METABOLIC SYNDROME DIABETOLOGY & Moreno et al. Diabetology & Metabolic Syndrome 2014, 6:84 http://www.dmsjournal.com/content/6/1/84 RESEARCH Open Access Metabolic profile response to administration of epigallocatechin-3-gallate in high-fat-fed mice Mayara Franzoi Moreno1*, Rachel De Laquila1,2, Marcos Hiromu Okuda1, Fábio Santos Lira3, Gabriel Inácio de Morais Honorato de Souza1, Cláudio Teodoro de Souza4, Monica Marques Telles5, Eliane Beraldi Ribeiro1, Claudia Maria Oller do Nascimento1 and Lila Missae Oyama1 Abstract Background: Obesity is associated with increased adipose tissue and glucose intolerance. High-fat diets (HFDs) are known to induce obesity and increase proinflammatory adipokines. The consumption of green tea may improve the health of obese individuals because it contains a potent antioxidant that has effects on body weight, energy expenditure and serum cholesterol concentrations. Methods: We examined the effects of epigallocatechin-3-gallate (EGCG) (50 mg/kg body weight per day) or saline after 30 or 60 days of treatment. Mice were distributed into four groups: 1) NS: normolipidic diet receiving saline; 2) NE: normolipidic diet receiving EGCG; 3) HFS: high-fat diet receiving saline; 4) HFE: high-fat diet receiving EGCG. Results: We observed that administration of a HFD plus EGCG treatment for 60 days reduced delta weight, the relative weights of the mesenteric adipose tissue (MES), retroperitonial adipose tissue (RET), epididymal adipose tissue (EPI), the sum of the adipose tissues (SAT), reduced triacylglycerol (TG) and improved both high-density lipoprotein (HDL) cholesterol levels and the adiponectin/STA ratio when compared with HFS. Conclusions: Our results suggest that the chronic administration of EGCG (60 days) promoted a significant improvement in glucose tolerance, decreased adipose tissue deposits, weight mass, TG and HDL-C only when associated with high-fat diet treatment. Keywords: High-fat diet, Adipokines, Adipose tissue, Inflammation Introduction Obesity is characterised by excessive fat accumulation and a resultant imbalance in energy intake and expend- iture. Notably, HFD is the mainstream food habit of modern society and is one of the major factors contrib- uting to obesity [1]. The obese state is characterised by low-grade systemic inflammation, primarily resulting from increased adipocyte size and the recruitment of macro- phages into the white adipose tissue (WAT) [2]. Adipose tissue is an endocrine organ that releases protein factors, known as adipokines which include hormones implicated in energy balance (e.g. leptin and adiponectin), glucose tolerance and insulin sensitivity (adiponectin and resistin) * Correspondence: mayaramoreno@hotmail.com 1Departamento de Fisiologia, Universidade Federal de São Paulo-EPM, São Paulo, SP, Brasil Full list of author information is available at the end of the article © 2014 Moreno et al.; licensee BioMed Centra Commons Attribution License (http://creativec reproduction in any medium, provided the or Dedication waiver (http://creativecommons.or unless otherwise stated. and classical cytokines (e.g. TNF-α, interleukin-6 and 10) [3,4]. Several strategies have been adopted for treatment and prevention of obesity, as well as for associated conditions, such as insulin resistance, inflammation and hypertension. Nutritional interventions, including calorie restriction, vitamin supplementation, fresh green tea or extract con- sumption are often utilised to promote weight loss [2,5-7]. Green tea (Camellia sinensis) is one of the most widely consumed beverages in the world and experimental studies indicate that the use of green tea extract is useful for obesity treatment and prevention [6,8,9]. Among the many polyphenols present in green tea, epigallocatechin- 3-gallate (EGCG) is the most active form. It is suggested that the action of EGCG occurs in the sympathetic ner- vous system, specifically by inhibiting the enzyme catechol-O-methyl transferase, which degrades norepin- ephrine. Thus, EGCG could exert regulatory functions on l Ltd. This is an Open Access article distributed under the terms of the Creative ommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and iginal work is properly credited. The Creative Commons Public Domain g/publicdomain/zero/1.0/) applies to the data made available in this article, mailto:mayaramoreno@hotmail.com http://creativecommons.org/licenses/by/2.0 http://creativecommons.org/publicdomain/zero/1.0/ Moreno et al. Diabetology & Metabolic Syndrome 2014, 6:84 Page 2 of 7 http://www.dmsjournal.com/content/6/1/84 sympathetic activation and lypolysis. Furthermore, in ani- mal studies, we observed a reduction of blood glucose and insulin after the consumption of green tea [10]. Friedrich et al. [11] showed that the anti-obesity ef- fects of EGCG can be explained by decreased food di- gestibility and nutrient absorption, ultimately resulting in increased post-prandial fat oxidation and reduced in- corporation of dietary lipids into tissues. In addition, Li et al. [12] suggested that EGCG protects rats from free fatty acid (FFA)-induced insulin resistance. However, few studies have analysed the inflammatory status of the WAT of HFD-fed mice and the balance of anti- and pro-inflammatory adipokines (IL-10 and TNF- α). In this study, we examined the effects of EGCG con- sumption on the glycemic and lipid profiles, as well as the expression of TNF-α, adiponectin, IL-10 and NF-κB p65 in the mesenteric WAT of HFD-fed mice. Materials and methods Animals and treatment The research committee of Universidade Federal de São Paulo approved all procedures in this study (protocol n° 2009/1796). Three weeks old male Swiss mice were pur- chased from Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia, and kept under controlled conditions of light (12 h light–dark cycle with lights on at 6 am) and temperature (22 ± 1°C). After one week of acclimation, mice were distributed in four groups: 1) NS: normolipidic diet treatment with saline; 2) NE: normolipidic diet treatment with EGCG; 3) HFS: high-fat diet treatment with; 4) HFE: high-fat diet treat- ment with EGCG. Throughout the experimental period, the animals were maintained in collective cages and had ad libitum access to food and water. The diets were prepared according to Table 1 Macronutrients and micronutrients composition of th Nutrients Normolip Carbohydrates (g) 720.7 Carbohydrates (kcal) 75.8% Protein (g) 140 Protein (kcal) 14.7% Lipids (g) 40* Lipids (kcal) 9.5% Fiber (g) 50 Vitamin mix (g) 10 Mineral mix (g) 35 L-Cysteine (g) 1.8 Choline bitartrate (g) 2.5 Tert-butylhydroquinone (mg) 14 Energy value 3.8 kcal/g *40 g soybean oil; **312 g lard plus 40 g soybean oil. the recommendations of the American Institute of Nu- trition. The normolipidic groups and the high-fat groups were fed with formulation of the AIN-93G diet for rapid growth [13] (Table 1). A green tea extract, TEAVIGO® (DSM Nutritional Products, Swiss), containing >90% of EGCG (50 mg/kg body weight per day) or saline was administered daily through gavage. Two experimental groups were conducted, one for 30 and another for 60 days. Oral Glucose Tolerance Test (OGTT) In both experiments (30 or 60 days) all animals were given 12 hours of fast. Initially, the baseline blood was collected to assess basal glucose concentration from the tail vein. Then a glucose solution (1.4 g/kg of body weight) was administrated by gavage. Blood samples were collected again after 15, 30, 45, 60 and 120 minutes to obtain the glycemic curve. Sample collection At the end of each experiment (30 or 60 days), mice were euthanized by decapitation without sedation. Blood was collected and the serum fraction was extracted and stored at −80°C for further analysis. The adipose tissue depots: RET, MES and EPI, were dissected, weighed, im- mediately frozen in liquid nitrogen and stored at −80°C. Lipid profile measurements Labtest® commercial kits were used to assess serum total cholesterol (TC), HDL-cholesterol and triacylglycerol (TG). The samples were analysed using an enzymatic method. Analysis of TNF-α, Adiponectin and IL-10 levels Adipose tissue samples were carefully rinsed in ice-cold 0.9% NaCl to remove any blood contaminants, snap e normolipidic and high-fat diet AIN-93G (g/kg diet) idic diet (g/kg) High fat diet (g/kg) 408.7 30.5% 140 10.5% 352** 59% 50 10 35 1.8 2.5 14 5.36 kcal/g Moreno et al. Diabetology & Metabolic Syndrome 2014, 6:84 Page 3 of 7 http://www.dmsjournal.com/content/6/1/84 frozen in liquid nitrogen, and stored at −80°C. Frozen tissue (0.1 g – 0.3 g) was homogenized in RIPA buffer (0.625% Nonidet P-40, 0.625% sodium deoxycholate, 6.25 mM sodium phosphate and 1 mM ethylenediamine tetraacetic acid at pH 7.4) containing 10 mg/ml protease inhibitor cocktail (Sigma–Aldrich, St. Louis, Missouri). Homogenates were centrifuged at 12,000 g for 10 min at 4°C. The supernatant was saved and protein concen- tration was determined by the Bradford assay (Bio-Rad, Hercules, CA) using bovine serum albumin (BSA) as a standard. Quantitative assessment of TNF-α, adiponectin and IL-10 levels in adipose tissue was done with ELISA (DuoSet ELISA, R & D Systems, Minneapolis, MN). The TNF-α, adiponectin and IL-10 assay sensitivity was found to be 5.0 pg/ml in the range of 31.2 - 2000 pg/ml. The intra- and inter-assay variability of the TNF-α, adi- ponectin and IL-10 kits were, respectively, 2.2-4.8%, 3.4- 6.7% and 4.9-9.5%. The intra-assay variability of the TNF-α, adiponectin and IL-10 kit was 2.0–4.2%, and its inter-assay variability was of 3.3 -6.4%. All samples were run as duplicates, and the mean value was reported. Protein analysis by western blotting After euthanasia, the MES was dissected and homoge- nized in 1.0 mL of solubilization buffer at 4°C [1% Tri- ton X-100, 100 mm Tris–HCl (pH 7.4), 100 mm sodium pyrophosphate, 100 mm sodium fluoride, 10 mm EDTA, 10 mm sodium orthovanadate, 2.0 mm phenylmethylsul- fonyl fluoride (PMSF), and 0.1 mg aprotinin/mL] with a Polytron (model 713 T; Fisatom Equipamentos Científi- cos, São Paulo, SP/Brazil). Insoluble material was re- moved by centrifugation for 30 min at 9,000 × g in a 70. Ti rotor (Beckman, Fullerton, CA, USA) at 4°C. The protein concentration of the supernatants was per- formed by the BCA assay (Bio-Rad, Hercules, CA, USA). Proteins were denatured by boiling (5 min) in a Laemmli sample buffer 10 containing 100 mM DTT, run on 8, 10 or 12% SDS-PAGE in a Bio-Rad miniature slab gel apparatus. The electrotransfer of proteins from gels to nitrocellu- lose membranes was performed for ~1.30 h/4gels at 15 V (constant) in a Bio-Rad semi-dry transfer appar- atus. Nonspecific protein binding to the nitrocellulose was reduced by preincubation for 2 h at 22°C in block- ing buffer (5% nonfat dry milk, 10 mM Tris, 150 mM NaCl and 0.02% Tween 20). The nitrocellulose mem- branes were incubated overnight at 4°C with antibodies against NFκBp65 and alpha-tubulin obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA), diluted in 1:1000 with blocking buffer supplemented with 1% BSA and then washed for 30 min in blocking buffer without BSA. The blots were subsequently incubated with peroxidase-conjugated secondary antibody for 1 h at 22°C. For evaluation of protein loading, membranes were stripped and reblotted with an anti-alpha-tubulin antibody as appropriate. Specific bands were detected by chemiluminescence and visualization/capture was performed by exposure of the membranes to RX films. B and intensities were quantified by optical densitometry of developed autoradiographs (Scion Image software- Scion Corporation, Frederick, Md., USA). Statistical analysis All results are presented as means ± standard error of the mean (SEM). Statistical significances were assessed using two-way analysis of variance (ANOVA) followed by Tukey test as a post hoc analysis to identify signifi- cant differences among the groups. Differences were considered significant when p < 0.05. Results Body mass and tissue weight Significant differences in body mass and tissue weight were found only in the 60-day treatment groups. Differ- ences in the delta weight (g) were observed between the HFS and NS groups (p < 0.0001), the HFE and NE groups (p = 0.042) and the HFS group (p = 0.0036). The relative weight of the EPI in the HFS group was significantly higher than the NS group (p < 0.0001) and the HFE group was significantly higher than the HFS group (p = 0.0098). The relative weights of RET, MES and SAT were higher in the HFS group compared to the NS and HFE groups and the HFE was higher than the NE group (p ≤ 0.02) (Table 2). Biochemical and hormonal serum analyses During the 30-day treatment period, only the TC values differ between the groups, in the HFS group were lower than in the NS group (p =0.016), and the HFE group had a higher concentration compared to the HFS group (p < 0.001). On the other hand, during the 60-day treatment period, we observed that TG in the HFE group was sig- nificantly lower than in the NE group (p = 0.047). There were no differences in TC between groups. HDL levels were lower in the HFS group compared to the NS group (p = 0.0031) and higher in the HFE group compared to the HFS group (p = 0.0012). There was a significant de- crease in serum levels of insulin in the HFE group com- pared to the HFS group (p = 0.022). We observed that adiponectin in the NS group in- creased in comparison to the NE (p = 0.008) and HFS groups (p < 0.0001) and that adiponectin in the HFE group decreased in comparison to the NE group (p = 0.009). In addition, the adiponectin/SAT ratio was lower in the HFS group comparison to the NS group (p = 0.001) and higher Table 2 Body and adipose tissue weights during two treatment periods (30 days or 60 days) in the experimental groups Parameters 30 days of treatment (n = 5-7 per group) 60 days of treatment (n = 11-13 per group) N S NE HFS HFE N S NE HFS HFE Initial weight (g) 31.7 ± 0.8 32.9 ± 1.1 31.5 ± 0.8 29.8 ± 1.1 26.7 ± 0.8 26.4 ± 0.5 28.9 ± 0.8 24.7 ± 1.0 Final weight (g) 37.8 ± 3.1 34.8 ± 5.7 36.7 ± 5.6 37.7 ± 6.0 32.7 ± 1.0 31.4 ± 0.8 42.5 ± 1.2* 33.8 ± 1.8$ Delta weight (g) 6.2 ± 1.3 3.3 ± 1.4 5.8 ± 1.3 8.6 ± 1.4 6.1 ± 0.8 5.0 ± 0.8 13.7 ± 1.5* 9.1 ± 1.0**$ EPI (g) 1.49 ± 0.2 1.15 ± 0.2 1.52 ± 0.2 1.67 ± 0.3 0.8 ± 0.1 0.7 ± 0.1 2.0 ± 0.2* 1.0 ± 0.2$ EPI (%) 3.80 ± 0.7 3.11 ± 0.7 3.82 ± 0.7 4.05 ± 0.9 2.5 ± 0.3 2.2 ± 0.2 4.6 ± 0.4* 3.2 ± 0.3$ RET (g) 0.38 ± 0.1 0.35 ± 0.1 0.47 ± 0.1 0.43 ± 0.1 0.3 ± 0.04 0.2 ± 0.02 0.6 ± 0.1* 0.4 ± 0.1$ RET (%) 0.96 ± 0.2 0.98 ± 0.2 1.19 ± 0.2 1.07 ± 0.2 0.8 ± 0.1 0.6 ± 0.1 1.4 ± 0.1* 1.1 ± 0.1**$ MES (g) 0.55 ± 0.1 0.51 ± 0.1 0.60 ± 0.1 0.68 ± 0.1 0.3 ± 0.05 0.2 ± 0.03 0.8 ± 0.1* 0.4 ± 0.1$ MES (%) 1.39 ± 0.2 1.42 ± 0.3 1.51 ± 0.2 1.67 ± 0.3 0.8 ± 0.1 0.8 ± 0.1 1.9 ± 0.2* 1.4 ± 0.2**$ SAT (g) 2.41 ± 0.3 2.02 ± 0.3 2.60 ± 0.3 2.78 ± 0.5 1.3 ± 0.2 1.1 ± 0.1 3.5 ± 0.3* 1.8 ± 0.2$ SAT (%) 6.16 ± 0.7 5.51 ± 0.5 6.53 ± 0.6 6.79 ± 0.9 3.2 ± 0.6 3.6 ± 0.3 7.4 ± 0.8* 4.4 ± 0.7**$ Data are mean ± SEM. *Different from NS. **Different from NE. $Different from HFS. Moreno et al. Diabetology & Metabolic Syndrome 2014, 6:84 Page 4 of 7 http://www.dmsjournal.com/content/6/1/84 in the HFE group comparison to the HFS group (p = 0.009) (Table 3). Cytokines in mesenteric adipose tissue In both treatment periods (30 or 60 days), no differences were observed in mesenteric cytokines levels between any of the groups (data not shown). OGTT Glucose tolerance, as measured by OGTT, did not differ between any of the groups in the 30-day treatment period. However, in the 60-day treatment groups, we ob- served differences between the HFS group and the NS group (p = 0.003) as well as between the HFE group and the NE group (p = 0.001) (Figure 1). Quantification of inflammatory proteins Western blot analysis shows that there were no differ- ences in NF-kBp65 protein levels between any of the groups treated for 30 days. Table 3 Triacylglycerol (TG), Total Cholesterol (TC), High Densi adiponectin /sum of adipose tissue (SAT) concentrations during experimental groups Parameters 30 Days of Treatment (n = 5-7 per group) N S NE HFS HF TG (mg/dL) 169.8 ± 19.1 166.8 ± 9.9 152.0 ± 8.5 137 TC (mg/dL) 174.7 ± 7.5 176.5 ± 5.0 155.7 ± 7.5* 181 HDL-C (mg/dL) 36.21 ± 2.2 40.99 ± 4.8 39.32 ± 4.5 36. Insulin (ng/mL) 0.6 ± 0.1 0.5 ± 0.1 0.3 ± 0.04 0.4 Adiponectin (μg/mL) - - - - Adiponectin/SAT - - - - Data are mean ± SEM (n = 5-8 per group); *Different from NS. **Different from NE. $ However, NF-kB p65 expression in the MES was in- creased in the NE group compared to the other groups after the 60-day treatment period (p < 0.005) (Figure 2). Discussion The pathogenesis of obesity and metabolic diseases is as- sociated with intake of a high-fat diet. Chronic systemic inflammation directly contributes to the development of obesity [14]. Therefore, suppressing chronic inflamma- tion may be a good strategy to prevent and/or treat obesity. Interestingly, previous studies suggest that the positive impacts of polyphenols could work through their ability to suppress chronic inflammation [15,16]. Based on this knowledge, an alternative strategy, such as phytother- apy treatment, may benefit modern obesity therapies. In this experimental study, we attempted to induce metabolic changes in mice by administration of a high- fat diet and then compared the outcomes of treatment with EGCG or saline over either 30-day or 60-day pe- riods. Our results show that the treatment of Swiss mice with a high-fat diet for 30 days did not increase weight ty Lipoprotein - Cholesterol (HDL- C), insulin, adiponectin, two treatment periods (30 days our 60 days) in the 60 Days of Treatment (n = 11-13 per group) E N S NE HFS HFE .6 ± 5.3 174.0 ± 16.1 184.0 ± 14.4 145.1 ± 5.4 136.6 ± 5.4** .6 ± 6.5$ 176.7 ± 6.4 212.0 ± 11.4 194.9 ± 13.6 178.1 ± 8.6 89 ± 1.9 42.1 ± 1.3 44.4 ± 0.8 37.0 ± 1.0* 42.6 ± 1.7$ ± 0.05 1.2 ± 0.2 1.0 ± 0.3 1.9 ± 0.2 0.8 ± 0.1$ 3.4 ± 0.2 2.5 ± 0.2* 1.8 ± 0.2* 1.6 ± 0.1** 99.8 ± 14.8 91.6 ± 8.6 30.1 ± 5.4* 33.9 ± 5.2$ Different from HFS. Figure 1 Oral Glucose Tolerance Test (OGTT) during two treatment periods (30 days our 60 days) in the experimental groups. *Different from NS (p = 0.003); ** different from HFE (p = 0.001). Moreno et al. Diabetology & Metabolic Syndrome 2014, 6:84 Page 5 of 7 http://www.dmsjournal.com/content/6/1/84 gain, relative tissue weights or induce significant changes in OGTT or inflammatory profile. The experimental period of 30 days may be too short to show significant changes in these parameters. On the other hand, we demonstrated that a 60-day treatment period of high-fat diet and EGCG treatment was effective in triggering inflammatory processes such as a decrease in adiponectin and induced changes in glu- cose homeostasis, weight mass, TG and HDL-C. The anti-obesity effects of green tea are most likely due to its capacity to elevate thermogenesis and fat oxidation Figure 2 Quantification of NF-kB p65 protein levels during the two tr groups. *Different from NS compared to the others groups (p = 0.005). [17]. Thus, we hypothesised that EGCG treatment reduces body-fat mass by changes in cytokine production and pro-inflammatory molecule protein levels. Our study is in agreement with other reports in the literature that clinical studies have shown that the consumption of EGCG is connected with weight loss [18]. Our results contrast with those currently expected once the literature evidences the increase in adiponectin with weight loss [19]. However, some studies also showed no in- crease in adiponectin with weight loss but still found an im- provement in insulin resistance, agreeing with the findings eatment periods (30 days our 60 days) in the experimental Moreno et al. Diabetology & Metabolic Syndrome 2014, 6:84 Page 6 of 7 http://www.dmsjournal.com/content/6/1/84 of this study [20,21]. It is possible that weight loss can lead to increased insulin sensitivity in other ways independent of the action of adiponectin, such as mobilisation of intracellu- lar lipid content of the liver [22]. When we analysed serum adiponectin levels we ob- served that the high-fat diet was effective in reducing serum adiponectin levels and that EGCG contributed to the decrease in both, normolipidic as in high-fat diet. However, when we need to consider the amount of fat in each mouse and adjust the serum adiponectin level with the SAT, the results showed that EGCG improved the adiponectin concentration. In another study, green tea extract added to the diet (1%) did not alter the concentrations of adiponectin [23] in adult mice fed a high-fat diet for 12 weeks. Addition of 1.2% green tea extract into the water decreased adipo- nectin concentrations in mice after eight weeks of treat- ment; however [24]. By adjusting our results for adiponectin/STA we observed that the negative effect of green tea disappears. The change in adiponectin could be explained by the reduced fat mass in the HFS and HFE groups since this is a cytokine that is produced by adipocytes. EGCG treatment caused decreased adipose tissue de- posits corresponding to the improvement of insulin levels in the HFE group. Several studies have shown that weight loss is associated with decreased insulin resist- ance and inflammatory markers [25]. These findings demonstrate that EGCG may be a novel, plant-derived compound capable of reducing the risk of HFD-induced glucose intolerance. Fu et al. [26] examined the potential (−)-epigallocate- chin gallate (EGCG, 0.05% in drinking water) on effect- ively delaying the onset of type 1 diabetes (T1D) in non- obese diabetic (NOD) mice. Mice supplemented with EGCG had significantly higher plasma insulin levels and survival rates compared with the control animals. EGCG had no significant effects on food or water intake or body weight in mice, suggesting that the glucose-lowering effect was not due to an alteration in these parameters. While EGCG did not modulate insulinemia, it did elevate the levels of the circulating anti-inflammatory cytokine IL-10 in NOD mice. These findings demonstrate that EGCG may be a novel, plant-derived compound capable of redu- cing the risk of T1D. Obesity and insulin resistance are associated with low grade chronic systemic inflammation [27]. In the present study, we observed no changes in the cytokine profile during either treatment period (30 days or 60 days). However, we did observe differences in delta weight, relative RET, MES, EPI and SAT weights, adiponectin levels and glucose homeostasis. We hypothesise that these differences will become more pronounced in a longer trial period (>60 days). We see the normocaloric diet with EGCG increased phosphorylation of the p65 subunit from the NF-kB complex in MES. These are unexpected effects of the administration of EGCG. Perhaps the consumption of antioxidants from the EGCG caused an adverse effect on the NE group. In summary, our data demonstrated that the adminis- tration of EGCG (60 days) promoted a significant im- provement in glucose tolerance, decreased adipose tissue deposits, weight mass, TG and HDL-C only when asso- ciated with high-fat diet treatment. More studies are re- quired to better understand the mechanism of this effect and to further elucidate the role of EGCG in reversing the inflammatory effects triggered by a high-fat diet. Abbreviations HFDs: High-fat diets; EGCG: Epigallocatechin-3-gallate; NS: Normolipidic diet treatment with saline; NE: Normolipidic diet treatment with EGCG; HFS: High- fat diet treatment with; HFE: High-fat diet treatment with EGCG; TNF- α: Tumor necrosis factor alpha; ELISA: Enzyme-linked immunosorbent assay; NF-κB: Nuclear factor kappa B; WAT: White adipose tissue; MES: Mesenteric adipose tissue; RET: Retroperitonial adipose tissue; SAT: Sum of adipose tissues; TG: Triacylglycerol; FFA: Free fatty acid; TC: Total cholesterol; HDL- C: High density lipoprotein - Cholesterol. Competing interests The authors declare that they have no competing interests. Authors’ contribution MFM and RDL - designed the study, carried out the experiments, performed the statistical analysis and drafted the manuscript. MHO, FSL, GIMHS, CTS, MMT - helped to carried out the experiments, revised and helped to draft the manuscript. EBR - helped to draft the manuscript. CMON - conceive the study, participated in its design, and helped to draft the manuscript. LMO - conceive the study, participated in its design, and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements This work was supported by FAPESP (grants n° 2009/14373-8), and FAINC. Author details 1Departamento de Fisiologia, Universidade Federal de São Paulo-EPM, São Paulo, SP, Brasil. 2Faculdades Integradas Coração de Jesus – FAINC, Santo André, SP, Brasil. 3Immunometabolism Research Group, Department of Physical Education, Universidade Estadual Paulista, UNESP, Presidente Prudente, SP, Brazil. 4Laboratory of Exercise Biochemistry and Physiology, Health Sciences Unit, University of Southern Santa Catarina, Criciúma, SC, Brazil. 5Departamento de Ciências Biológicas, Universidade Federal de São Paulo-Campus Diadema, Diadema, SP, Brasil. Received: 27 January 2014 Accepted: 6 August 2014 Published: 12 August 2014 References 1. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Abstract Background Methods Results Conclusions Introduction Materials and methods Animals and treatment Oral Glucose Tolerance Test (OGTT) Sample collection Lipid profile measurements Analysis of TNF-α, Adiponectin and IL-10 levels Protein analysis by western blotting Statistical analysis Results Body mass and tissue weight Biochemical and hormonal serum analyses Cytokines in mesenteric adipose tissue OGTT Quantification of inflammatory proteins Discussion Abbreviations Competing interests Authors’ contribution Acknowledgements Author details References << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /PageByPage /Binding /Left /CalGrayProfile (Dot Gain 20%) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.4 /CompressObjects /Tags /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /DetectCurves 0.1000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails true /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams true /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true /PreserveFlatness true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts true /TransferFunctionInfo /Apply /UCRandBGInfo /Preserve /UsePrologue false /ColorSettingsFile () /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /CropColorImages true /ColorImageMinResolution 300 /ColorImageMinResolutionPolicy /OK /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 300 /ColorImageDepth -1 /ColorImageMinDownsampleDepth 1 /ColorImageDownsampleThreshold 1.50000 /EncodeColorImages true /ColorImageFilter /DCTEncode /AutoFilterColorImages true /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /CropGrayImages true /GrayImageMinResolution 300 /GrayImageMinResolutionPolicy /OK /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /GrayImageResolution 300 /GrayImageDepth -1 /GrayImageMinDownsampleDepth 2 /GrayImageDownsampleThreshold 1.50000 /EncodeGrayImages true /GrayImageFilter /DCTEncode /AutoFilterGrayImages true /GrayImageAutoFilterStrategy /JPEG /GrayACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /GrayImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000GrayACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000GrayImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasMonoImages false /CropMonoImages true /MonoImageMinResolution 1200 /MonoImageMinResolutionPolicy /OK /DownsampleMonoImages true /MonoImageDownsampleType /Bicubic /MonoImageResolution 1200 /MonoImageDepth -1 /MonoImageDownsampleThreshold 1.50000 /EncodeMonoImages true /MonoImageFilter /CCITTFaxEncode /MonoImageDict << /K -1 >> /AllowPSXObjects false /CheckCompliance [ /None ] /PDFX1aCheck false /PDFX3Check false /PDFXCompliantPDFOnly false /PDFXNoTrimBoxError true /PDFXTrimBoxToMediaBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXSetBleedBoxToMediaBox true /PDFXBleedBoxToTrimBoxOffset [ 0.00000 0.00000 0.00000 0.00000 ] /PDFXOutputIntentProfile (None) /PDFXOutputConditionIdentifier () /PDFXOutputCondition () /PDFXRegistryName () /PDFXTrapped /False /CreateJDFFile false /Description << /ARA /BGR /CHS /CHT /CZE /DAN /DEU /ESP /ETI /FRA /GRE /HEB /HRV /HUN /ITA /JPN /KOR /LTH /LVI /NLD (Gebruik deze instellingen om Adobe PDF-documenten te maken voor kwaliteitsafdrukken op desktopprinters en proofers. 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