Article pubs.acs.org/JAFC
Pterostilbene, a Dimethyl Ether Derivative of Resveratrol, Reduces Fat Accumulation in Rats Fed an Obesogenic Diet Saioa Gómez-Zorita,†,§ Alfredo Fernández-Quintela,†,§ Arrate Lasa,†,§ Leixuri Aguirre,†,§ Agnes M. Rimando,# and María P. Portillo*,†,§ †
Nutrition and Obesity Group, Department of Nutrition and Food Science, University of the Basque Country (UPV/EHU) and Lucio Lascaray Research Center, 01006 Vitoria, Spain § CIBERobn Physiopathology of Obesity and Nutrition, Institute of Health Carlos III (ISCIII), Spain # U.S. Department of Agriculture, Agricultural Research Service, Natural Products Utilization Research Unit, University, Mississippi, United States ABSTRACT: The current study aimed to demonstrate the effects of pterostilbene in rats fed an obesogenic diet. For this purpose, pterostilbene was administered at doses of 15 mg/kg body weight/day (PT15 group) or 30 mg/kg body weight/day (PT30 group) for 6 weeks. Pterostilbene reduced adipose tissue mass −15.1% (PT15) and −22.9% (PT30). In this tissue, it decreased malic enzyme (−39.4 and −49.5% for PT15 and PT30 groups, respectively) and fatty acid synthase (−45 and −53.4% for PT15 and PT30) activities. Acetyl-CoA carboxylase activity was reduced and AMPK activity was increased only in the PT30 group. In the liver, pterostilbene (PT30) reduced malic enzyme (−29.5%) and glucose-6-P dehydrogenase (−43.2%) activities and increased carnitine palmitoyltransferase-1a (37.5%) and acyl-coenzyme A oxidase (42.5%) activities. This increased oxidative capacity was not associated with increased mitochondriogenesis. Among biochemical serum parameters, only insulin was modified by pterostilbene (−31.6%) in the PT15 group. The amounts of pterostilbene in serum and tissues from rats in the PT30 group were in not all cases 2-fold greater than those found in the PT15 group. In conclusion, pterostilbene shows antiobesity properties due, at least in part, to reduced lipogenesis in adipose tissue and increased fatty acid oxidation in liver. KEYWORDS: adipose tissue, bioavailability, fatty acid oxidation, lipogenesis, pterostilbene
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INTRODUCTION Obesity and overweight are major public health concerns that are spreading throughout the world, afflicting not only adults but also many children and adolescents. Moreover, obesity is associated with several chronic diseases, such as diabetes, strokes, and hypertension.1 Considerable efforts are being made to identify naturally occurring molecules that are active and safe when administered orally and can be employed for obesity management, using a broad range of in vivo and in vitro methodologies.2 Phenolic compounds make up one of the groups of molecules that have been most frequently studied in recent years. In this context, resveratrol has been reported to decrease triacylglycerols in cultured adipocytes3−5 and to reduce adipose tissue in various animal models, such as mice,6 rats,7 and primates.8 However, resveratrol has a very low bioavailability as a consequence of its rapid metabolism in the gut and liver.9 This poor bioavailability is highly dependent on its free hydroxyl groups, making it susceptible to glucuronidation and sulfation.10 Due to this fact, the scientific community is looking for other resveratrol-related molecules showing higher bioavailability. In this context, pterostilbene, a dimethyl ether derivative of resveratrol that is less metabolized than resveratrol, is a good candidate.11 Pterostilbene is a phenolic compound biologically classified as a phytoalexin, which is part of a plant’s defense system and is synthesized in response to pathogen infection, as well as to excessive ultraviolet exposure. Pterostilbene is known to have © 2014 American Chemical Society
diverse benefits in the prevention and treatment of several diseases, including cancer,12 dyslipidemia,13 diabetes,14 and cardiovascular and cognitive function degeneration.15 However, the potential effects of pterostilbene on body fat accumulation have not been studied yet. In this scenario, the aim of the present study was to determine the effect of pterostilbene on body fat in rats fed an obesogenic diet. The analysis of several mechanisms of action underlying this effect was also assessed.
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MATERIALS AND METHODS
Chemicals. 3T3-L1 preadipocytes were supplied by American Type Culture Collection (Manassas, VA, USA). Pterostilbene (purity > 98%) and resveratrol (purity > 98%) were obtained from Bertin Pharma (Montigny le Bretonneux, France). Dulbecco’s modified Eagle medium (DMEM) was purchased from Life Technologies (Grand Island, NY, USA). Infinity Triglycerides reagent for triacylglyceride content and BCA reagent for protein determination were obtained from Thermo Scientific (Rockford, IL, USA). Antibodies for phosphorylated acetyl-CoA carboxylase (ACC1) (Ser 79), ACC1, phosphorylated-adenosine monophosphate-activated protein kinase (AMPK) (Thr 172), and AMPK were purchased form Cell Signaling Technology (Beverly, MA, USA). Trizol for RNA isolation was acquired from Invitrogen (Carlsbad, CA, USA). Primers for gene Received: Revised: Accepted: Published: 8371
March 19, 2014 July 31, 2014 August 1, 2014 August 1, 2014 dx.doi.org/10.1021/jf501318b | J. Agric. Food Chem. 2014, 62, 8371−8378
Journal of Agricultural and Food Chemistry
Article
Table 1. Primer Sequences, Annealing Temperatures, and Primer Concentrations for PCR Amplification of Each Gene Studied genea
sense primer
antisense primer
annealing (°C)
concn (nM)
FAS ATGL HSL IL6 CPT-1a CD36 TFAM COX-II β-actin
5′-AGCCCCTCAAGTGCACAGTG-3′ 5′-CACTTTAGCTCCAAGGATGA-3′ 5′-CCCATAAGACCCCATTGCCTG-3′ 5′-TCCTACCCCAACTTCCAATGCTC-3′ 5′-CGGTTCAAGAATGGCATCATC-3′ 5′-GGTGTGCTCAACAGCCTTATC-3′ 5′-GCTTCCAGGGGGCTAAGGAT-3′ 5′-AACAATTCTCCCAGCTGTCATTC-3′ 5′-TCTATGAGGGCTACGCTCTCC-3′
5′-TGCCAATGTGTTTTCCCTGA-3′ 5′-TGGTTCAGTAGGCCATTCCT-3′ 5′-CTGCCTCAGACACACTCCTG-3′ 5′-TTGGATGGTCTTGGTCCTTAGCC-3′ 5′-TCACACCCACCACCACGAT-3′ 5′-TTATGGCAACCTTGCTTATG-3′ 5′-CCCAATCCCAATGACAACTC-3′ 5′-AGTCAAAGCATAGGTCTTCATAGTC-3′ 5′- CACGCTCGGTCAGGATCTTC-3′
60.0 60.0 60.0 60.0 60.0 62.1 60.0 60.0 60.0
300 900 300 300 300 300 300 300 300
a
FAS, fatty acid synthase; ATGL, adipose triglyceride lipase; HSL, hormone sensitive lipase; IL6, interleukine 6; CPT, carnitine palmitoyltransferase; CD36, cluster of differentiation 36; TFAM, mitochondrial transcription factor; COX-II, cytochrome c oxidase subunit II.
expression were purchased from Integrated DNA Technologies, Inc. (Leuven, Belgium). For pterostilbene analysis, β-glucuronidase (from Escherichia coli, Type IX-A) and sulfatase (from abalone entrails, Type VIII) were acquired from Sigma-Aldrich (St. Louis, MO, USA), and bis[trimethylsilyl]-trifluoroacetamide/dimethylformamide (BSTFA/ DMF, 1:1), the derivation reagent, was obtained from Pierce Biotechnology, Inc. (Rockford, IL, USA). All other reagents and chemicals were of the highest purity grade available and from Sigma-Aldrich. Cell Culture. 3T3-L1 preadipocytes were cultured in DMEM containing 10% fetal bovine serum (FBS). Two days after confluence (day 0), the cells were stimulated to differentiate with DMEM containing 10% FBS, 10 μg/mL insulin, 0.5 mM isobutylmethylxanthine (IBMX), and 1 μM dexamethasone for 2 days. On day 2, the differentiation medium was replaced by FBS/DMEM (10%) medium containing 10 μg/mL insulin and from day 4 onward by FBS/DMEM (10%) medium containing 0.2 μg/mL insulin. This medium was changed every 2 days until cells were harvested. All media contained 1% penicillin/streptomycin (10000 U/mL), and the media for differentiation and maturation contained 1% (v/v) biotin and panthothenic acid. Cells were maintained at 37 °C in a humidified 5% CO2 atmosphere. For the adipocyte treatment, cells grown in 6-well plates were incubated with pterostilbene or resveratrol at 1 μM (diluted in 95% ethanol) for 24 h on day 12 after differentiation. By that day, >90% of cells were mature adipocytes with visible lipid droplets. This cell treatment was carried out in triplicate. Adipocyte, Adipose Tissue, and Liver Triacylglycerol Content. For triacylglycerol extraction, cells were washed extensively with phosphate-buffered saline (PBS) and incubated three times with 800 μL of hexane/isopropanol (2:1). The total volume was then evaporated under nitrogen, and the pellet was resuspended in 200 μL of Triton X-100 in 1% distilled water. Afterward, triacylglycerols were solubilized by a sonicator, and the content was measured using Infinity Triglycerides reagent. For protein determination, cells were lysed in 0.3 N NaOH and 0.1% SDS. Protein measurements were performed using the BCA reagent. Adipose tissue and liver total lipids were extracted following the method described by Folch et al.16 The lipid extract was dissolved in isopropanol and triacylglycerol content, was measured using a commercial kit (BioSystems, Barcelona, Spain). Cytotoxicity Assay. Cell viability was assessed in cells treated for 24 h with 1 μM pterostilbene or resveratrol by the neutral red assay (TOX4 kit, Sigma-Aldrich, St. Louis, MO, USA). Animals, Diets, and Experimental Design. The experiment was conducted with 27 male Wistar rats (6 weeks old) with an initial body weight of 180 ± 2 g purchased from Harlan Ibérica (Barcelona, Spain) and took place in accordance with the University of the Basque Country’s guide for the care and use of laboratory animals (reference protocol approval CUEID CEBA/30/2010). The rats were individually housed in polycarbonate metabolic cages (Techniplast Gazzada, Guguggiate, Italy) and placed in an air-conditioned room (22 ± 2 °C)
with a 12 h light−dark cycle. After a 6 day adaptation period, rats were randomly divided into three experimental groups of nine animals each and fed a commercial obesogenic diet, high in sucrose (20.0%) and fat (22.5%) (Harlan Ibérica, TD.06415). Pterostilbene (purity = 99.9%) was synthesized according to a published procedure.15 This polyphenol was added to the diet in amounts that assured doses of 15 mg/kg body weight/day (PT15 group) or 30 mg/kg body weight/ day (PT30 group). All animals had free access to food and water. Food intake and body weight were measured daily. At the end of the experimental period (6 weeks), animals were sacrificed, after an overnight fasting, under anesthesia (chloral hydrate) by cardiac exsanguination. White adipose tissue from different anatomical locations (perirenal, epididymal, mesenteric, and subcutaneous) and liver were dissected and weighed. Serum was obtained from blood samples after centrifugation (1000g for 10 min at 4 °C). All samples were stored at −80 °C until analysis. Serum Parameters. Serum insulin, triacylglycerols, and nonesterified fatty acids (NEFAs), as well as aspartate aminotransferase (AST/GOT) and alanine aminotransferase (ALT/GPT) activities, were measured using a commercial kits (BioSystems, Barcelona, Spain; EZRMI-13K, Linco, St. Charles, MO, USA; Roche Diagnostics GmbH, Mannheim, Germany; and Spinreact, Barcelona, Spain, respectively). An ELISA kit was used for interleukin 6 (IL6) determination (Quantikine ELISA, R&D Systems, Abingdon, UK). Enzyme Activities in White Adipose Tissue and Liver. For lipogenic enzyme analysis, samples of subcutaneous adipose tissue (1 g) or liver (0.5 g) were homogenized in 5 mL of buffer (pH 7.6) containing 150 mM KCl, 1 mM MgCl2, 10 mM N-acetylcysteine, and 0.5 mM dithiothreitol. After centrifugation at 100000g for 40 min at 4 °C, the supernatant fraction was used for quantitation of enzyme activities. Fatty acid synthase (FAS), glucose-6-phosphate dehydrogenase (G6PDH), malic enzyme (ME), and lipoprotein lipase (LPL) activities were measured as previously described.17 Enzyme activities were expressed either as nanomoles of NADPH consumed (FAS) or as nanomoles of NADPH produced (G6PDH and ME) per minute per milligram of protein. Protein was assessed using the Bradford method.18 Carnitine palmitoyltransferase-1a (CPT-1a) and acyl-coenzyme A oxidase (ACO) activities in liver were assessed in the mitochondrial/ peroxisomal fraction as previously described.16 CPT-1a activity was expressed as nanomoles of CoA formed per minute per milligram of protein and ACO activity as nanomoles of NADH formed per minute per milligram of protein. Extraction and Analysis of RNA and Quantification by Reverse Transcription Polymerase Chain Reaction (Real-Time RT-PCR). RNA samples were extracted from cells by using Trizol, according to the manufacturer’s instructions. The integrity of the RNA extracted from all samples was verified and quantified using an RNA 6000 Nano Assay (Thermo Scientific, Wilmington, DE, USA). RNA samples were then treated with DNase I kit (Applied Biosystems, Foster City, CA, USA) to remove any contamination with genomic DNA. One microgram of total RNA in a total reaction volume of 20 μL was reverse transcribed using the iScript cDNA Archive Kit (Applied 8372
dx.doi.org/10.1021/jf501318b | J. Agric. Food Chem. 2014, 62, 8371−8378
Journal of Agricultural and Food Chemistry
Article
Table 2. Final Body Weight, Food Intake, Body Weight Gain, Adipose Tissue Weight, Liver Weight, and Liver Triacylglycerol Content of Rats Fed the Experimental Diets for 6 Weeksa control final body weight (g) food intake (g/day) body weight gain (g) adipose tissue weights epididymal (g) perirenal (g) mesenteric (g) subcutaneous (g) PR + MS (g) E + PR + MS (g) total (g) liver weight (g) triacylglycerols (mg/g)
PT15
382 ± 5 17.6 ± 0.3 202 ± 10 11.8 13.5 6.02 16.8 18.8 36.0 47.5
± ± ± ± ± ± ±
387 ± 9 17.7 ± 0.5 206 ± 9
0.9 0.8 a 0.4 a 1.1 a 3.1 a 3.1 a 3.0a
10.5 12.0 5.19 12.6 17.2 30.1 40.3
9.40 ± 0.4 33.1 ± 4.9
± ± ± ± ± ± ±
0.9 1.1ab 0.4 ab 1.2 b 1.3 b 2.5 b 3.0b
10.0 ± 0.3 30.9 ± 3.0
PT30
ANOVA
376 ± 9 17.5 ± 0.3 200 ± 6
NS NS NS
± ± ± ± ± ± ±
NS P< P< P< P< P= P
