Article pubs.acs.org/JAFC
Toona sinensis Leaf Extract Inhibits Lipid Accumulation through Upregulation of Genes Involved in Lipolysis and Fatty Acid Oxidation in Adipocytes Hung-Wen Liu, Yue-Tseng Tsai, and Sue-Joan Chang* Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan S Supporting Information *
ABSTRACT: Toona sinensis leaf (TSL) has been shown to lower plasma triacylglycerol levels and diminish the size of visceral fat cells in vivo. The molecular mechanism of TSL ethanol extract (TSL-E) on lipid metabolism in 3T3-L1 adipocytes was investigated in this study. Oil Red O staining as well as immunoblotting, real-time PCR, and dual-Luciferase reporter system were performed to investigate the effect of TSL-E on lipid accumulation and the regulation of lipid metabolism, respectively. In addition, active compounds in the TSL-E were analyzed by HPLC. TSL-E significantly decreased lipid accumulation, stimulated free fatty acid (FFA) release, and up-regulated peroxisome proliferator-activated receptor-α (PPARα) and genes involved in peroxisomal (acyl-CoA oxidase) and mitochondrial (uncouple protein 3) fatty acid oxidation. TSL-E also up-regulated cytoplasmic triacylglycerol hydrolysis gene (adipose triglyceride lipase) and genes related to fatty acid oxidation (AMP-activated protein kinase, acetyl-CoA carboxylase, carnitine palmitoyltransferase I, PPARγ, and adiponectin). The major constituents directly inducing PPARα transactivity in TSL-E are gallic acid, rutin, palmitic acid, linoleic acid, and α-linolenic acid. These results indicate that the inhibitory effect of TSL-E on lipid accumulation was through PPARα activation and further up-regulation of PPARα-mediated genes plus up-regulation of cytoplasmic genes involved in lipid catabolism. KEYWORDS: peroxisome proliferator-activated receptor alpha (PPARα), Toona sinensis Roem (T. sinensis), lipolysis
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peroxidation,15 against H2O2-induced oxidative stress, and DNA damage in epithelial Madin−Darby canine kidney (MDCK) cells,16 anti-LDL glycative activity,17 and improvement of learning and memory through the reduction of lipid peroxidation and β-amyloid plaques in the brains of senescence-accelerated mice.18 In addition to a wide spectrum of health beneficial activities, TSL also significantly lowers plasma triacylglycerol levels and diminishes the size of visceral fat cells in high fat diet induced obese mice.19 The precise mechanism underlying the lipid lowering effects of TSL through PPARα activation remains unclear. In the present study, differentiated 3T3-L1 adipocytes were used to investigate the molecular mechanism of the TSL ethanol extract (TSL-E) on PPARα mediated lipid metabolism. We hypothesized that TSL-E with lipid metabolism-modulating effects up-regulates genes associated with peroxisomal and mitochondrial fatty acid oxidation and cytoplasmic triglyceride hydrolysis. To test our hypothesis and elucidate the underlying mechanism of TSL-E on the regulation of lipid metabolism in 3T3-L1 adipocytes, we examined its effects on lipid accumulation, PPARα target gene expression, and PPARα transcriptional activation. Furthermore, high-performance liquid chromatography (HPLC) analysis was used to identify major constituents responsible for PPARα activation in TSL-E.
INTRODUCTION Dysregulation of lipid metabolism leading to lipid accumulation inside the body is a major risk factor for chronic diseases including diabetes, cardiovascular diseases, and hypertension.1−4 To reduce the risk of these diseases, it is important to ameliorate the dysregulation of lipid metabolism to diminish deposition of extra fat. Peroxisome proliferator-activated receptors (PPARs), PPARα, PPARδ, and PPARγ, a family of ligand-activated transcription factors, are involved in the regulation of glucose and lipid metabolism, inflammation, and cell differentiation.5−8 Among these three nuclear receptors, PPARα predominantly expressed in metabolically active tissues including liver, heart, skeletal muscle, kidney, and brown adipose tissue7,9 plays a central role in peroxisomal and mitochondrial fatty acid oxidation. The activation of PPARα up-regulates the transcription of target genes for fatty acid oxidation such as uncouple protein-1 (UCP1), carnitine palmitoyltransferase I (CPT-1), and acyl-CoA oxidase (ACO).10,11 Most interestingly, recent studies indicate that the activation of PPARα enhances fatty acid oxidation in white adipose tissue and decreases the levels of circulating lipid profiles in obese diabetic patients.12,13 Therefore, stimulating PPARα activity using PPARα agonist or herbal product-derived PPARα activator could modulate lipid catabolism in adipocytes. TSL is extensively used as a vegetable in Asia and currently considered as a functional food. TSL improves health outcomes including antioxidant properties with effective protection for atherogenesis,14 strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities with inhibitory effects on lipid © 2014 American Chemical Society
Received: Revised: Accepted: Published: 5887
February 12, 2014 May 28, 2014 June 2, 2014 June 2, 2014 dx.doi.org/10.1021/jf500714c | J. Agric. Food Chem. 2014, 62, 5887−5896
Journal of Agricultural and Food Chemistry
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Article
(Invitrogen, Grand Island, NY, USA) according to the manufacturer’s instructions. mRNA levels of PPARα, ACO, UCP3, CPT1, PPARγ, adiponectin, AMP-activated protein kinase (AMPK), acetyl-CoA carboxylase (ACC), and adipose triglyceride lipase (ATGL) in 3T3L1 adipocytes were quantified using StepOnePlus real-time systems (Applied Biosystems, Foster City, CA, USA). Primer sequences are shown in the Supporting Information (Table 1). Real-time PCR was performed using a Fast Start SYBR Green master mix kit (Applied Biosystems). The PCR reaction included the following components: each primer at a concentration of 10 μM, diluted cDNA template, and Fast Start SYBR Green master mix and running 40 cycles. Each cDNA sample was run in triplicate, and GAPDH primers as an internal control were included in each run to correct sample-to-sample variation and to normalize mRNA levels. The relative mRNA level was calculated according to the comparative ΔΔCt method. HPLC Analysis. Phytochemical compounds in TSL-E were analyzed by using an HPLC system consisting of a UV detector (Jasco UV1575, Jasco, Easton, MD, USA), an autosampler (Gilson 234, Gilson, Middleton, WI, USA), and a pump (Hitachi L-7100, Hitachi, Tokyo, Japan). Data were analyzed by chromatography EC2000 data system (Analab Corp., Taipei, Taiwan). The chromatography was performed on an Ascentis C18 column (250 mm × 4.6 mm i.d.). The mobile phase was a gradient prepared from (A) 100% methanol with 0.05% TFA and (B) 20% methanol with 0.05% TFA. A gradient was used as 0−9 min, 0−15% B; 9−12 min, 15% B; 12−17 min, 15−20% B; 17−35 min, 20−25% B; 35−55 min, 25−53% B; 55−65 min, 53% B. The flow rate was 1 mL/min at room temperature, and detection was performed at 280 nm. To determine fatty acids in TSL-E, fatty acid was derivatized as described by Wood and Lee.20 Fatty acid standards and TSL-E were converted to fatty acid p-bromophenacyl ester by reacting with αbromoacetophenone and triethylamine. Bromophenacyl esters of fatty acids were used as standards for comparison of retention times to the derivatized TSL-E. Analytes were dissolved in a mixture of acetonitrile/acetone (1:1; v/v). The chromatographic elution was ACN + THF (99:1; v/v) at a flow rate of 1 mL/min. The injection volume was 20 μL, detection was at 258 nm, and all experiments were under room temperature. Calibration curves of fatty acids (palmitic acid, linoleic acid, α-linolenic acid; Sigma-Aldrich, St. Louis, MO, USA) and phytochemicals (gallic acid, catechin, methyl gallate, rutin, and quercetin; Sigma-Aldrich) were obtained by triplicate measurements on a single standard at increasing concentration. Plasmids and Transfection Assay. The plasmids for PPARαligand binding domain (LBD) luciferase report assay, pBIND-PPARαLBD and pG5luc-TK, were obtained from the Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Taiwan.21 HepG2 (human hepatocellular carcinoma cell line) cells were seeded (5 × 104 cells/well) in 12well cell culture plates in DMEM supplemented with 10% FBS. After 24 h, transfections were performed using transfection reagent Lipofectamine 2000 (Life Technologies Corp., Carlsbad, CA, USA) according to the manufacturer’s instruction. Cells were treated with bezafibrate, TSL-E, palmitic acid, linoleic acid, α-linolenic acid, gallic acid, methyl gallate, catechin, rutin, and quercetin for 24 h. Following treatment, cells were lysed, and firefly and renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Fitchburg, WI, USA). Bezafibrate was used as a positive control for PPARα activation. The results were expressed as fold difference compared to the Con group. Statistical Analysis. Results are expressed as means ± standard error (SEM). When only the Con and TSL-E groups were involved, statistical analysis of the difference between the two groups was determined by using Student’s t test (SigmaPlot 12.0). For multiple comparisons, one-way ANOVA was used to determine the statistical significance of the differences among the groups. ANOVA was followed by post hoc assessment by Student−Newman−Keuls method correction for multiple comparisons (SigmaPlot 12.0). A P value of
