Activation of the constitutive androstane receptor inhibits gluconeogenesis without affecting lipogenesis or fatty acid synthesis in human hepatocytes

Highlights

• Novel hCAR activators were identified by computational and biological approaches.

• The role of hCAR in hepatic energy metabolism was examined.

• hCAR activators repress gluconeogenesis but not lipogenesis and fatty acid synthesis.

• Human and mouse CAR exhibit differential effects on energy metabolism.

Abstract

Objective

Accumulating evidence suggests that activation of mouse constitutive androstane receptor (mCAR) alleviates type 2 diabetes and obesity by inhibiting hepatic gluconeogenesis, lipogenesis, and fatty acid synthesis. However, the role of human (h) CAR in energy metabolism is largely unknown. The present study aims to investigate the effects of selective hCAR activators on hepatic energy metabolism in human primary hepatocytes (HPH).

Methods

Ligand-based structure–activity models were used for virtual screening of the Specs database (www.specs.net) followed by biological validation in cell-based luciferase assays. The effects of two novel hCAR activators (UM104 and UM145) on hepatic energy metabolism were evaluated in HPH.

Results

Real-time PCR and Western blotting analyses reveal that activation of hCAR by UM104 and UM145 significantly repressed the expression of glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, two pivotal gluconeogenic enzymes, while exerting negligible effects on the expression of genes associated with lipogenesis and fatty acid synthesis. Functional experiments show that UM104 and UM145 markedly inhibit hepatic synthesis of glucose but not triglycerides in HPH. In contrast, activation of mCAR by 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, a selective mCAR activator, repressed the expression of genes associated with gluconeogenesis, lipogenesis, and fatty acid synthesis in mouse primary hepatocytes, which were consistent with previous observations in mouse model in vivo.

Conclusion

Our findings uncover an important species difference between hCAR and mCAR in hepatic energy metabolism, where hCAR selectively inhibits gluconeogenesis without suppressing fatty acid synthesis.

Implications

Such species selectivity should be considered when exploring CAR as a potential therapeutic target for metabolic disorders.

Introduction

Xenobiotic receptors, such as the constitutive androstane receptor (CAR, NR1I3), are responsible for both controlling the expression of genes associated with all phases of drug metabolism and transport as well as defending against stress caused by xenobiotics. CAR is most notably known as the transcriptional regulator for a number of drug-metabolizing enzymes, such as cytochrome P450 (CYP) 2B6 and CYP3A4, UDP glucuronosyltransferase 1A1 and sulfotransferase 2A1, as well as drug transporters including organic anion transporting polypeptide 2 and multidrug resistance protein 1 (Qatanani and Moore, 2005, Timsit and Negishi, 2007, Tolson and Wang, 2010). Collectively, up-regulation of these metabolizing enzymes and membrane transport proteins not only alters the clearance of xenobiotic compounds, such as prescription drugs and environmental chemicals, but also influences the excretion of endobiotic metabolites such as bilirubin and bile acids (Kakizaki et al., 2009, Yamamoto et al., 2003). Activation of CAR may represent a precursor for potential drug–drug interactions by accelerating the metabolism of co-administered medications, often leading to decreased therapeutic efficacy or enhanced toxicity (Wang and LeCluyse, 2003). In addition to these well-established roles of CAR in xenobiotic metabolism and clearance, accumulating evidence reveals that CAR has also emerged as a coordinate factor that modulates diverse liver functions under physiological and pathophysiological conditions including energy homeostasis, insulin signaling, cell proliferation and apoptosis, inflammation, and tumor development (Gao and Xie, 2010, Huang et al., 2005, Moreau et al., 2008, Yamamoto et al., 2004). A need for further understanding of the endobiotic roles of CAR is evident.

Hepatic energy homeostasis is maintained by comprehensive physiological mechanisms that balance the formation and oxidation of lipids, fatty acids, and glucose in the liver. The correlation between CAR activation and energy metabolism was initially observed by Negishi and colleagues in comparison of the global gene expression profiles between wild-type and CAR knockout mice (Ueda et al., 2002). Notably, other than the induction of typical drug-metabolizing enzymes and transporters, treatment with phenobarbital (PB), a prototypical CAR activator, profoundly repressed the expression of a group of genes associated with energy metabolism in a CAR-dependent manner (Ueda et al., 2002). Subsequent studies revealed that through cross-talk with forkhead box O1 or peroxisome proliferator activated receptor γ-coactivator 1α, activation of CAR leads to attenuated gluconeogenesis or increased energy expenditure in mouse liver (Kodama et al., 2004). Activation of CAR also compromised the liver X receptor interaction with the promoter region of the sterol regulatory element binding protein 1 (SREBP-1), a master lipogenic regulator (Zhai et al., 2010). Under high fat diet challenge or in leptin-deficient (ob/ob) mice, activation of CAR significantly ameliorated hyperglycemia and improved insulin sensitivity, while such beneficial effects were absent in CAR knockout mice (Dong et al., 2009, Gao et al., 2009). On the other hand, CAR-null mice appear to be defective in fasting adaptation and exhibit greater weight lose under calorie restriction (Maglich et al., 2004). It is noteworthy, however, that current conception regarding the role of CAR in energy metabolism was drawn predominantly from studies using either animal models in vivo or cultured cells with ectopic expression of mouse (m) CAR. Although human (h) CAR and its rodent counterparts share several common characteristics, significant species differences between these receptors exist. For instance, 6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehydeO-(3,4-dichlorobenzyl)oxime (CITCO), the selective hCAR activator, cannot bind or activate mCAR (Maglich et al., 2003), while 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP), the most potent mCAR agonist, does not activate hCAR (Tzameli et al., 2000). Moreover, while activation of mCAR was essential for TCPOBOP- and PB-mediated tumor promotion in mice, activation of hCAR by CITCO appears to be associated with cell cycle arrest and enhanced apoptosis in human brain tumor stem cells (Chakraborty et al., 2011, Yamamoto et al., 2004). As such, direct extrapolation of conclusions drawn from animal models to humans is unsafe. The role of hCAR in energy homeostasis is yet to be elucidated.

Recent studies have demonstrated that docking and pharmacophore-based virtual screening of chemical databases combined with cell-based biological assays are effective in identifying new compounds as modulators of nuclear receptors including hCAR (Lynch et al., 2013, Pan et al., 2011). Utilizing this combined approach, here, we have retrieved 144 compounds as potential hCAR activators by virtual screening of the Specs database (www.specs.net) based on their structure–activity features. Of the 144 lead compounds, ten hCAR activators that exhibited equal or greater potency than CITCO were identified by cell-based luciferase assays. Subsequently, the role of hCAR in hepatic energy metabolism was evaluated in cultured human primary hepatocytes (HPH) using CITCO and a number of newly identified hCAR activators. Importantly, we have uncovered a significant species difference between the effects of hCAR and mCAR on energy homeostasis. Unlike mCAR, activation of hCAR selectively inhibits hepatic gluconeogenesis by repressing the expression of glucose-6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) without significantly affecting lipogenesis or the synthesis of fatty acids.