Recently accumulated evidence has revealed a

Recently, accumulated evidence has revealed a close link of FXR to gluconeogenesis. For example, activation of FXR by its agonist GW4064 improved hyperglycemia in db/db mice by repressing gluconeogenesis [15], and FXR agonist CDCA decreased transactivation activity of key gluconeogenic genes by inducting the expression of small heterodimer partner (SHP) [16]. Interestingly, similar to phosphoenolpyruvate carboxykinase (PEPCK) gene, FXR mRNA level in the liver of mice was induced during fasting and decreased after refeeding [17], while pronounced reduction of PEPCK gene expression was observed by refeeding in FXR−/− mice [18]. These results thus imply that FXR is involved in glucose metabolism during fasting-refeeding transition. In addition, in FXR−/− mice, the level of hepatic gluconeogenic gene expression was decreased after fasting, while blood glucose level and the hepatic glucose production in response to pyruvate challenge were declined, indicative of the restrained gluconeogenesis during FXR deficiency [18,19]. Currently, several assumptions have been proposed for gluconeogenesis modulation in response to FXR deficiency. For example, it is suggested that FXR regulates hepatic gluconeogenesis through glucocorticoid receptor in the transition from unfed to fed state or FXR controlled gluconeogenesis in an indirect way by regulating PPARα and tribbles homolog 3 (TRB3) [19,20]. Besides, an intestinal FXR-ceramide signaling axis has been also proved to regulate hepatic gluconeogenesis [21]. Notably, treatment of rat hepatoma BML-277 or primary hepatocytes by FXR agonist CDCA or GW4064 sharply stimulated the mRNA levels of glucose 6-phosphatase (G6Pase) and PEPCK, and FXR antagonist guggulsterone antagonized CDCA or GW4064-induced promotion of G6Pase and PEPCK [20]. Besides, FXR antagonist NDB effectively decreased gluconeogenic gene expression in the liver of db/db mice [22]. All results have clearly demonstrated the regulation of FXR antagonist against gluconeogenesis although the underlying mechanism remains obscure. Here, we determined a new specific FXR antagonist HS218 (N-benzyl-N-(3-(tert- butyl)-4-hydroxyphenyl)-2,4-dichlorobenzamide) (Fig. 1A), and identified that HS218 inhibited FXR-dependently gluconeogenesis by inhibiting the FXR-induced increase of promoter activity of the key gluconeogenic gene PGC-1α, followed by the repression of PGC-1α and PPARα. Our work might be the first report on the mechanism underlying the regulation of FXR antagonist against gluconeogenesis, and HS218 is expected to be a new lead compound for anti-T2DM drug discovery.
Materials and Methods
Results
Discussion FXR is closely related to glucose homeostasis [43]. It is activated by the increased enterohepatic circulation of bile acids under feeding leading to the inhibition of glycolysis and promotion of glycogen storage [17], while rise of insulin level after feeding represses FXR expression resulting in the redirection of the glucose to glycolysis [44]. Thus, in FXR deletion mice, short-term fasting can induce hypoglycemia due to the impaired glycogenolysis [44]. In long-term fasting, there are reports that activation of FXR by its agonists could inhibit gluconeogenesis and reduce blood glucose in db/db and C57 mice [15,16,45]. However, there is still a controversy over whether FXR activation may inhibit gluconeogenesis, because FXR expression is induced by PGC-1α during fasting, and gluconeogenesis is inhibited in FXR-knockout mice [18,19]. In addition, it has been also determined that FXR knockout efficiently improves blood glucose homeostasis in ob/ob mice [46,47], thus supporting that FXR inhibition might be considered a potential therapeutic strategy for T2DM. Currently, there have been published reports on FXR antagonists functioning in improving glucose metabolism. For example, glycine-β-muricholic acid inhibited FXR signaling in the intestine and reduced blood glucose in obese mice [48]; guggulsterone antagonized the GW4064 or CDCA-induced promotion of G6Pase and PEPCK mRNA levels and reduced hepatic gluconeogenesis in hepatocytes [20]. Besides, we have recently reported that FXR small molecule antagonist NDB could reduce gluconeogenic gene expression in the liver of db/db mice [22]. Here, we determined that FXR antagonist HS218 as a NDB derivative could FXR-dependently inhibit gluconeogenesis and improve glucose homeostasis in both db/db and HFD/STZ-induced T2DM mice. All results have suggested that FXR antagonist should find its promising application in anti-T2DM drug discovery.