Article Cite This: J. Agric. Food Chem. 2018, 66, 10964−10976
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Oleanolic Acid Inhibits Liver X Receptor Alpha and Pregnane X Receptor to Attenuate Ligand-Induced Lipogenesis Yen-Ning Lin,†,∥ Hsiao-Yun Chang,‡,∥ Charles C. N. Wang,¶,∥ Fang-Yi Chu,† Hsin-Yi Shen,† Chao-Jung Chen,§ and Yun-Ping Lim*,†,◊,⊥ †
Department of Pharmacy, College of Pharmacy, China Medical University, Taichung, Taiwan Department of Biotechnology, Asia University, Taichung, Taiwan ¶ Department of Bioinformatics and Medical Engineering, Asia University, Taichung, Taiwan § Proteomics Core Laboratory, Department of Medical Research, China Medical University Hospital, Taichung, Taiwan ◊ Department of Internal Medicine, China Medical University Hospital, Taichung, Taiwan ⊥ Department of Medical Research, China Medical University Hospital, Taichung, Taiwan
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S Supporting Information *
ABSTRACT: Liver X receptor α (LXRα) controls important biological and pathophysiological processes such as lipid homeostasis. Inhibiting LXRα transactivation may beneficial in the treatment of nonalcoholic fatty liver disease (NAFLD), which is one of the main causes of liver diseases and hyperlipidemia. Oleanolic acid (OA) is a naturally occurring triterpenoid found in many plants. It has several beneficial effects on biological pathways; however, the mechanisms underlying its effects on LXRα are unclear. Therefore, we evaluated the effects of OA on T0901317-induced LXRα activation and explored whether OA can attenuate hepatic lipogenesis. The results showed that OA significantly decreased the promoter activities of LXR response element and sterol regulatory element binding protein-1c (SREBP-1c). It also decreased the mRNA and protein expression of LXRα target genes. These resulted in reduced hepatocellular lipid content. Our results also revealed that the overall binding pose of OA is similar to the X-ray pose of T0901317. Furthermore, OA stimulated AMP-activated protein kinase phosphorylation in hepatic cells. Additionally, it increased small heterodimer partner-interacting leucine zipper protein (SMILE) but decreased steroid receptor coactivator-1 (SRC-1) recruitment to the SREBP-1c promoter region. OA also enhanced LXRαmediated induction of reverse cholesterol transport (RCT)-related gene, ATP-binding cassette transporter (ABC) A1, and ABCG1 expression in intestinal cells. It was found that OA increased the binding of SRC-1 but decreased SMILE recruitment to the ABCG1 gene promoter region. Furthermore, it reduced valproate- and rifampin-induced LXRα- and pregnane X receptormediated lipogenesis, respectively, which indicates its potential benefit in treating drug-induced hepatic steatosis. The results also show that OA is liver-specific and can be selectively repressed of lipogenesis. Moreover, it preserves and enhances LXRαinduced RCT stimulation. The results show that OA may be a promising treatment for NAFLD. Additionally, it can be used in the development of LXRα agonists to prevent atherosclerosis. KEYWORDS: oleanolic acid, liver X receptor α, pregnane X receptor, lipogenesis, reverse cholesterol transport
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INTRODUCTION Liver X receptors (LXRs) are members of the ligandedactivated nuclear receptor (NR) superfamily. LXRα (NR1H3) is highly expressed in organs that are responsible for cholesterol and metabolic homeostasis, such as the liver, which participates in the transport, synthesis, and storage of lipids.1 However, LXRβ (NR1H2) is expressed ubiquitously.1 LXRs bind to ligands and form heterodimers with retinoid X receptor (RXR, NR2B1). The heterodimers then bind to LXR response element (LXREs). The LXREs comprise sequences consisting of two nucleotide direct repeats (DRs) of the hexanucleotide motif −AGGTCA, separated by four nucleotides, referred to as a DR4 element. These LXREs in the promoter regions of LXRs’ target genes are responsible for the induction of target gene expression.1 LXRα activates the hepatic lipogenic pathway mainly through transcriptional activation via the LXRE in the promoter region (TGACCG−CCAG−TAACCC) of sterol regulatory element binding protein-1c (SREBP-1c). This also © 2018 American Chemical Society
induces the expression of a series of target genes, such as acetylCoA carboxylase (ACC), ATP citrate lyase (ACLY), fatty acid (FA) elongase (FAE), FA synthase (FAS), and stearoyl-CoA desaturase (SCD).2,3 In addition, the hepatic expression levels of LXRα, SREBP-1c, ACC, FAS, and SCD are significantly upregulated in nonalcoholic fatty liver disease (NAFLD) patients.4 T0901317 is a synthetic compound that was developed through structure−activity relationship studies, and it stimulates LXRα and causes hepatic steatosis and hypertriglyceridemia.2,5 NAFLD is a common and growing health problem worldwide; it affects 11−46% of the adult population.6 It is a clinicopathophysiological alteration that causes abnormal liver function and is characterized by triglyceride (TG) accumulation in Received: Revised: Accepted: Published: 10964
June 27, 2018 September 11, 2018 September 16, 2018 October 15, 2018 DOI: 10.1021/acs.jafc.8b03372 J. Agric. Food Chem. 2018, 66, 10964−10976
Article
Journal of Agricultural and Food Chemistry
oils, fruits (such as apple, pomegranate, and dates), and medicinal plants (such as Crataegus pinnatifida Bunge, Aralia chinensis L. var. nude, and Eclipta alba).20 It has several promising pharmacological activities such as hepatoprotective, antiinflammatory, antioxidant, and anticancer activities.21 OA has also been demonstrated to improve abnormal lipid metabolism, hypertriglyceridemia, and fatty liver in rodents.22,23 In the present study, the effects of OA on the activities of LXRα and PXR mediated by LXRα and PXR ligands were investigated as these have not been reported yet.
hepatic cells. Over 10% of NAFLD patients develop nonalcoholic steatohepatitis (NASH), which involves inflammatory cell infiltration and ballooning of hepatocytes.6 Some patients with NASH progressively develop liver cirrhosis and hepatocellular carcinoma if the metabolic dysfunction is not reversed. In spite of the high incidences of NAFLD and NASH, there are no approved therapies to treat them. LXR knockout mice have been shown to be resistant to obesity and hepatic lipid accumulation when they are fed a high-fat diet.7 Therefore, it is likely that LXR antagonists can be used to fight against NAFLD. Synthetic LXRα agonists such as T0901317 are beneficial on antiatherosclerotic agents.5 They can induce LXRα-mediated reverse cholesterol transport (RCT) by inducing the expression of ATP-binding cassette transporter (ABC) A1 and G1 in peripheral tissues.8 They are reported to reduce cholesterol absorption and accelerate fecal cholesterol excretion.8 However, they cause several unwanted effects such as hepatic steatosis, which is increased via lipogenic activation. Thus, developing LXRs agonists into antiatherogenic agents is challenging. Drug-induced fatty liver is one of the causes of adverse drug reactions (ADRs). Furthermore, it is reported that dysregulated lipid metabolism via interactions of drugs with key regulators of lipid homeostasis is exemplified by members of the NR family other than LXRs and pregnane X receptor (PXR, NR1I2).9,10 PXR is activated by various ligands, including insecticides, pesticides, nutrients, and therapeutic compounds, via heterodimerization with RXR.11 Additionally, it coordinates the transcriptional control of several xenobiotic metabolizing and transporter systems. It is therefore involved in a number of important processes that determine the fate of xenobiotics.12 Moreover, it protects the body from the effects of xenobiotics, toxicants, and potentially hazardous materials through metabolism and transportation mechanisms. It is also involved in lipid metabolism through FA uptake and lipogenesis. Additionally, it inhibits FA catabolism (β-oxidation) via several mechanisms.9,10 Thus, alterations in PXR function result in the development of hepatic steatosis. Rifampin (RIF) is a potent activator of PXR in humans. It causes hepatic steatosis in patients with tuberculosis13,14 and steatotic formations in the rat liver.15 AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that consists of a catalytic subunit (α) and two regulatory subunits (β and γ).16 It has been shown to act as a “metabolic sensor” in cellular homeostasis under environmental and nutritional stress conditions.17 Activation of AMPK results in inhibition of energy-consuming biosynthetic pathways, such as FA and sterol synthesis, via suppression of SREBP-1c, ACC, and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) expressions; however, the ATP-producing catabolic pathway is activated.16 Transcriptional activation or repression are regulated by NR coregulators, which may be coactivators or corepressors.18 Coactivators of the p160 family, such as steroid receptor coactivator-1 (SRC-1), interact with the activation function 2 (AF2) domain of the ligand-binding domain (LBD) of NRs. However, small heterodimer partner-interacting leucine zipper protein (SMILE), which belongs to the basic region leucine zipper family, to inhibit T0901317-induced LXRα transcriptional activity by competing with coactivator SRC-1. This results in decreased SREBP-1c, FAS, and ACC gene expression.19 Moreover, the expression of SMILE gene may be induced through the liver AMPK pathway.19 Oleanolic acid (OA; 3β-hydroxyolean-12-en-28-oic acid) is a pentacyclic triterpenoid found in Beta vulgaris L., virgin olive
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MATERIALS AND METHODS
Chemicals. OA (purity ≥97%, catalog no. O5504), T0901317 (T090, catalog no. T2320), Oil Red O (catalog no. O0625), valproate (VPA, catalog no. P4543), rifampin (RIF, catalog no. R3501), and compound C (catalog no. P5499) were purchased from SigmaAldrich (St. Louis, MO). The chemicals were prepared as stock solutions at appropriate concentrations using dimethyl sulfoxide (DMSO) or ultrapure water as the solvent before use. Cell Culture. HepG2 cells were obtained from the Food Industry Research and Development Institute (FIRDI, Hsinchu, Taiwan) and used for transient transfection of reporter gene assays in this study, whereas LS174T cells were obtained from CLS Inc. (Cell Lines Service GmbH, Eppelheim, Germany). Both cell lines were maintained in Minimum Essential Medium-alpha (αMEM; Gibco BRL) modification containing 10% fetal bovine serum (FBS; Gibco BRL, Grand Island, NY) and freshly added 1× L-GlutaMAX (Thermo Fisher Scientific, Waltham, MA) without antibiotics. Frozen single-use HepaRG cells were purchased from Thermo Fisher Scientific (Waltham, MA) for the study. The cells were thawed, directly seeded into a 10 cm culture dish, maintained in William’s E medium (Sigma-Aldrich, St. Louis, MO), and supplemented with 10% FetalCloneIII serum (Hyclone, GE Healthcare, Chicago, IL). Next, freshly prepared 50 μM hydrocortisone hydrogen succinate, 5 μg/mL human insulin, and 1× L-GlutaMAX were added to the cells. The culture was maintained for 2 weeks until the cells became confluent, after which the medium was replaced with fresh medium. Next, 2% DMSO was added to the cells, after which the culture was maintained for 2 weeks for the induction of differentiated hepatocyte-like properties. All the cells were cultured at 37 °C in a 5% CO2 humidified incubator. Cytotoxicity assays were carried out as previously described.24 Transient Transfection and Reporter Assay. Luciferase (Luc) assay was carried out as described previously.25 HepG2 cells were transfected using PolyJET transfection reagent (SignaGen Laboratories, Rockville, MD) under the manufacturer’s instructions. Reporter assays included human full-length LXRα25 and PXR26 expression vector, promoter constructs, including 3× LXRE-Luc,1 SREBP-1cLuc,27 S14-Luc,28 and SCD-Luc10 reporter construct. β-galactosidase reporter plasmid was used for normalizing transfection efficiency. After 6−7 h of transfection, treatments with drugs were performed for 24 h. Cell lysates were extracted later and analyzed for the activities of luciferase and the internal control β-galactosidase. The luciferase detection kit used was purchased from Promega (Madison, WI). RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). In order to determine gene expression, cellular RNA was extracted using Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) according to the manufacturer’s instructions. One microgram of total RNA was reversed-transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Thermo Fisher Scientific Inc., Waltham, Massachusetts). The resulting cDNA was amplified by qRT-PCR using Luminaris Color HiGreen qPCR Master Mix (ThermoFisher Scientific, Waltham, MA) and StepOnePlus Real-Time PCR System. The sequences of the specific gene primers are listed in Table 1. The results were normalized to individual β-actin mRNA levels. Western Blotting. Total cell lysates were isolated using RIPA buffer (containing 10 mM Tris-Cl, pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 140 mM NaCl, and addition of 1 mM PMSF before use) or with 10965
DOI: 10.1021/acs.jafc.8b03372 J. Agric. Food Chem. 2018, 66, 10964−10976
Article
Journal of Agricultural and Food Chemistry Table 1. Sequences of PCR Primers gene
species
forward primer (5′-3′)
reverse primer (5′-3′)
hSREBP-1c hFAS hSCD hACLY hACC hFAE hABCA1 hABCG1 hS14 hActin
human human human human human human human human human human
CGC TCC TCC ATC AAT GAC AA ACA TCA TCG CTG GTG GTC TG CCG ACG TGG CTT TTT CTT CT GTG TGG ACG TGG GTG ATG TG CTC TTG ACC CTG GCT GTG TAC TAG TTC CGA GTC TCC CGG AAG T GAC ATC GTG GCG TTT TTG G CGG TGC TCT CAT CCC TTT CA TAT TTG CTC TGG CCC TTG CT CCT GGC ACC CAG CAC AAT
TGC AGA AAG CGA ATG TAG TCG AT GGA GCG AGA AGT CAA CAC GA GCG TAC TCC CCT TCT CTT TGA C TTG ATG TCC TCA GGA TTC AGT TTC TGA GTG CCG TGC TCT GGA T ACA GCC CAT CAG CAT CTG AGT CGA GAT ATG GTC CGG ATT GC GGT GTA CAG CCC GTC TTC CA GGT CGC CAA GTA AGA GGG TG GCC GAT CCA CAC GGA GTA CT
were pelleted by centrifugation at 7 500g for 20−30 s, resuspended in 400 μL of buffer A [10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, and with 1× protease inhibitor cocktail], and left on ice for 10 min. The nuclei were pelleted by centrifuging the suspensions at 7 500g for 20−30 s, after which they were resuspended in 100 μL of buffer C [20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.2 mM EDTA, 420 mM NaCl, and 25% (v/v) glycerol, with 1× protease inhibitor cocktail]. 1× protease inhibitor cocktail contained 0.5 mM dithiothreitol (DTT), 2 μg/mL leupeptin, 1 mM orthovanadate, 2 μg/mL pepstatin A, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The pellets were incubated on ice for 20 min. Nuclei extracts were obtained by centrifuging the suspensions obtained at 7 500g for 30 min. The extracts were then stored at −80 °C until they were analyzed. DNA Affinity Precipitation Assay (DAPA). DAPA was used to identify transcription factors, coregulator interactions, and binding to specific promoter response elements. It was also used to evaluate the binding effects of the various drugs. Biotinylated oligonucleotides covering SREBP-1c LXRE were synthesized and used as probes. Nuclear extracts (500 μg) from treated HepaRG and LS174T cells were exposed to T090 (10 μM) and/or OA (10 or 20 μM) for 24 h. They were then incubated with the biotinylated probes (2 μg each) and 20 μL of 50% slurry streptavidin-agarose beads (Sigma-Aldrich, St. Louis, MO). The mixture was incubated at room temperature for 1 h at 6 rpm/h. Next, the beads were pelleted and rinsed three times with 1× ice-cold PBS. Bound protein complexes were eluted using 1× SDS-PAGE sampling buffer. Western blotting was then performed using specific antibodies against SRC-1, SMILE, and β-actin. Statistical Analysis. Data have been presented as mean ± standard error. Multiple comparisons were evaluated by analysis of variance followed by least significant difference multiple comparison procedure. P values
