[Show abstract][Hide abstract] ABSTRACT: The solute carrier family 13 member 5 (SLC13A5) is a sodium-coupled transporter that mediates cellular uptake of citrate, which plays important roles in the synthesis of fatty acids and cholesterol. Recently, the pregnane X receptor (PXR, NR1I2) initially characterized as a xenobiotic sensor, has been functionally linked to the regulation of various physiological processes that are associated with lipid metabolism and energy homeostasis. Here, we show that the SLC13A5 gene is a novel transcriptional target of PXR and altered expression of SLC13A5 affects lipid accumulation in human liver cells. The prototypical PXR activator rifampicin markedly induced the mRNA and protein expression of SLC13A5 in human primary hepatocytes. Utilizing cell-based luciferase reporter assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation assays, we identified and functionally characterized two enhancer modules located upstream of the SLC13A5 gene transcription start site, that are associated with regulation of PXR-mediated SLC13A5 induction. Functional analysis further revealed that rifampicin can enhance lipid accumulation in human primary hepatocytes; and knockdown of SLC13A5 expression alone leads to significant decrease of the lipid content in HepG2 cells. Overall, our results uncover SLC13A5 as a novel target gene of PXR and may contribute to drug-induced steatosis and metabolic disorders in humans.
The American Society for Pharmacology and Experimental Therapeutics.
Full-text · Article · Jan 2015 · Molecular pharmacology
[Show abstract][Hide abstract] ABSTRACT: Nanocarriers have recently emerged as an attractive platform for the delivery of various types of therapeutics including anticancer agents. Previously, we developed an improved TPGS delivery system (PEG5K-VE2) which demonstrated improved colloidal stability and greater in vivo antitumor activity. Nevertheless, the application of this system is still limited by a relatively low drug loading capacity (DLC). In this study we report that incorporation of a fluorenylmethyloxycarbonyl (Fmoc) motif at the interfacial region of PEG5K-VE2 led to significant improvement of the system through the introduction of an additional mechanism of drug/carrier interaction. Doxorubicin (DOX) could be effectively loaded into PEG5K-Fmoc-VE2 micelles at a DLC of 39.9%, which compares favorably to most reported DOX nanoformulations. In addition, PEG5K-Fmoc-VE2/DOX mixed micelles showed more sustained release of DOX in comparison to the counterpart without Fmoc motif. MTT assay showed that PEG5K-Fmoc-VE2/DOX exerted significantly higher levels of cytotoxicity over DOX, Doxil as well as PEG5K-VE2/DOX in PC-3 and 4T1.2 cells. A cytotoxicity assay with NCI/ADR-RES, a drug resistant cell line, suggested that PEG5K-Fmoc-VE2 may have the potential to reverse multidrug resistance, which was supported by its inhibition of P-gp ATPase. Pharmacokinetic (PK) and biodistribution studies showed an increased half-life in blood circulation and more effective tumor accuulation for DOX formulated in PEG5K-Fmoc-VE2 micelles. More importantly, DOX-loaded PEG5K-Fmoc-VE2 micelles showed an excellent safety profile with a MTD (~ 30 mg DOX/kg) that is about 3 times as much as that for free DOX. Finally, superior antitumor activity was demonstrated by PEG5K-Fmoc-VE2/DOX in both drug-sensitive (4T1.2 and PC-3) and drug-resistant (KB 8-5) tumor models compared to DOX, Doxil, and PEG5K-VE2/DOX.
Full-text · Article · Dec 2014 · Journal of Controlled Release
[Show abstract][Hide abstract] ABSTRACT: The constitutive androstane receptor (CAR) is initially defined as a xenobiotic nuclear receptor that protects the liver from injury. Detoxification of damaging chemicals is achieved by CAR-mediated induction of drug-metabolizing enzymes and transporters. More recent research has implicated CAR in energy metabolism, suggesting a therapeutic potential for CAR in metabolic diseases, such as type 2 diabetes and obesity. A better understanding of the mechanisms by which CAR regulates energy metabolism will allow us to take advantage of its effectiveness while avoiding its side effects. This review summarizes the current progress on the regulation of CAR nuclear translocation, upstream modulators of CAR activity, and the crosstalk between CAR and other transcriptional factors, with the aim of elucidating how CAR regulates glucose and lipid metabolism.
Preview · Article · Dec 2014 · Acta Pharmacologica Sinica
[Show abstract][Hide abstract] ABSTRACT: Hormonal homeostasis is essential for a variety of physiological and pathological processes. Elimination and detoxification of xenobiotics, such as drugs introduced into the human body, could disrupt the balance of hormones due to the induction of drug metabolizing enzymes (DMEs) and transporters. Pregnane X receptor (PXR, NR1I2) functions as a master xenobi-otic receptor involved in drug metabolism and drug-drug interactions by its coordinated tran-scriptional regulation of phase I and phase II DMEs and transporters. Recently, increasing evidences indicate that PXR can also mediate the endocrine disruptor function and thus impact the integrity of the endocrine system. This review focuses primarily on the recent advances in our understanding of the function of PXR in glucocorticoid, mineralocorticoid, androgen and estrogen homeostasis. The elucidation of PXR-mediated drug-hormone interactions might have important therapeutic implications in dealing with hormone-dependent diseases and safety assessment of drugs.
[Show abstract][Hide abstract] ABSTRACT: Liver X receptors (LXRs) are nuclear receptors that play an essential role in lipid and cholesterol metabolism. Emerging studies indicate a potential function for LXRs in regulating dendritic cell (DC)-dependent immune responses; however, the role of LXRs in DC differentiation is largely unknown. Here, we report that LXRα regulates the differentiation of mouse GM-CSF-derived DCs. Activation or overexpression of LXRα significantly enhanced myeloid DC differentiation from mouse bone marrow (BM) cells, while siRNA-mediated knockdown of LXRα suppressed DC differentiation. In addition, we demonstrated that LXR agonist-programmed DCs showed an increased capacity for stimulating T-cell proliferation. Mechanistic studies showed that activation of LXR could inhibit the phosphorylation of STAT3 and downregulate the expression of its target, S100A9, an important negative regulator of myeloid DC differentiation. We also found that Histone deacetylase (HDAC) inhibition interfered with the effect of LXR on STAT3 signaling via acetylation of STAT3. Chromatin immunoprecipitation assays confirmed that LXR activation and HDAC inhibition balanced the recruitment of STAT3 to the S100A9 promoter, which involved distinct post-translational modifications of STAT3. In conclusion, our observations demonstrated a novel role for LXRα in GM-CSF-derived DC differentiation and revealed the underlying mechanism.
No preview · Article · Apr 2014 · Molecular Immunology
[Show abstract][Hide abstract] ABSTRACT: Altered expression of miR-29b is implicated in the pathogenesis and progression of liver fibrosis. We and others previously demonstrated that miR-29b down-regulates the expression of several extracellular-matrix (ECM) genes including Col 1A1, Col 3A1 and Elastin via directly targeting their 3’-UTRs. However, whether or not miR-29b plays a role in the post-translational regulation of ECM biosynthesis has not been reported. Heat shock protein 47 (HSP47) and lysyl oxidase (LOX) are known to be essential for ECM maturation. In this study we have demonstrated that expression of HSP47 and LOX was significantly up-regulated in culture-activated primary rat hepatic stellate cells (HSCs), TGF-β stimulated LX-2 cells and liver tissue of CCl4-treated mice, which was accompanied by a decrease of miR-29b level. In addition, over-expression of miR-29b in LX-2 cells resulted in significant inhibition on HSP47 and LOX expression. Mechanistically, miR-29b inhibited the expression of a reporter gene that contains the respective full-length 3’-UTR from HSP47 and LOX gene, and this inhibitory effect was abolished by the deletion of a putative miR-29b targeting sequence from the 3’-UTRs. Transfection of LX-2 cells with miR-29b led to abnormal collagen structure as shown by electron-microscopy, presumably through down-regulation of the expression of molecules involved in ECM maturation including HSP47 and LOX. These results demonstrated that miR-29b is involved in regulating the post-translational processing of ECM and fibril formation.
Full-text · Article · Apr 2014 · Biochemical and Biophysical Research Communications
[Show abstract][Hide abstract] ABSTRACT: The estrogen sulfotransferase (EST/SULT1E1) is known to catalyze the sulfoconjugation and deactivation of estrogens. The goal of this study is to determine whether and how EST plays a role in human adipogenesis. By using human primary adipose derived stem cells (ASCs) and whole fat tissues from the abdominal subcutaneous fat of obese and non-obese subjects, we showed that the expression of EST was low in pre-adipocytes but increased upon differentiation. Overexpression and knockdown of EST in ASCs promoted and inhibited differentiation, respectively. The pro-adipogenic activity of EST in humans was opposite to the anti-adipogenic effect of the same enzyme in rodents. Mechanistically, EST promoted adipogenesis by deactivating estrogens. The pro-adipogenic effect of EST can be recapitulated by using an estrogen receptor (ER) antagonist or ERα knockdown. In contrast, activation of ER in ASCs inhibited adipogenesis by decreasing the recruitment of the adipogenic peroxisome proliferator activated receptor γ (PPARγ) onto its target gene promoters, whereas ER antagonism increased the recruitment of PPARγ to its target gene promoters. Linear regression analysis revealed a positive correlation between the expression of EST and body mass index (BMI), as well as a negative correlation between the ERα expression and BMI. We conclude that EST is a pro-adipogenic factor, which may serve as a druggable target to inhibit the turnover and accumulation of adipocytes in obese patients.
Preview · Article · Feb 2014 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: Bedaquiline is a recently approved drug for treatment of multi-drug resistant tuberculosis. Adverse cardiac and hepatic drug reactions of bedaquiline have been noted in clinical practice. The current study investigated bedaquiline metabolism in human hepatocytes using a metabolomic approach. Bedaquiline N-demethylation via cytochrome P450 3A4 (CYP3A4) was confirmed as the major pathway in bedaquiline metabolism. Besides CYP3A4, we found that both CYP2C8 and CYP2C19 contributed to bedaquiline N-demethylation. The Km values of CYP2C8, CYP2C19 and CYP3A4 in bedaquiline N-demethylation were 13.1, 21.3 and 8.5 μM, respectively. We also identified a novel metabolic pathway of bedaquiline that produced an aldehyde intermediate. In summary, this study extended our knowledge of bedaquiline metabolism, which can be applied to predict and prevent drug-drug interactions and adverse drug reactions associated with bedaquiline.
Full-text · Article · Feb 2014 · Drug metabolism and disposition: the biological fate of chemicals
[Show abstract][Hide abstract] ABSTRACT: The steroid sulfatase (STS)-mediated de-sulfation is a critical metabolic mechanism that regulates the chemical and functional homeostasis of endogenous and exogenous molecules. In this report, we first showed that the liver expression of Sts was induced in both the high-fat diet (HFD) and ob/ob models of obesity and type 2 diabetes and during the fed to fasting transition. In defining the functional relevance of STS induction in metabolic disease, we showed that over-expression of STS in the liver of transgenic mice alleviated HFD and ob/ob models of obesity and type 2 diabetes, including reduced body weight, improved insulin sensitivity, and decreased hepatic steatosis and inflammation. Interestingly, STS exerted its metabolic benefit through sex-specific mechanisms. In female mice, STS may have increased hepatic estrogen activity by converting biologically inactive estrogen sulfates to active estrogens and consequently improved the metabolic functions; whereas ovariectomy abolished this protective effect. In contrast, the metabolic benefit of STS in males may have been accounted for by the male-specific decrease of inflammation in white adipose tissue and skeletal muscle, as well as a pattern of skeletal muscle gene expression that favors energy expenditure. The metabolic benefit in male STS transgenic mice was retained after castration. Treatment with the STS substrate estrone sulfate also improved metabolic functions in both the HFD and ob/ob models. Our results have uncovered a novel function of STS in energy metabolism and type 2 diabetes. Liver-specific STS induction or estrogen/estrogen sulfate delivery may represent a novel approach to manage metabolic syndrome.
Full-text · Article · Feb 2014 · Journal of Biological Chemistry
[Show abstract][Hide abstract] ABSTRACT: Sulfotransferase (SULT)-mediated sulfation represents a critical mechanism in regulating the chemical and functional homeostasis of endogenous and exogenous molecules. The cholesterol sulfotransferase SULT2B1b catalyzes the sulfoconjugation of cholesterol to synthesize cholesterol sulfate (CS). In this study, we showed that the expression of Sult2B1b in the liver was induced in obese mice and during the transition from the fasted to the fed state, suggesting that the regulation of SULT2B1b is physiologically relevant. CS and SULT2B1b inhibited gluconeogenesis by targeting the gluconeogenic hepatocyte nuclear factor 4α (HNF4α) in both cell cultures and transgenic mice. Treatment of mice with CS or transgenic overexpression of the CS-generating SULT2B1b in the liver inhibited hepatic gluconeogenesis and alleviated metabolic abnormalities in both the diet-induced obesity (DIO) and ob/ob mice. Mechanistically, CS and SULT2B1b inhibited gluconeogenesis by suppressing the expression of acetyl-CoA synthetase (Acss), leading to decreased acetylation and nuclear exclusion of HNF4α. Our results also suggested leptin as a potential effector of SULT2B1b in improving metabolic functions. We conclude that SULT2B1b and its enzymatic byproduct CS are important metabolic regulators that control glucose metabolism, suggesting CS as a potential therapeutic agent and SULT2B1b as a potential therapeutic target for metabolic disorders.
No preview · Article · Nov 2013 · Molecular and Cellular Biology
[Show abstract][Hide abstract] ABSTRACT: To investigate the effects of three natural product compounds, carapin, santonin and isokobusone, on the activity of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) in induction of drug-metabolizing enzymes and inhibition of inflammation.
The monkey kidney-derived fibroblast (CV-1) cells and human embryonic kidney HEK293 cells were used for transient transfection and luciferase reporter gene assays. Human primary hepatocytes and primary hepatocytes from wild type, PXR-/-, and hPXR transgenic mice were used to study the induction of drug-metabolizing enzymes and the implication of these compounds in inflammation.
Carapin, santonin and isokobusone activated both PXR and CAR in transient transfection and luciferase reporter gene assays. Mutagenesis studies showed that two amino acid residues, Phe305 of the rodent PXR and Leu308 of the human PXR, are critical for the recognition of these compounds by PXR. Importantly, the activation of PXR and CAR by these compounds induced the expression of drug-metabolizing enzymes in primary human and mouse hepatocytes. Furthermore, activation of PXR by these compounds inhibited the expression of inflammatory mediators in response to lipopolysaccharide (LPS). The effects of these natural compounds on drug metabolism and inflammation were abolished in PXR-/- hepatocytes.
Our results show that carapin, santonin and isokobusone activate PXR and CAR and induce drug-metabolizing enzymes. In addition, these compounds inhibited the expression of inflammatory mediators in response to LPS through the activation of PXR.
Full-text · Article · Jul 2013 · Pharmaceutical Research
[Show abstract][Hide abstract] ABSTRACT: Farnesoid X receptor (FXR) has important roles in maintaining bile acid and cholesterol homeostasis. Here we report that the antiparasitic drug ivermectin is a ligand for nuclear FXR. We identify ivermectin using a high-throughput compound library screening and show that it induces the transcriptional activity of the FXR with distinctive properties in modulating coregulator recruitment. The crystal structure of ivermectin complexed with the ligand-binding domain of FXR reveals a unique binding mode of ivermectin in the FXR ligand-binding pocket, including the highly dynamic AF-2 helix and an expanded ligand-binding pocket. Treatment of wild-type mice, but not of FXR-null mice, with ivermectin decreases serum glucose and cholesterol levels, suggesting that ivermectin regulates metabolism through FXR. Our results establish FXR as the first mammalian protein targeted by ivermectin with high selectivity. Considering that ivermectin is a widely used clinical drug, our findings reveal a safe template for the design of novel FXR ligands.
Full-text · Article · Jun 2013 · Nature Communications
[Show abstract][Hide abstract] ABSTRACT: Non-alcoholic steatohepatitis (NASH) is a liver disorder that still demands improved treatment. Understanding the pathogenesis of NASH will help to develop novel approaches to prevent or treat this disease. In this study, we revealed a novel function of the aryl hydrocarbon receptor (AhR) in NASH. Transgenic or pharmacological activation of AhR heightened animal sensitivity to NASH induced by the methionine and choline deficient (MCD) diet, which was reasoned to be due to increased hepatic steatosis, production of reactive oxygen species (ROS), and lipid peroxidation. Mechanistically, the increased ROS production in AhR-activated mouse liver was likely a result of a lower superoxide dismutase 2 (SOD2) activity and compromised clearance of ROS. Activation of AhR induced TCDD-inducible poly (ADP-ribose) polymerase (TiPARP) gene expression, depleted NAD+, deactivated the mitochondrial sirtuin deacetylase 3 (Sirt3), increased SOD2 acetylation, and thereby decreased SOD2 activity. We also showed that Sirt3 ablation sensitized mice to NASH, whereas adenoviral overexpression of Sirt3 alleviated the NASH phenotype in AhR transgenic mice. We conclude that activation of AhR sensitizes mice to NASH by facilitating both the "first hit" of steatosis and the "second hit" of oxidative stress. Our results suggest that the use of AhR antagonists might be a viable approach to prevent and treat NASH. Manipulation of the expression or activity of Sirt3 may also represent a novel approach to manage NASH.
Full-text · Article · Mar 2013 · Molecular and Cellular Biology