most bile alcohols with a side chain containing hydroxyl group(s) were highly efficacious agonists for TGR5 comparable to its most potent ligand in the naturally occurring bile acid, lithocholic acid. However, the abilities of the bile alcohols to activate TGR5 varied with the position and number of the hydroxyl substituent in the side chain. Additionally, the conformation of the steroidal nucleus of bile alcohols is also important for its activity as a TGR5 agonist. Thus, we have provided new insights into the structure-activity relationships of bile alcohols as TGR5 agonists.

Fig. 1.

Structures of bile alcohols. A: [1], 5β-cholestane-3α,7α,12α-triol (THC); [2], 5β-cholestane-3α,7α,12α,26-tetrol; [3], 5β-cholestane-3α,7α,12α,25-tetrol; [4], (24R)-5β-cholestane-3α,7α,12α,24-tetrol; [5], (24S)-5β-cholestane-3α,7α,12α,24-tetrol; [6], (23R)-5β-cholestane-3α,7α,12α,23-tetrol; [7], (23S)-5β-cholestane-3α,7α,12α,23-tetrol; [8], (22R)-5β-cholestane-3α,7α,12α,22-tetrol; [9], (22S)-5β-cholestane-3α,7α,12α,22-tetrol; [10], 24-nor-5β-cholane-3α,7α,12α,23-tetrol; [11], 5β-cholane-3α,7α,12α,24-tetrol; [12], 26,27-dinor-5β-cholestane-3α,7α,12α,25-tetrol; [13], 27-nor-5β-cholestane-3α,7α,12α,26-tetrol; [14], 23,24-dinor-5β-cholane-3α,7α,22-triol; [15], 24-nor-5β- cholane-3α,7α,23-triol; [16], 5β-cholane-3α,7α,24-triol; [17], 26,27-dinor-5β-cholestane-3α,7α,25-triol; [18], 27-nor-5β-cholestane-3α,7α,26-triol; [19], 5β-cholestane-3α,7α,12α,26,27-pentol; [20], 5β-cholestane-3α,7α-diol; [21], 5β-cholestane-3α,7α,26-triol; [22], 5β-cholestane-3α,7α,26,27-tetrol. B: [23], 5α-cholestane-3α,7α,12α-triol; [24], 5α-cholestane-3α,7α,12α,26-tetrol; [25], 5α-cholestane-3α,7α,12α,26,27-pentol; [26], 5α-cholestane-3α,7α,26-triol; [27], 5α-cholestane-3α,7α,26,27-tetrol. The compounds in the figure are indicated by the bracketed numbers.

Fig. 2.

Effect of the presence of a hydroxyl group at different positions in the side chain of THC on TGR5 activation in a cellular transactivation assay. Hek293T cells were transfected with CRE-luciferase reporter constructs and a human TGR5 expression plasmid. The cells were exposed to the vehicle alone or 0.1–10 μM of the indicated bile alcohols. Firefly luciferase activity in the cell extract was normalized to Renilla luciferase activity and expressed as the fold induction relative to vehicle-exposed cells. The values are the means ± SD of three experiments. The compounds in the figure are indicated by the bracketed numbers. *: The parameter was not used for the calculation of EC₅₀ value.

Fig. 3.

Effect of length of steroid side chain of bile alcohol on TGR5 activation. TGR5 activation was evaluated using the luciferase reporter assay as described in the legend to Fig. 2. The data represent the mean ± SD of three determinations. The compounds in the figure are indicated by the bracketed numbers. *: The parameter was not used for the calculation of EC₅₀ value.

Fig. 4.

Effect of A/B ring juncture conformation of bile acids on TGR5 activation. The TGR5 activation ability in a series of both 5α- (allo-) and 5β-bile acids was evaluated using the luciferase reporter assay, as described in the legend to Fig. 2. The data represent the mean ± SD of three determinations. *: The parameter was not used for the calculation of EC₅₀ value.

Fig. 5.

Effect of the number of hydroxyl groups in the side chain of bile alcohols and their A/B ring juncture conformation on TGR5 activation. The TGR5 activation ability of a series of bile alcohols with the hydroxyl group in the 3α-, 7α-, and 12α-positions (A) and in the 3α- and 7α-positions (B) of their steroid nucleus was evaluated using the luciferase reporter assay, as described in the legend to Fig. 2. The data represent the mean ± SD of three determinations. The compounds in the figure are indicated by the bracketed numbers. *: The parameter was not used for the calculation of EC₅₀ value.

Fig. 6.

hTGR5-transfected HEK293T cells produced cAMP after stimulation with LCA or bile alcohols. HEK293T cells were transfected with an hTGR5 expression plasmid. Then, the cells were stimulated with vehicle (DMSO) only or LCA or various bile alcohols at 37°C for 30 min. cAMP production and the protein concentration were then determined. The data represent the mean ± SD of three determinations. The compounds in the figure are indicated by the bracketed numbers. *: P < 0.05; **: P < 0.01 versus vehicle (DMSO)-treated cells.