Eur J Nutr (2016) 55:341–348 DOI 10.1007/s00394-015-0854-9
ORIGINAL CONTRIBUTION
The combination of resveratrol and quercetin enhances the individual effects of these molecules on triacylglycerol metabolism in white adipose tissue Noemí Arias · M. Teresa Macarulla · Leixuri Aguirre · Iñaki Milton · María P. Portillo
Received: 24 September 2014 / Accepted: 31 January 2015 / Published online: 11 February 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Purpose The aim of this study was to analyze whether the combination of resveratrol and quercetin showed additive or synergic effects on body fat accumulation and triacylglycerol metabolism in adipose tissue from rats fed an obesogenic diet. Methods Rats were divided into four dietary groups: a control group and three groups each treated with either resveratrol (15 mg/kg/day; RSV), quercetin (30 mg/kg/day; Q), or both (15 mg resveratrol/kg/day and 30 mg quercetin/kg/day; RSV + Q) for 6 weeks. White adipose tissues from several anatomical locations were dissected. Serum parameters were analyzed by using commercial kits. The activities of fatty acid synthase and heparin-releasable lipoprotein lipase (HR-LPL) were measured using spectrophotometric and fluorimetric methods, and protein expression of acetyl-CoA carboxylase (ACC), adipose tissue triglyceride lipase (ATGL), and hormone-sensitive lipase (HSL) by western blot. Results The administration of either resveratrol or quercetin separately did not induce significant reductions in adipose tissue weights. By contrast, the combination of both molecules led to a significant reduction in all the fat depots analyzed. The percentage of reduction in each tissue was
N. Arias · M. T. Macarulla · L. Aguirre · I. Milton · M. P. Portillo (*) Nutrition and Obesity Group, Department of Nutrition and Food Science, Faculty of Pharmacy and Lucio Lascaray Research Center, University of the Basque Country (UPV/EHU), Paseo de la Universidad 7, 01006 Vitoria, Spain e-mail:
[email protected] N. Arias · M. T. Macarulla · L. Aguirre · M. P. Portillo CIBER de Fisiopatología de la Obesidad y Nutrición (CIBERobn), Instituto de Salud Carlos III, Vitoria, Spain
greater than the calculated additive effect. HR-LPL activity was reduced in RSV and RSV + Q groups. The activity of HSL was not modified. By contrast, ACC was inhibited and ATGL increased only by the combination of both polyphenols. Conclusion The results obtained demonstrate a synergistic effect between resveratrol and quercetin and suggest that when these molecules are combined, a great number of metabolic pathways involved in adipose tissue triacylglycerol accumulation are affected. Keywords Resveratrol · Quercetin · Obesity · White adipose tissue · Rat
Introduction The prevalence of obesity in Western societies has increased dramatically in the recent years [1–3]. As a result, scientific research is constantly looking for new biomolecules which occur naturally in foodstuffs and plants and which can be effective in the prevention or treatment of this disease. In this context, considerable attention has been paid to polyphenols, and beneficial effects have been found. A general consensus concerning the body fat-lowering effect of resveratrol in mice and rats exists [4, 5]. Several studies have also shown this effect in primates [6, 7]. As far as quercetin is concerned, although controversial results have been reported, several authors have found decreases in body fat after quercetin treatment in mice and rats [8, 9]. No data for primates are available. In human beings, a small number of studies have been reported to date. Yoshino et al. [10] observed no changes in body fat in nonobese, postmenopausal women treated with resveratrol
13
342
at a dose of 75 mg/day, for 12 weeks. The lack of effects may be due to the fact that subjects were not obese. No changes in body fat were also found by Crandall et al. [11] in adults aged 65 years and older, with a mean body mass index of 29 ± 5 kg/m2, treated with 1, 1.5 or 2 g/day resveratrol for 4 weeks, and by Poulsen et al. [12] in healthy obese subjects treated with resveratrol, at a dose of 500 mg/ day, for 4 weeks. In these cases, the lack of effect could be due to the short treatment period. Finally, there is a study conducted in overweight–obese subjects treated with resveratrol at a dose of 3,000 mg/day for 8 weeks in which no changes in total adipose tissue were observed [13]. In this case, it is important to point out that the dose of resveratrol used was very high. There are data in the literature showing that low doses of resveratrol are sometimes more effective than higher doses [14, 15]. By contrast, Timmers et al. [16] found a reduction in adipocyte size in subjects treated with resveratrol at a dose of 150 mg/day for 30 days. In summary, the reported studies do not allow a clear conclusion concerning the effects of resveratrol in humans to be reached, and further studies are needed in this field of research. No clinical studies have been addressed to assess the anti-obesity effect of quercetin. In the vast majority of the studies reported, these molecules have been investigated as monotherapies. However, the use of combinations of natural products to achieve additive or synergistic effects might be an interesting approach. As far as polyphenols and obesity are concerned, few studies have investigated the effects of combinations of these biomolecules, and the results reported have been obtained in cultured cells. Thus, Yang et al. [17] showed that resveratrol and quercetin caused an enhanced increase in apoptosis in mature adipocytes compared with the predicted additive response. Also in maturing pre-adipocytes, they observed that the combination of these molecules caused an enhanced inhibition of adipogenesis, compared with the predicted additive response. The same group reported similar results by combining quercetin, resveratrol, and genistein [18, 19]. Herranz-López et al. [20] observed that the effect on adipogenesis of a standardized Hibiscus sabdariffa extract, rich in several polyphenols, was higher than the sum of its parts. These studies provide interesting and promising results, which should be checked under in vivo conditions. It is important to remember that the potential interactions in absorption and metabolism among biomolecules cannot be analyzed in cultured cells. Furthermore, the doses used in in vitro studies are frequently far higher than the concentrations of these biomolecules found in blood and tissues in in vivo studies, and this makes it difficult to extrapolate results. The aim of the present study was to analyze whether the combination of resveratrol, a stilbene abundant in red
13
Eur J Nutr (2016) 55:341–348
wine, grapes, berries, and nuts [21], and quercetin, a flavonoid found in abundance in onions, broccoli, tomatoes, apples, and berries [22], might show additive or synergic effects on body fat accumulation and triacylglycerol metabolism in adipose tissue of rats fed an obesogenic diet.
Materials and methods Animals, diets, and experimental design The experiment was conducted with thirty-six male 6-week-old Wistar rats purchased from Harlan Ibérica (Barcelona, Spain) and took place in accordance with the institution’s guide for the care and use of laboratory animals (CUEID CEBA/30/2010). Rats were individually housed in polycarbonate metabolic cages (Techniplast Gazzada, Buguggiate, 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 four dietary groups of nine animals each: a control group (C), a group treated with 15 mg resveratrol/kg body weight/ day (RSV), a group treated with 30 mg quercetin/kg body weight/day (Q), and a group treated with both 15 mg resveratrol/kg body weight/day and 30 mg quercetin/kg body weight/day (RSV + Q). All animals were fed a commercial obesogenic diet (OpenSource Diets Inc., Ref. D12451M), which provided 4.73 kcal/g, for 6 weeks. The composition of this diet was as follows: 233 g/kg of casein, 3.5 g/kg of l-cysteine, 85 g/ kg of cornstarch, 117 g/kg of maltodextrin, 201 g/kg of sucrose, 207 g/kg of lard, 29 g/kg of soy oil, 58 g/kg of cellulose, 12 g/kg of a mineral mixture, 15 g/kg de dibasic phosphate calcium, 12 g/kg of a vitamin mixture, and 2 g/ kg of choline bitartrate. Resveratrol was supplied by Monteloeder (Elche, Spain) and quercetin by Sigma (St. Louis, MO, USA). Resveratrol, quercetin, and the combination of both polyphenols were added to the diet as previously reported [23] in order to ensure the above-mentioned doses. Briefly, taking into account that the rats started eating as soon as the daily diet was replaced, the polyphenol solutions were added to the surface of the diet contained in food boxes in the metabolic cages. All animals had free access to food and water. Food intake and body weight were measured daily. At the end of the experimental period, animals were killed under anesthesia (chloral hydrate) by cardiac exsanguinations after a 12-h fasting period. White adipose tissue from four anatomical locations (perirenal, epididymal, mesenteric, and subcutaneous) was dissected, weighed, and immediately frozen in liquid nitrogen. For serum collection, blood was allowed to clot at 4 °C before centrifugation
Eur J Nutr (2016) 55:341–348
(1,000 g for 10 min, at 4 °C). All samples were stored at −80 °C until analysis. Serum parameters Commercial kits were used for serum parameter assessment: triacylglycerols (Ref. 1001313 Spinreact, Barcelona, Spain), total cholesterol (Ref. 11505 Biosystem Barcelona, Spain), HDL cholesterol (Ref. 1001095 Spinreact, Barcelona, Spain), free fatty acids (Ref. 11383175001 Roche Diagnostics GmbH, Mannheim, Germany), and glucose (Ref. 11503 Biosystem, Barcelona, Spain). Lipoprotein lipase and fatty acid synthase activities For heparin-releasable lipoprotein lipase (HR-LPL) activity determination, 500 mg of perirenal adipose tissue were incubated (37 °C, 45 min) in 400 μL of Krebs–Ringerphosphate buffer, which contains 100 mL of NaCl 0.15 M, 4 mL of KCl 0.15 M, 3 mL of CaCl2 0.10 M, 1 mL of MgSO4 0.15 M, 21 mL of phosphate buffer 50 mM, and 0.26 mg of heparin sodium (pH 7.4). Then, enzyme activity was assessed following the method described by Del Prado et al. [24], with minor modifications [25]. Briefly, samples (100 µL) of incubation medium were incubated (5 min, 37 °C) with a buffer containing dibutyril fluorescein (10 μg/mL), with or without NaCl (2.5 M). Fluorescence was measured, and HR-LPL activity was calculated by subtracting non-LPL lipolytic activity in the presence of NaCl from the total lipolytic activity, determined without NaCl. Results were expressed as nmol oleate released per minute per gram of tissue. For fatty acid synthase (FAS) activity assay, 1 g of adipose tissue was homogenized in 5 mL of a buffer (pH 7.6) containing 150 mM KCl, 1 mM MgCl2, 10 mM N-acetylcysteine and 0.5 mM dithiothreitol. After centrifuging at 100,000 g for 40 min at 4 °C, the supernatant fraction was used for quantification of enzyme activity. FAS activity was measured as previously described [26], from the rate of malonyl-CoA-dependent NADPH oxidation. Briefly, homogenate samples were incubated (5 min, 37 °C) in a buffer containing NADPH (150 μM) and malonyl-CoA (70 μM). NADPH was measured spectrophotometry by reading absorbance at 340 nm, and results were expressed as nmol of NADPH consumed per minute per milligram of protein. Western blot of acetyl‑CoA carboxylase, adipose triglyceride lipase, and hormone‑sensitive lipase For acetyl-CoA carboxylase (ACC), adipose triglyceride lipase (ATGL), and hormone-sensitive lipase (HSL) activity assessment, 100 mg of each perirenal adipose tissue
343
samples were homogenated in 500 µL of cellular PBS (pH 7.4), containing protease inhibitors (100 mM phenylmethylsulfonyl fluoride and 100 mM iodoacetamide). Homogenates were centrifuged at 500 g for 10 min at 4 °C. Protein concentrations in homogenates were measured by Bradford method [27] using bovine serum albumin as standard. Immunoblot analyses were performed using 20 µg of perirenal extracts separated by electrophoresis in a 10 % (for ACC) and 7.5 % (for lipases) SDS–polyacrylamide gel and transferred to PVDF membranes. The membranes were then blocked with 5 % caseine PBS-Tween buffer for 2 h at room temperature. Subsequently, they were blotted with the appropriate antibodies overnight at 4 °C. Protein levels were detected via specific antibody for ATGL (Santa Cruz Biotech, CA, USA), HSL, pHSL, ACC and pACC (Cell Signaling) (1:1,000), and polyclonal mouse β-actin antibody (1:5,000) (Sigma, St. Louis, MO, USA). Afterward, polyclonal anti-goat for ATGL, anti-rabbit for ACC, pACC, HSL and HSL phosphorylated in Ser660, and anti-mouse for β-actin (Sigma, St. Louis, MO, USA) antibodies (1:5,000) were incubated for 2 h at room temperature, and ATGL, HSL, and ACC were measured. After antibody stripping, the membranes were blocked and then incubated with a pHSL, pACC, and β-actin and measured again. The bound antibodies were visualized by an ECL system (Thermo Fisher Scientific Inc., Rockford, IL, USA) and quantified by a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). Specific bands were identified by using a standard loading buffer (Precision Plus protein standards dual color; Ref. 161-0374 Bio-Rad), and β-actin was used to normalize the results. Statistical analysis Results are presented as mean ± standard error of the means. Statistical analysis was performed using SPSS 20.0 (SPSS Inc. Chicago, IL, USA). Normal distribution of data was confirmed by Shapiro–Wilks test, and then, they were analyzed by using one-way ANOVA followed by Newman Keuls post hoc test. Statistical significance was set up at the P
