Investigation of triamterene as an inhibitor of the TGR5 receptor: identification in cells and animals.

BACKGROUND: G-protein-coupled bile acid receptor 1 (GPBAR1, also known as TGR5) has been shown to participate in glucose homeostasis. In animal models, a TGR5 agonist increases incretin secretion to reduce hyperglycemia. Many agonists have been developed for clinical use. However, the effects of TGR5 blockade have not been studied extensively, with the exception of studies using TGR5 knockout mice. Therefore, we investigated the potential effect of triamterene on TGR5. METHODS: We transfected the TGR5 gene into cultured Chinese hamster ovary cells (CHO-K1 cells) to express TGR5. Then, we applied a fluorescent indicator to examine the glucose uptake of these transfected cells. In addition, NCI-H716 cells that secrete incretin were also evaluated. Fura-2, a fluorescence indicator, was applied to determine the changes in calcium concentrations. The levels of cyclic adenosine monophosphate (cAMP) and glucagon-like peptide (GLP-1) were estimated using enzyme-linked immunosorbent assay kits. Moreover, rats with streptozotocin (STZ)-induced type 1-like diabetes were used to investigate the effects in vivo. RESULTS: Triamterene dose dependently inhibits the increase in glucose uptake induced by TGR5 agonists in CHO-K1 cells expressing the TGR5 gene. In cultured NCI-H716 cells, TGR5 activation also increases GLP-1 secretion by increasing calcium levels. Triamterene inhibits the increased calcium levels by TGR5 activation through competitive antagonism. Moreover, the GLP-1 secretion and increased cAMP levels induced by TGR5 activation are both dose dependently reduced by triamterene. However, treatment with KB-R7943 at a dose sufficient to block the Na+/Ca2+ exchanger (NCX) failed to modify the responses to TGR5 activation in NCI-H716 cells or CHO-K1 cells expressing TGR5. Therefore, the inhibitory effects of triamterene on TGR5 activation do not appear to be related to NCX inhibition. Blockade of TGR5 activation by triamterene was further characterized in vivo using the STZ-induced diabetic rats. CONCLUSION: Based on the obtained data, we identified triamterene as a reliable inhibitor of TGR5. Therefore, triamterene can be developed as a clinical inhibitor of TGR5 activation in future studies.

Constitutive androstane receptor-mediated changes in bile acid composition contributes to hepatoprotection from lithocholic acid-induced liver injury in mice.

Pharmacological activation of the constitutive androstane receptor (CAR) protects the liver during cholestasis. The current study evaluates how activation of CAR influences genes involved in bile acid biosynthesis as a mechanism of hepatoprotection during bile acid-induced liver injury. CAR activators phenobarbital (PB) and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) or corn oil (CO) were administered to C57BL/6 wild-type (WT) and CAR knockout (CAR-null) mice before and during induction of intrahepatic cholestasis using the secondary bile acid, lithocholic acid (LCA). In LCA-treated WT and all the CAR-null groups (excluding controls), histology revealed severe multifocal necrosis. This pathology was absent in WT mice pretreated with PB and TCPOBOP, indicating CAR-dependent hepatoprotection. Decreases in total hepatic bile acids and hepatic monohydroxy, dihydroxy, and trihydroxy bile acids in PB- and TCPOBOP-pretreated WT mice correlated with hepatoprotection. In comparison, concentrations of monohydroxylated and dihydroxylated bile acids were increased in all the treated CAR-null mice compared with CO controls. Along with several other enzymes (Cyp7b1, Cyp27a1, Cyp39a1), Cyp8b1 expression was increased in hepatoprotected mice, which could be suggestive of a shift in the bile acid biosynthesis pathway toward the formation of less toxic bile acids. In CAR-null mice, these changes in gene expression were not different among treatment groups. These results suggest CAR mediates a shift in bile acid biosynthesis toward the formation of less toxic bile acids, as well as a decrease in hepatic bile acid concentrations. We propose that these combined CAR-mediated effects may contribute to the hepatoprotection observed during LCA-induced liver injury.

Effect of inhibition of protein synthesis on cholestasis induced by taurolithocholate, lithocholate, and a manganese-bilirubin combination in the rat.

Taurolithocholate, lithocholate, and a manganese-bilirubin combination produced a rapid reduction in bile flow after i.v. injection in the rat. The effect was diminished or blocked completely by pretreating the animals with cycloheximide or ethionine, known inhibitors of protein synthesis. The injection sequence and time period between administration of the inhibitor of protein synthesis and the cholestatic agent influenced the degree to which they modulated the cholestatic effect. The results indicate that uninterrupted protein synthesis is required for the expression of maximal reduction of bile flow by taurolithocholate, lithocholate, and a manganese-bilirubin combination.

Supplemental administration of 1 alpha-hydroxyvitamin D3 inhibits promotion by intrarectal instillation of lithocholic acid in N-methyl-N-nitrosourea-induced colonic tumorigenesis in rats.

The effect of 1 alpha-hydroxyvitamin D3 [1 alpha(OH)D3] on promotion by intrarectal instillation of lithocholic acid (LC) in N-methyl-N-nitrosourea (MNU)-induced colonic tumorigenesis was studied in a rodent model. Ninety-two female F344 rats received intrarectal injection of 2.5 mg of MNU twice in one week followed by 1 mg of LC or its vehicle alone three times weekly for 48 weeks. Those which received LC were given a concomitant intragastric administration of 0.04 micrograms of 1 alpha(OH)D3 or its vehicle alone three times weekly. In the group receiving MNU alone (n = 30) five rats bore colonic tumors; in the MNU + LC group (n = 32) 15 and in the MNU + LC + 1 alpha(OH)D3 group (n = 30) six rats bore colonic tumors (MNU + LC versus MNU + LC + 1 alpha(OH)D3 group, P less than 0.05). These results indicated that promotion of MNU-induced colonic tumorigenesis by LC was suppressed by supplemental administration of 1 alpha(OH)D3.