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ScienceDirect Journal of Nutritional Biochemistry xx (2014) xxx – xxx
Green tea extract improves high fat diet-induced hypothalamic inflammation, without affecting the serotoninergic system☆ Marcos H. Okuda⁎, Juliane C.S. Zemdegs, Aline A. de Santana, Aline B. Santamarina, Mayara F. Moreno, Ana C.L. Hachul, Bruno dos Santos, Claudia M. Oller do Nascimento, Eliane B. Ribeiro, Lila M. Oyama Departmento de Fisiologia, Disciplina de Fisiologia da Nutrição, Universidade Federal de São Paulo, São Paulo, SP, Brazil
Received 22 January 2014; received in revised form 23 May 2014; accepted 28 May 2014
Abstract To investigate possible mechanisms of green tea’s anti-obesity and anti-diabetic effects in the hypothalamus, the central regulator of metabolism, of mice fed with high-fat diet (HFD), we analyzed proteins of the toll-like receptor 4 (TLR4) pathway and serotoninergic proteins involved in energy homeostasis. Thirtyday-old male Swiss mice were fed with HFD rich in saturated fat and green tea extract (GTE) for 8 weeks. After that, body weight and mass of fat depots were evaluated. Oral glucose tolerance test was performed 3 days prior to euthanasia; serum glucose, insulin and adiponectin were measured in fasted mice. Hypothalamic TLR4 pathway proteins, serotonin receptors 1B and 2C and serotonin transporter were analyzed by Western blotting or enzyme-linked immunosorbent assay. A second set of animals was used to measure food intake in response to fluoxetine, a selective serotonin reuptake inhibitor. Mice fed with HFD had increased body weight and mass of fat depots, impaired oral glucose tolerance, elevated glucose and insulin and decreased adiponectin serum levels. TLR4, IκB-α, nuclear factor κB p50 and interleukin 6 were increased by HFD. Concomitant GTE treatment ameliorated these parameters. The serotoninergic system remained functional after HFD treatment despite a few alterations in protein content of serotonin receptors 1B and 2C and serotonin transporter. In summary, the GTE attenuated the deleterious effects of the HFD investigated in this study, partially due to reduced hypothalamic inflammation. © 2014 Elsevier Inc. All rights reserved. Keywords: Green tea extract; High-fat diet; Hypothalamus; Neuroinflammation; Serotonin
1. Introduction Obesity is an inflammatory disease, characterized by increased synthesis and secretion of pro-inflammatory cytokines, such as interleukin 6 (IL-6) and tumour necrosis factor α (TNF-α), and decreased anti-inflammatory signals, including adiponectin . One of the major causes of obesity is chronic positive energy balance, which varies according to extrinsic factors (e.g., food palatability and portion size) and intrinsic factors (e.g., satiety and hunger) . The hypothalamus is the central regulator of energy homeostasis, and it receives nutritional, hormonal and neural information about the metabolic and nutritional status from the body . It also
Grants, sponsors and funding sources: DSM Nutritional Products Ltd., Basel, Switzerland, granted green tea extract. CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, 2009/14373-8, 2011/16199-5 and 2012/03713-5) financially supported the project. ⁎ Corresponding author. Universidade Federal de São Paulo Disciplina de Fisiologia da Nutrição, Rua Botucatu 862, Edifício de Ciências Biomédicas 2nd Floor, Postal Code 04023-062, São Paulo/SP, Brazil. Tel./fax: +55-1155764765. E-mail address: [email protected]
(M.H. Okuda). http://dx.doi.org/10.1016/j.jnutbio.2014.05.012 0955-2863/© 2014 Elsevier Inc. All rights reserved.
regulates glucose homeostasis directly through the autonomic nervous system and indirectly by regulating insulin and glucagon secretion . Dysfunction in these mechanisms may lead to weight gain and ultimately to obesity . An important mechanism involved in hypothalamic dysfunction is neuroinflammation, which is observed in diet-induced obesity, and one of the key features is resistance to insulin  and leptin . The toll-like receptor 4 (TLR4) pathway mediates the inflammatory process induced by diet rich in saturated fats, leading to proinflammatory cytokines synthesis and secretion , and inhibition of downstream signals of the TLR4 pathway — such as inhibitor of κB kinase β/nuclear factor κB (IKKβ/NF-κB) — in the hypothalamus prevented dietinduced obesity, as well as insulin and leptin resistance . Prevention of obesity costs much less than its treatment and daily consumption of green tea (Camellia sinensis) is an interesting approach, as it induces weight loss or weight maintenance in adult subjects . We demonstrated that green tea extract (GTE) reduced mass of adipose tissue depots and attenuated the mesenteric adipose tissue inflammatory response to high-fat diet (HFD) . Moreover, other researchers demonstrated that polyphenols from green tea interacts with the TLR4 pathway [12,13]. One of the regulatory mechanisms of energy homeostasis in the hypothalamus involves the serotoninergic system. Its pharmacological stimulation induces anorexia in both humans and rodents [14,15],
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whereas its inhibition increases food intake . Moreover, serotonin signalling crosstalks with insulin and leptin signalling, two important metabolic factors that also regulate energy homeostasis . The physiological effects of serotonin (5-hydroxytryptamine, 5HT) are mediated by specific receptors, and the serotonin receptors 1B and 2C (5-HT1BR and 5-HT2CR, respectively) are involved in the control of food intake. Mice deficient in 5-HT1BR  or 5-HT2CR display hyperphagia , but only the latter develops obesity. Chronic consumption of HFD impairs the anorexigenic effects of serotonin by reduced post-prandial insulin and serotonin in extracellular regions of the hypothalamus  and altered turnover of serotonin . We investigated the anti-inflammatory effects of GTE in the hypothalamus of HFD-fed mice. In addition, we analyzed the effects on the hypothalamic serotoninergic system, which contributes to energy homeostasis and may be altered by HFD. 2. Methods and materials All procedures were approved by the ethics committee of Universidade Federal de São Paulo (protocol no. CEP 0883/11). 2.1. Experimental protocol Three-week-old male Swiss mice were purchased from Centro de Desenvolvimento de Modelos Experimentais para Medicina e Biologia and given 1 week of acclimation in our facility, under controlled 12-h light–dark cycle (lights on at 6:00), temperature (24±1°C) and free access to standard chow food pellets and tap water. After that, mice were distributed semi-randomly into the following groups so that the mean initial body weight was similar among groups (within group weight variability of 4.3g, maximum): control diet+vehicle (control), control diet+GTE (control-GTE), high-fat diet+vehicle (HFD) and high-fat diet+GTE (HFD-GTE). As a daily routine (from 8:00 to 10:00), all mice were weighed in order to calculate the amount of GTE for each mouse (50mg/kg of body mass/day), then GTE was freshly prepared in tap water, and the GTE (0.1ml, different concentrations) or vehicle (0.1ml) was administered po by a single gavage, using a feeding needle. Treatments started when mice were 4 weeks old and carried on for a total period of 8 weeks. The first treatment group (84 in total) was used for the oral glucose tolerance test (OGTT), performed 3 days before the end of the eighth week of treatment. We treated another group (42 in total) under the same conditions to evaluate food intake response to fluoxetine injection. Both procedures are described below. 2.2. Diets The control diet (4% soybean oil) and HFD (4% soybean oil; 31.2% lard) were manufactured in-house (see Table 1 for diet composition). The GTE (TEAVIGO) was a kind gift from DSM Nutritional Products Ltd., Basel, Switzerland, and contained N90% of epigallocatechin-3-gallate (EGCG), 0.0% caffeine, ≤10ppm of heavy metals, ≤3ppm of arsenic and ≤1ppm of lead, as informed by the provider (product code 5009227, lot no. UQ00522013, analysis no. 01815600). 2.3. OGTT The OGTT was performed 3 days before euthanasia, from 8:00 to 11:00. After an overnight fast, basal glycemia was measured from the tail vein using a glucometer (Accu-Check, Roche Diagnóstica Brasil Ltda., São Paulo, SP, Brazil). Immediately after the first measure, glucose (1.5g/kg)  was administered po, using with a feeding
Table 1 Composition of the control diet and HFD (modified AIN-93), growth (G) or maintenance (M)
Corn starch, % Casein, % Soya oil, % Lard, % Cellulose, % Vitamin mix, % Mineral mix, % L-Cystine, % Choline bitartrate, % Butylhydroquinone, g/kg Energy, kJ/g
Control diet (G/M)
62.95/72.07 20.0/14.0 7.0/4.0 – 5.0 1.0 3.5 0.3/0.18 0.25 0.014/0.008 16.5/15.9
40.92/40.87 13.95/14.0 7.0/4.0 28.08/31.2 5.0 1.0 3.5 0.3/0.18 0.25 0.014/0.008 22.4/22.4
needle, and glycemia was measured again after 15, 30, 60, 90 and 120min to obtain the area under the glycemic curve.
2.4. Food intake in response to fluoxetine To investigate the effects of HFD and GTE treatment on the serotoninergic system, we used fluoxetine, a selective serotonin reuptake inhibitor that induces hypophagia . At the last week of treatment, mice were individualized and fasted for 6 h (12:00 to 18:00). Four days before euthanasia, at 17:30, intraperitoneal (ip) injection of saline (0.9%) or fluoxetine (10mg/kg of body weight) was performed. Thirty minutes later, food pellets were presented, and after 2 h, food intake was measured. The selected dose was based on studies by others [23,24], and we performed a pilot study with different doses of fluoxetine (1, 5 or 10mg/kg of body weight), and the hypophagic effect was only observed with the 10mg/kg dose. All mice served as their own control and were kept individualized until euthanasia.
2.5. Tissue sampling To eliminate any stress caused by the OGTT or the fluoxetine test, euthanasia was carried out after 3 days of these procedures. During this interval, treatments were not interrupted. At the end of the eighth week of treatment, mice (10–12h of overnight fast) were weighed and euthanatized by decapitation. The hypothalami were dissected, frozen in liquid nitrogen and stored at −80°C for further analysis. Epididymal (E-WAT), retroperitoneal (R-WAT) and mesenteric (M-WAT) white adipose tissues (WAT) were dissected and weighed. Relative mass of WATs was calculated as % of total body weight and the sum of the three WAT pads (∑-WAT) was also calculated.
2.6. Serum analysis Trunk blood was collected, centrifuged to obtain serum and stored at −80°C for further analysis. The serum glucose, triacylglycerol, total cholesterol and high-density lipoprotein cholesterol concentrations were measured using a commercial enzymatic colorimetric kit (Labtest, Lagoa Santa, MG, Brazil). The insulin and adiponectin serum concentrations were quantified using specific enzyme-linked immunosorbent assay (ELISA) kits (Merck Millipore, Billerica, MA, USA).
2.7. Protein extraction and Western blotting The hypothalami were homogenized in protein extraction buffer (Tris–HCl, 0.1M, pH7.5; aprotinin, 0.1mg/ml; sodium pyrophosphate, 0.1M; sodium fluoride, 0.1M; EDTA, 0.01M; sodium orthovanadate, 0.01M; and PMSF, 0.002M), mixed with Triton X100 (10%) and kept on ice for 30min and centrifuged (12,000g/40min/4°C). The supernatant was collected and protein content was analyzed with a Bradford assay kit (Bio-Rad, Hercules, CA, USA). Proteins were denatured at 100°C/5min in Laemmli buffer (bromophenol blue, 0.1%; sodium phosphate, 1M; glycerol, 50%; SDS, 10%; and DTT, 0.2M). Samples were loaded (25μg of total protein) and separated in SDS-PAGE gels (10%) in a Bio-Rad miniature slab gel apparatus. The electrotransfer of proteins from gels to nitrocellulose membranes was performed for ~1.5h/4 gels at 15V (constant) in a Bio-Rad semi-dry transfer apparatus, in transfer buffer containing methanol (20%) and SDS (0.02%). The membranes were rinsed thoroughly with wash buffer (Tris–HCl, 0.01M; NaCl, 0.15M; and Tween 20, 0.02%), blocked with bovine serum albumin (BSA) buffer (wash buffer with BSA, 1%) for 2 h and incubated with primary antibodies (1:1000) overnight at 4°C in blocking buffer. The membranes were washed 3 times for 10min and incubated with horseradish peroxidase-conjugated secondary antibodies (1:5000 to 1:10,000) for 1 h at room temperature. The membranes were rinsed 3 times for 10min and chemiluminescence (Thermo Fisher Scientific, Waltham, MA, USA) were visualized in a gel documentation system (Alliance 4.7, UVitec, Cambridge, UK). Band intensities were calculated with Scion Image (Scion Corporation 184.108.40.206) and standardized with β-tubulin. The following primary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA): TLR4 (sc-10741), MyD88 (sc-11356), TRAF6 (sc-7221), p-IKKα/β (sc-21661), total IKKβ (sc-34673), total IκB-α (sc-847), p-NF-κB p50 (sc-101744), total NF-κB p50 (sc-53744), p-NF-κB p65 (sc-101748), total NF-κB p65 (sc-71675), 5HT1BR (sc-1460-P), 5-HT2CR (sc-10802) and serotonin transporter (sc-13997). Anti-β-tubulin (#2146) was purchased from Cell Signalling Technology (Danvers, MA, USA). Secondary antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA).
2.8. ELISA Hypothalamic homogenate was obtained exactly as described above, using the same protein extraction buffer. Quantitative assessment of TNF-α and IL-6 proteins was carried out using ELISA kit (DuoSet ELISA, R&D Systems, Minneapolis, MN, USA) following the recommendations of the manufacturer. The results obtained were then standardized for total protein content.
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2.9. Statistical analysis Distribution assumptions were verified by the Shapiro–Wilk test; homogeneity, by the Levene’s test; and sphericity, by the Mauchly’s test. Data were standardized to z-score values when needed, and values exceeding three standard scores were considered as outliers. Two-way analysis of variance was performed, followed by Bonferroni’s post hoc test for group comparisons when needed. For the fluoxetine injection test, paired t test was performed. Alpha value of 0.05 was considered. Biological relevance (described in Ref. ) was considered using Cohen’s criteria for effects size d and η2: 0.2≤db0.5 and 0.01≤η2b0.06, small effect; 0.5≤db0.8 and 0.06≤η2b0.14, medium effect; 0.8≤d and 0.14≤η2, large effect . The effect size was used to help data analysis, and the effect size is indicated when appropriate. All data are expressed as mean±standard error.
3. Results 3.1. Body and adipose tissues weight At the end of the treatment, the HFD group had increased body mass (Pb.001, η2=0.232, large effect) and relative masses of E-WAT (P=.008, η2=0.097, medium effect), R-WAT (P=.02, η2=0.077, medium effect), M-WAT (Pb.001, η2=0.18, large effect) and ∑WAT (P=.002, η2=0.13, medium effect), in comparison with the control group. GTE treatment prevented the deleterious effect of the HFD, indicated by reduced body mass (P=.001, η2=0.11, medium effect), relative masses of M-WAT (P=.026, η2=0.062, medium effect) and ∑-WAT (P=.048, η2=0.05, small effect) and a small effect in E-WAT (P=.064, η2=0.046) (Table 2). 3.2. OGTT The intake of the HFD augmented the area under the glycemic curve, indicating impairment of glucose tolerance, regardless of the GTE treatment (HFD vs. control, P=.016, η2=0.126, medium effect; HFD-GTE vs. control-GTE, P=.004, η2=0.187, large effect). The GTE intervention had a small biological effect (control-GTE vs. control, P=.178, η2=0.037; HFD-GTE vs. HFD, P=.3, η2=0.022) (Fig. 1). 3.3. Serum analysis The HFD altered serum glucose, insulin and adiponectin. In the HFD groups, glucose was higher (HFD vs. control, Pb.001, η2=0.365, large effect; HFD-GTE vs. control-GTE, P=.022, η2=0.132, medium effect), while the increase in insulin was discrete, with a small effect (HFD vs. control, P=.21, η2=0.042). GTE treatment reduced glucose (P=.022, η2=0.13, medium effect) and insulin (P=.009, η2=0.2, large effect) in comparison with the HFD group. On the other hand, adiponectin was reduced in HFD groups (HFD vs. control, Pb.001, η 2=0.46, large effect; HFD-GTE vs. control-GTE, P=.002, η2=0.13, medium effect); GTE treatment decreased adiponectin (control-GTE vs. control, P=.001, η 2=0.14, large effect) (Table 3). 3.4. Western blotting and ELISA of the TLR4 pathway The HFD increased protein expression of IκB-α (P=.041, η2= 0.107, medium effect), when compared to control diet. Furthermore, biologically relevant increases were observed for TLR4 (P=.097, η2= 0.077, medium effect) and NF-κB p50 (P=.196, η2=0.079, medium effect) in the HFD group. The GTE treatment increased expression of IκB-α (P=.003, η2=0.241, large effect) when compared to control. Furthermore, the HFD-GTE group had decreased expression of IKKβ (Pb.001, η2=0.464, large effect) and biologically relevant decreases in NF-κB p50 (P=.113, η2=0.121, medium effect) and NF-κB p65 (P=.311, η2=0.044, small effect), compared to the HFD group (Fig. 2A–E). To evaluate the inflammatory status further, we also analyzed IL-6 concentrations in the hypothalamus. The HFD promoted biologically relevant increase of IL-6 (HFD vs. control, P=.051, η2=0.17, large
effect), and the GTE attenuated this effect (HFD-GTE vs. HFD, P=.386, η2=0.033, small effect) (Fig. 2F). 3.5. Food intake in response to fluoxetine and expression of serotoninergic proteins We used fluoxetine to evaluate the HFD and GTE treatment effect on the serotoninergic system. Paired t test was used, and we observed that the hypophagic effect of fluoxetine in all groups was large (control, Pb.001, d=2.3; control-GTE, P=.009, d=1.7; HFD, Pb.001, d=3.4; and HFD-GTE, Pb.001, d=2.5) (Fig. 3A). In regard of proteins from the serotoninergic system, the HFD, when compared to control, did not alter expression of 5-HT1BR (P= .873, η2=0.001), while it increased 5-HT2CR (P=.033, η2=0.141, large effect), and a biologically relevant increase in serotonin transporter (5-HTT) was observed (P=.138, η2=0.247, large effect). The GTE treatment increased expression of 5-HT2CR (control-GTE vs. control, P=.007, η2=0.236, large effect). When combined with HFD, it decreased 5-HT2CR (HFD-GTE vs. HFD, P=.037, η2=0.132, medium effect) and relevantly reduced 5-HTT (P=.315, η2=0.104, medium effect) and increased 5-HT1BR (HFD-GTE vs. HFD, P=.2, η2=0.063, medium effect) (Fig. 3B–D).
4. Discussion Metabolic syndrome is characterized by abnormalities that predispose to cardiovascular diseases and type 2 diabetes mellitus, and it is associated with obesity and diet induced inflammation . We recently demonstrated that, in rats, GTE attenuated the low-grade inflammation in M-WAT induced by a diet rich in saturated fats . In the present study, we sought new mechanisms by which GTE exerts its beneficial effects during HFD feeding, focusing on the central regulator of metabolism, the hypothalamus. The HFD increased body and fat pads weight and concomitant treatment with GTE attenuated these responses to HFD, which is in line with works using different types of GTE [28,29]. A possible explanation was demonstrated by the work of Friedrich et al. , in which mice fed with HFD rich in corn oil and supplemented with GTE had reduced intestinal fat absorption and increased fat oxidation, resulting in decreased fat depots. Studies showed that HFD induced glucose intolerance and insulin resistance in WAT  and hypothalamus . In the present work, the HFD increased the area under the glycemic curve, glycemia and a modest increase in serum insulin, which indicates impaired glucose control. Conversely, treatment with GTE ameliorated these parameters, and this is in line with other studies that showed lower fasting and post-prandial serum glucose and insulin concentrations [28,29,33]. A hyperinsulinemic–euglycemic clamp and expression of phosphorylated proteins of its signalling pathway would complement the analysis. Table 2 Body mass and relative mass of adipose tissues (expressed as percentage of total body mass)
Initial body mass, g (n=15–17) Final body mass, g (n=15–17) Epididymal WAT (n=15–17) Retroperitoneal WAT (n=14–17) Mesenteric WAT (n=14–17) Sum of WAT (n=14–17)
26.0±0.7 30.5±0.9 2.51±0.3 0.79±0.1 0.88±0.1 4.69±0.4
25.7±0.5 29.4±0.9 2.31±0.2 0.65±0.1 0.79±0.1 3.76±0.4
27.0±0.9 39.9±1.6 ⁎ 4.03±0.4 ⁎ 1.21±0.1 ⁎ 1.78±0.2 ⁎ 7.02±0.7 ⁎
24.0±0.7 33.5±1.2 † 3.14±0.2 1.00±0.1 1.28±0.1 † 5.57±0.4 †
Results are expressed as mean±S.E.M. ⁎ Pb.05 for HFD vs. control. †
Pb.05 for HFD-GTE vs. HFD.
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Fig. 1. OGTT and area under the glycemic curve after 60days of HFD and GTE treatment. Results are expressed as mean±S.E.M. *Pb.05 for HFD vs. control; #Pb.05 for HFD-GTE vs. control-GTE. n=8–10/group.
Adiponectin, an adipocyte-derived hormone that increases insulin sensitivity  and has anti-inflammatory effects, is inversely correlated with overweight/obesity, and weight loss increases its serum concentrations . In addition, HFD is associated with reduced protein and mRNA expression of adiponectin in adipose tissue . In our study, the HFD group had lower concentrations of this hormone. However, GTE treatment did not prevent this decrease, corroborating with a study by Shirai and Suzuki  in which they treated mice with HFD and GTE in food pellets (1%) for 12 months. A recent study by Tian et al.  showed that green tea polyphenols prevented weight gain induced by HFD and maintained circulating adiponectin concentrations by modulating peroxisome proliferatoractivated receptor γ, the master regulator of adiponectin gene transcription. However, in the cited study, mice treated with both HFD and green tea polyphenols had the same body weight and relative mass of visceral adipose tissues as the control group; therefore, the effects of green tea polyphenols on adiponectin mRNA and protein expression could be attributed to weight maintenance, rather to tea polyphenols . We further looked into HFD-induced inflammation, in which NF-κB plays a pivotal role. Activation of IKKβ in the hypothalamus induces leptin and insulin resistance through expression of suppressor of cytokine signalling 3, a common inhibitor of these hormones . In the present study, the HFD slightly increased protein expression of TLR4, IκB-α and NF-κB p50. In support, recent publications showed that HFD increased protein expression of phosphorylated IκB-α and JNK, as well as TNF-α in mice , and mRNA expression of TLR4, NF-κB, TNF-α, IL-6 and IL-1β in rats .
Table 3 Serum glucose, insulin and adiponectin
Glucose, mg/dl (n=5–8) Insulin, ng/ml (n=8–9) Adiponectin, μg/ml (n=8)
111.2±1.6 1.48±0.3 3.39±0.2
110.9±2.4 1.20±0.3 2.50±0.2 ⁎
128.1±2.0 ⁎ 1.9±0.2 1.79±0.2 ⁎
118.3±4.4 # † 0.95±0.2 † 1.63±0.1 #
Results are expressed as mean±S.E.M. ⁎ Pb.05 for control-GTE or HFD vs. control. # Pb.05 for HFD-GTE vs. control-GTE. † Pb.05 for HFD-GTE vs. HFD.
Although we did not measure phosphorylated proteins of the TLR4 pathway, we analyzed hypothalamic IL-6 content, a target gene of the NF-κB, and indeed, the HFD increased IL-6 concentration. Studies both in vivo [41–43] and in vitro [44,45] have linked EGCG as a suppressor of the TLR/NF-κB pathway. One of the mechanisms involves binding of EGCG to 67-kDa laminin receptor, which induces expression of Tollip (toll interacting protein), a negative regulator of TLR4 signalling. In turn, reduced synthesis of pro-inflammatory cytokines TNF-α, IL-1β and IL-6, as well as nuclear translocation of NF-κB p65, is observed [12,44]. Our data show that GTE reduced hypothalamic inflammatory signalling by reducing the expression of IKKβ, NF-κB monomers p50 and p65 and IL-6. In addition, glucose tolerance was ameliorated. Although other works indicate anti-inflammatory activity of herbal extracts [7,46], to our knowledge, we are the first to report anti-inflammatory effects of a GTE rich in EGCG in the hypothalamus of mice fed with HFD. Of note, the GTE increased expression of NF-κB p50 in the control group, but so did its inhibitory protein, IκB-α, and IL-6 was slightly reduced, suggesting that the increase in NF-κB p50 was not relevant to the inflammatory status. We also investigated the role of hypothalamic serotonin signalling, which can crosstalk with insulin signalling through phosphoinositide 3-kinase . The 5-HT2CR is involved in glucose homeostasis, in part by increasing insulin sensitivity, as demonstrated by pharmacological  and genetic  studies. Moreover, 5-HT2CR  and 5-HT1BR  activity reduces food intake, and alterations in their activity may lead to weight gain. While the pro-inflammatory effects of saturated fats are well known , the outcome in the serotoninergic system is far less explored. After a fluoxetine injection, food intake was reduced in all groups, indicating that the serotoninergic system remained functional. The larger effect observed in the HFD groups can be explained by the difference in energy density of the diets. The 5-HT2CR stimulates POMC neurons, whereas 5-HT1BR is involved in inhibition of NPY/AgRP neurons , while 5-HTT is responsible for the reuptake of serotonin from the synaptic cleft, and mice lacking 5-HTT develop insulin and leptin resistance and glucose intolerance . In the HFD group, increased 5-HTT may result in lower serotonin availability at synapses, and the increase in 5-HT2CR could be an attempt to compensate it, as suggested by Huang et al. . In contrast, the HFD-GTE group had increased 5-HT1BR, but not 5-HTT and 5-HT2CR. Considering that 5-HT2CR are expressed by anorexigenic POMC neurons, and 5-HT1BR are expressed by orexigenic NPY/AgRP neurons , the alterations observed may have different mechanisms or thresholds. Despite these differences, the hypophagia induced by fluoxetine changes was not affected by the HFD, indicating that the serotoninergic system was well adapted to the treatments and remained functional. The higher energy intake observed after saline injection may partly explain the increased body weight of the HFD group. It is possible that other cues to the food intake control are down- or up-regulated, such as NPY, AgRP, ghrelin (hyperphagia-related peptides), cholecystokinin, leptin and insulin (hypophagia-related peptides). In addition to feeding control, the hypothalamus regulates energy expenditure and glucose homeostasis, and these functions may be impaired due to the HFDinduced inflammation . Finally, GTE treatment attenuated the inflammatory response but did not alter food intake, which is in line with other studies [54,55]. This indicates that the beneficial effects of GTE involve other mechanisms, including increased thermogenesis , increased fat oxidation and reduced nutrient absorption , and the anti-inflammatory effects discussed above. The present experiments demonstrated that the GTE had beneficial effects in the animals fed with the HFD, as shown by attenuation of hypothalamic inflammatory status and improvement of body energy and glucose homeostasis. No prominent effects of the
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Fig. 2. Analysis of TLR4 pathway proteins in the hypothalamus. (A–E) Expression of proteins of the TLR4 pathway (n per group indicated in the graphics). Anti-β-tubulin used as housekeeping, and results expressed as arbitrary units (fold of control). (F) Hypothalamic IL-6, as picograms per milligram of total protein content (n=4–5/group). (A–F) Results are expressed as mean±S.E.M. *Pb.05 for control-GTE or HFD vs. control, †Pb.05 for HFD-GTE vs. HFD and #Pb.05 for HFD-GTE vs. control-GTE.
HFD diet were observed on the serotonergic parameters evaluated and the GTE had an ambiguous action, as it increased 5-HT1BR while it decreased 5-HT2CR hypothalamic content in mice fed an HFD.
Acknowledgments We thank Valter Tadeu Boldarine and Mauro Cardoso Pereira for technical assistance.
Author Contributions References MHO and JCSZ were responsible for the design of the study; MHO conducted the study; AAS, ABS, MFM, ACLH, BS, MHO, CMON, EBR and LMO helped with experimental procedures, data analysis and discussion.
Conflict of Interest The authors declare no conflict of interest.
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Fig. 3. Analysis of the serotoninergic system. (A) Total energy intake in 2 h after fluoxetine (10mg/kg) or saline (0.9%) ip injection (n=10–11/group). (B–D) Protein expression of hypothalamic 5-HT1B, 5-HT2C and 5-HTT (n per group indicated in the graphics). Anti-β-tubulin used as housekeeping, and results expressed as arbitrary units (fold of control). (A–D) Results are expressed as mean±S.E.M. $Pb.05 for fluoxetine vs. saline, *Pb.05 for control-GTE or HFD vs. control, †Pb.05 for HFD-GTE vs. HFD and #Pb.05 for HFD-GTE vs. control-GTE.
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