Abstruse

Green tea (GT) and its components have been shown to possess antiobesity backdrop and the corresponding mechanisms of action are beingness investigated, given the epidemic proportions of obesity incidence. In the current work, nosotros used 12-mo-old male Wistar rats to exam the effect of half dozen mo of treatment with GT as the sole drinking beverage (52.eight ± 6.4 mL/d) on adipose tissue (AT). AT aromatase expression was adamant past Western blotting, plasma concentrations of 17β-estradiol and testosterone were adamant past RIA, and adipocyte size determined by measuring diameter in tissue sections. Proliferation and apoptosis were also assessed past Ki67 immunostaining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, respectively. Evaluations were made in subcutaneous (sc) AT and visceral (v) AT. Body weight increased over time in both groups (P < 0.001), but the increase was more pronounced in controls (P < 0.001) and nutrient and fluid intake did not influence that effect. At the cease of the experiment, aromatase expression increased in the AT (318.five ± 60.6% of control in scAT, P < 0.05, and 285.5 ± 82.nine% of command in vAT, P < 0.01). AT of GT-treated rats had a higher percentage of proliferating cells (204.1 ± xix.five% of control in scAT, P < 0.01, and 246.vi ± l.2% of control in vAT, P < 0.01) and smaller adipocytes (78.3 ± one.7% of command in scAT, P < 0.001, and 87.9 ± 3.2% of command in vAT, P < 0.05). GT too increased the number of apoptotic cells in vAT (320.4 ± 21.9% of command; P < 0.001). These results advise new mechanisms for GT on body weight and highlight its potential benefit to forestall or treat obesity and the metabolic syndrome.

Introduction

Obesity has become a major wellness problem equally a growing proportion of the population worldwide is overweight and very likely to get obese (one). One of the major concerns related to the increasing obesity incidence is its association with other pathological signs, including hypertension, dyslipidemia, insulin resistance, and glucose intolerance. The combination of these features constitutes the so-called metabolic syndrome that dramatically increases the gamble to develop cardiovascular disease or diabetes (2). Furthermore, visceral (v)7 fat accumulation is the chief characteristic of this syndrome, whereas subcutaneous (sc) fat is a minor contributor to obesity complications (3). Obesity is generally associated with systemic inflammation, with increased production of cytokines from inflammatory cells, such every bit macrophages, and adipocytes (iv,v). It has been shown that cytokine and hormone release profiles of adipose tissue (AT) from diverse locations are distinct, although the reasons for these differences are not fully antiseptic (6).

Estrogens can be produced in the AT from aromatization of circulating androgens by the action of aromatase (7) and its expression is higher in scAT in comparison to vAT (8). Show from estrogen deficiency models shows that estrogen signaling contributes to weight regulation and its absence is related to v obesity, insulin resistance, and glucose intolerance along with other features of the metabolic syndrome (ix,x). In men, aromatase mutations that give rise to lack of enzyme function are related with undetectable estrogen concentration and normal or high testosterone and gonadotropins associated with excess weight or obesity. Torso fat aggregating in the v region and alterations of lipid metabolism are present as well (11). In women, aromatase deficiency too results in developmental and metabolic alterations. Surprisingly, these disturbances are ameliorated past estrogen treatment (12). Furthermore, alterations in AT distribution and inflammatory status are common afterwards estrogen product ceases in menopause and may as well be reversed by hormone replacement treatment (thirteen). vAT has a relative estrogen insufficiency due to its lower expression of aromatase compared with scAT (7). Although estrogens influence body fat accumulation past altering appetite and energy expenditure, straight AT effects are possible, including lipoprotein lipase inhibition and hormone-sensitive lipase stimulation, besides as preadipocyte proliferation and differentiation alterations (14,xv). This hormone is thus likely to induce its antiobesity effects locally, every bit its tissue concentration may be ∼10 times higher than circulating concentrations (sixteen) and because AT accounts for most of the estrogen production in males and postmenopausal females (10).

The investigation of the antiobesity backdrop of food components is a popular field of enquiry, because information technology may pb to the discovery of new naturally occurring agents to foreclose or care for obesity. There are studies on green tea (GT) and GT components, especially catechins, demonstrating their effects on free energy expenditure, fatty oxidation, blood lipids, and preadipocyte differentiation and adipocyte apoptosis (17,18). Even so, to date, petty is known about its ability to interfere with estrogen synthesis in vivo, particularly in the AT. Aromatase activity inhibition by some GT components has been demonstrated on placental microsomes (19) and choriocarcinoma-derived JAR cells (20), only in vivo long-term studies are lacking. We have likewise recently found that red wine, a drinkable containing compounds able to inhibit aromatase activeness in cellular lines, could induce an increase in aromatase expression in animal models afterwards prolonged handling (21). Taking this data into business relationship, together with the known effects of locally produced estrogens, information technology is possible that GT components may modulate aromatase in the AT and that, as a consequence, alterations in tissue dynamics may occur. For this reason, we intended to make up one's mind the result of chronic consumption of GT on the expression of aromatase in the AT from different anatomical locations. Nosotros also sought to determine the effect of GT intake on adipocyte size, AT apoptosis, and proliferation. To approach this subject, we treated adult male Wistar rats with GT, performed evaluations in scAT and vAT, and measured circulating 17β-estradiol and testosterone concentrations.

Materials and Methods

Rats and treatments.

Ten 12-mo-onetime male Wistar rats weighing 918.3 ± 22.8 g were individually housed and maintained under standard temperature and light conditions (21–22°C; 12-h-light/-dark cycles). Rats were randomly assigned to two groups: control rats with admission to tap water and GT-treated rats with access to GT as the only liquid source. During the 6-mo handling, all rats had admission to standard laboratory animate being nutrient (Panlab S.L; 15,733 kJ/kg, 156 m/kg poly peptide, 28 g/kg fat, 48 1000/kg cellulose, 49 yard/kg mineral salts, 22.five mg/kg retinol equivalents, 0.375 mg/kg cholecalciferol equivalents, and 150 mg/kg α-tocopherol equivalents). Bottles containing the beverages were protected from low-cal to avoid oxidation of lite-sensitive components and beverages were renewed every 2–3 d with water (controls) or freshly prepared GT. We monitored advert libitum consumption of both food and fluid every other twenty-four hours and recorded rat weight every calendar week. To prepare GT infusion, 1 Fifty of boiling water (100°C) was dispensed over 3 tea bags (1.3 g/bag) for 5 min. The infusion was allowed to cool to room temperature before being transferred to the bottles and supplied to the rats. The tea used during the treatments was from the same batch and the limerick of the last beverage was determined afterward 3 randomly chosen infusion preparations. Tea composition in catechins was adamant using HPLC (22) and was equally follows: (–)-epigallocatechin-3-gallate (EGCG), 1.21 ± 0.256 mmol/50; (–)-epicatechin (EC), 0.84 ± 0.071 mmol/Fifty; EC-iii-gallate, 0.29 ± 0.067 mmol/L; (+)-gallocatechin-3-gallate, 0.07 ± 0.001 mmol/50. Hateful caffeine content was 0.52 ± 0.022 mmol/L, measured using GC-ion trap MS, every bit described (23). Animal procedures were according to the European Community guidelines (86/609/EEC) and the Portuguese Act (129/92) for the utilize of experimental animals.

Tissue collection and preparation.

At the finish of treatment, fed rats were anesthetized (intraperitoneal sodium pentobarbital; 80 mg/kg body weight) and perfused with saline at four°C. All rats were killed between 1000 and 1200. Samples from sc (inguinal) and v (mesenteric) AT were removed and frozen in liquid nitrogen for protein extraction. A fraction of each sample was immersed in 10% formalin solution for paraffin embedding and 4-μm-thick tissue sections were fabricated for histological assay, apoptosis determination, and immunohistochemistry.

Plasma 17β-estradiol and testosterone measurement.

Before perfusion, blood was drawn from the left ventricle into heparinized tubes and plasma fractions were frozen at −80°C until analysis. 17β-Estradiol and testosterone were measured with RIA using antiserum confronting 17β-estradiol and 125I-17β-estradiol (Coat-a-Count TKE2, Diagnostic Products) or antiserum against testosterone and 125I-testosterone (Testo-RIA-CT, Biosource), respectively. The commercial kits used to mensurate 17β-estradiol and testosterone were specific for these hormones and had very low reactivity with estrone or androstenedione. The method for measuring 17β-estradiol had a sensitivity of 29 pmol/Fifty; the detection limit was 174 nmol/L for the test measuring testosterone.

Measurement of adipocyte size.

Hematoxylin-eosin–stained tissue sections were observed and photographed under specimen identity occultation. We estimated adipocyte hateful bore using NIS Elements BR software (Nikon). Fifty to 100 adipocytes from v randomly selected unlike fields were measured for each sample.

Apoptosis assay.

Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling was used according to the manufacturer (Roche) in paraffin-embedded tissue sections. Cell nuclei were counterstained with 4′,6-diamidine-ii′-phenylindole dihydrochloride (DAPI; Roche). Slides were visualized under a fluorescence microscope (Nikon 50i) at 200× magnification. Apoptosis was adamant as the percentage of positive over total counted cells. Nosotros counted 200 nuclei from different section fields for each sample.

Determination of proliferation.

Proliferation was measured through immunohistochemical labeling of proliferation-characteristic nuclear poly peptide Ki67. This cellular marker is expressed in all agile phases of the cell cycle (G1, S, G2, and mitosis) and is absent in quiescent (G0) cells (24). Anti-Ki67 primary and fluorescein isothiocyanate-conjugated secondary antibodies (1:50 and 1:200 in four% bovine serum albumin, respectively; Santa Cruz Biotechnology) were used. Total nuclei were counterstained with DAPI and visualized at 200× magnification. Nuclei stained with anti-Ki67 antibody were counted in 5 randomly selected section fields. Total DAPI-stained nuclei were counted for the same optical fields. Proliferation is given as a percentage of proliferating cells over total cells. We counted 400–600 nuclei in scAT and 200–300 in vAT from different department fields for each sample.

Western blotting.

AT samples (0.viii–1 g) were incubated at 4°C for 2 h in i.5 mL of detergent protein extraction buffer (fifty mmol/Fifty Tris-HCl, pH 7.iv, 1% Triton X-100, 0.2% sodium deoxycholate, 0.2% sodium dodecylsulfate, 1 mmol/Fifty EDTA, ane mmol/L phenylmethylsulfonyl fluoride) containing 25 μL protease inhibitor cocktail (Sigma) as described (25). Each sample was incubated at 4°C with agitation for 2 h in the buffer and afterwards homogenized in a Teflon-glass homogenizer and centrifuged at 10,000 × g for 10 min. The infranatant protein solution was collected, quantified past bicinchoninic acrid protein analysis (Pierce), and stored at −80°C. Before SDS-Page separation, proteins were dissolved 1:1 in loading buffer (Bio-Rad Laboratories) containing ii% mercaptoethanol, denatured for 90 southward at 95°C, and twenty μk of protein was loaded per well. Anti-aromatase polyclonal antibody (1:200, Santa Cruz Biotechnology) and horseradish peroxidase-conjugated polyclonal antibody (1:k) were used followed past chemiluminescent detection. β-Actin principal antibody (ane:chiliad, Lab Vision) and secondary antibody hybridizations were conducted using the same procedure. Band intensity was determined using Gel Pro Analyzer (Media Cybernetics) and aromatase was normalized to β-actin expression (expressed in arbitrary units). Determinations were fabricated in membranes containing both control and GT-treated rat proteins in 2 separate experiments.

Statistical analyses.

Results are expressed as ways ± SEM. Mixed effects model with random intercept was used to appraise the progression of body weight with time (26 repeated measures of the body weight) adapted for the liquid and nutrient intake. This model allows the incorporation of both fixed (population-specific) and random (subject-specific) variables and assumes that the vector of repeated measures on each subject follows a linear regression model. Using the random intercept, it is tested if variability in subject-specific slopes can be ascribed to treatment. The differences between 2 groups were assessed past unpaired Student's t-test or Mann Whitney U-test when variance of measurements was significantly different. We used Fisher's exact test to make up one's mind the difference betwixt groups in 17β-estradiol measurements, comparing the number of detectable and undetectable measurements in controls and treated animals. Association betwixt outcome variables was determined with the data pooled from the 2 groups using Pearson or Spearman correlations for data with normal or nonnormal distributions, respectively, and AT measurements were compared within the aforementioned tissue. The normality of the distribution for each outcome variable was tested using Shapiro's examination. Statistical analyses were performed using Graph Pad Prism software (Graph Pad Software version iii.0) and SPSS (SPSS 12.0 for Windows). Differences were considered significant when P < 0.05.

Results

Trunk weight and food and fluid ingestion.

Torso weight did not differ between groups at the first of the experiment (Table ane) and increased gradually throughout treatment (Fig. 1). Food and fluid intakes as well did not differ betwixt the two groups (Table 1). However, body weights increased during each week of the written report (P < 0.001) and the increase in GT-treated rats (ii.83 ± 0.twenty g · wk−one) was less than in control rats (5.26 ± 0.25 g · wk−ane; P < 0.001). Food and fluid intakes did non bear upon the changes in torso weight (Table ii). After six mo of treatment, the weight gain of GT rats (64.8 ± 16.0 thou) was 47.5% that of controls (136.four ± 25.4 g; P < 0.05).

Effigy 1

Body weight throughout the 6 mo of treatment in control (C) and GT-treated rats. Results are given in mean and dotted lines represent 1 SEM, n = 5.

Body weight throughout the vi mo of treatment in control (C) and GT-treated rats. Results are given in mean and dotted lines stand for one SEM, due north = v.

FIGURE 1

Body weight throughout the 6 mo of treatment in control (C) and GT-treated rats. Results are given in mean and dotted lines represent 1 SEM, n = 5.

Body weight throughout the 6 mo of treatment in control (C) and GT-treated rats. Results are given in mean and dotted lines represent 1 SEM, due north = v.

TABLE 1

Initial and final trunk weights and nutrient and fluid intakes of GT and control rats 1

C GT
Body weight, g
    12 mo former 938 ± 32 897 ± 48
    18 mo old 1074 ± 46 963 ± 53
Food intake, grand/d 34 ± one 32 ± one
Fluid intake, mL/d 49 ± 5 53 ± 6
C GT
Trunk weight, g
    12 mo one-time 938 ± 32 897 ± 48
    18 mo sometime 1074 ± 46 963 ± 53
Food intake, one thousand/d 34 ± 1 32 ± 1
Fluid intake, mL/d 49 ± 5 53 ± half dozen

1

Values are means ± SEM, n = 5.

Tabular array 1

Initial and final trunk weights and food and fluid intakes of GT and control rats i

C GT
Body weight, k
    12 mo onetime 938 ± 32 897 ± 48
    xviii mo one-time 1074 ± 46 963 ± 53
Nutrient intake, thou/d 34 ± i 32 ± 1
Fluid intake, mL/d 49 ± 5 53 ± 6
C GT
Torso weight, g
    12 mo erstwhile 938 ± 32 897 ± 48
    18 mo old 1074 ± 46 963 ± 53
Food intake, one thousand/d 34 ± 1 32 ± 1
Fluid intake, mL/d 49 ± 5 53 ± six

1

Values are means ± SEM, n = five.

TABLE 2

Mixed model for the variation of body weight with time, adjusted for liquid and food intake (random-intercept), in GT and control rats

Parameter β SEM P-value
Intercept, g 893.3 48.3 <0.001
Time, wk two.8 0.two <0.001
GT, yard 0 one 0
Control, g 51.2 63.9 0.446
GT ten time, wk 0 1 0
C x time, wk 2.4 0.3 <0.001
Liquid intake, mL −0.1 0.2 0.707
Nutrient intake, mL 0.two 0.half-dozen 0.756
Parameter β SEM P-value
Intercept, k 893.3 48.iii <0.001
Time, wk 2.8 0.2 <0.001
GT, g 0 one 0
Control, g 51.2 63.9 0.446
GT x time, wk 0 ane 0
C x time, wk 2.4 0.3 <0.001
Liquid intake, mL −0.1 0.2 0.707
Food intake, mL 0.2 0.6 0.756

Tabular array 2

Mixed model for the variation of trunk weight with time, adapted for liquid and food intake (random-intercept), in GT and control rats

Parameter β SEM P-value
Intercept, g 893.three 48.iii <0.001
Time, wk 2.8 0.2 <0.001
GT, m 0 1 0
Control, g 51.2 63.9 0.446
GT x time, wk 0 1 0
C x fourth dimension, wk two.iv 0.three <0.001
Liquid intake, mL −0.one 0.2 0.707
Food intake, mL 0.two 0.half-dozen 0.756
Parameter β SEM P-value
Intercept, m 893.iii 48.3 <0.001
Fourth dimension, wk ii.8 0.2 <0.001
GT, 1000 0 1 0
Control, grand 51.2 63.9 0.446
GT x time, wk 0 1 0
C x time, wk 2.4 0.three <0.001
Liquid intake, mL −0.1 0.2 0.707
Food intake, mL 0.2 0.half dozen 0.756

Plasma 17β-estradiol and testosterone concentrations.

The plasma concentration of 17β-estradiol was undetectable in control rats, whereas that of GT-treated rats was 39.2 ± 10.4 pmol/L (P < 0.01). The plasma testosterone concentration was lower in GT-treated rats (790 ± 177 pmol/L) than in controls (2720 ± 769 pmol/Fifty; P < 0.01).

Aromatase expression.

Relative aromatase expression in homogenates from scAT was ∼213% higher than that of vAT (P < 0.05) in command rats (Fig. 2). Interestingly, GT treatment induced a marked increase in aromatase expression in both AT locations to 318.5 ± 60.six% of control in scAT (P < 0.05) and 285.5 ± 82.9% of control in vAT (P < 0.01).

FIGURE 2

Representative Western blots (A) and relative aromatase expression (B) in scAT and vAT from controls (C) or rats treated with GT for 6 mo. Aromatase expression is presented equally the aromatase:β-actin ratio. Results are means ± SEM, north = v. *Unlike from control, P < 0.05.

Representative Western blots (A) and relative aromatase expression (B) in scAT and vAT from controls (C) or rats treated with GT for vi mo. Aromatase expression is presented as the aromatase:β-actin ratio. Results are ways ± SEM, northward = 5. *Unlike from control, P < 0.05.

FIGURE ii

Representative Western blots (A) and relative aromatase expression (B) in scAT and vAT from controls (C) or rats treated with GT for 6 mo. Aromatase expression is presented as the aromatase:β-actin ratio. Results are ways ± SEM, n = v. *Unlike from control, P < 0.05.

Representative Western blots (A) and relative aromatase expression (B) in scAT and vAT from controls (C) or rats treated with GT for 6 mo. Aromatase expression is presented as the aromatase:β-actin ratio. Results are ways ± SEM, n = 5. *Dissimilar from command, P < 0.05.

Adipocyte size.

In command rats, the diameters of scAT (123.7 ± ii.3 μyard) and vAT adipocytes (113.3 ± 5.0 μm) did not differ. Chronic GT consumption resulted in a reduction in adipocyte mean diameter compared with controls, both in scAT (96.9 ± two.i μm; P < 0.05) and in vAT (99.half dozen ± 3.seven μchiliad; P < 0.01) (Fig. 3).

Effigy iii

Hematoxylin-eosin–stained AT sections (A) and adipocyte diameter (B) in scAT and vAT of controls (C) or rats treated with GT for 6 mo. In AT sections, smaller adipocytes are evident in GT-treated rats in both AT locations. Scale bar = 100 μm. Results are means ± SEM, n = 5. *Unlike from control, P < 0.05.

Hematoxylin-eosin–stained AT sections (A) and adipocyte bore (B) in scAT and vAT of controls (C) or rats treated with GT for half dozen mo. In AT sections, smaller adipocytes are evident in GT-treated rats in both AT locations. Calibration bar = 100 μk. Results are ways ± SEM, n = v. *Unlike from command, P < 0.05.

FIGURE 3

Hematoxylin-eosin–stained AT sections (A) and adipocyte diameter (B) in scAT and vAT of controls (C) or rats treated with GT for 6 mo. In AT sections, smaller adipocytes are evident in GT-treated rats in both AT locations. Scale bar = 100 μm. Results are means ± SEM, n = 5. *Dissimilar from command, P < 0.05.

Hematoxylin-eosin–stained AT sections (A) and adipocyte diameter (B) in scAT and vAT of controls (C) or rats treated with GT for 6 mo. In AT sections, smaller adipocytes are axiomatic in GT-treated rats in both AT locations. Scale bar = 100 μthousand. Results are means ± SEM, n = five. *Dissimilar from control, P < 0.05.

AT apoptosis and proliferation.

Apoptotic cell number in vAT was greater in GT-treated rats (24.1 ± 1.6%) than in controls (7.5 ± 1.4%) (P < 0.01) and tended to exist greater in scAT (P = 0.08). The number of apoptotic cells in the AT of control rats did not differ between depots. Moreover, GT treatment increased the number of proliferating cells both in scAT and vAT (P < 0.01). The per centum of proliferating cells too differed between the locations, equally the sc depot had more proliferating cells (P < 0.05). GT treatment raised proliferation in vAT to levels similar to those in control scAT, with virtually Ki67 immunostaining appearing in the stromal fraction of the tissue (Fig. iv).

FIGURE 4

Paraffin-embedded AT sections immunostained with Ki67 (A) and AT cell proliferation (B) in scAT and vAT from controls (C) or rats treated with GT for vi mo. Note that Ki67 immunostaining is present mainly in the stromal fraction. Scale bar = twoscore μm. For quantification, Ki67-labeled nuclei, also as full DAPI-stained nuclei, were counted in different randomly selected department fields. Results are means ± SEM, n = 5. *Unlike from control, P < 0.01.

Paraffin-embedded AT sections immunostained with Ki67 (A) and AT cell proliferation (B) in scAT and vAT from controls (C) or rats treated with GT for 6 mo. Note that Ki67 immunostaining is present mainly in the stromal fraction. Scale bar = 40 μthousand. For quantification, Ki67-labeled nuclei, every bit well as total DAPI-stained nuclei, were counted in different randomly selected section fields. Results are ways ± SEM, n = 5. *Different from control, P < 0.01.

FIGURE 4

Paraffin-embedded AT sections immunostained with Ki67 (A) and AT cell proliferation (B) in scAT and vAT from controls (C) or rats treated with GT for 6 mo. Annotation that Ki67 immunostaining is present mainly in the stromal fraction. Scale bar = 40 μm. For quantification, Ki67-labeled nuclei, as well every bit full DAPI-stained nuclei, were counted in different randomly selected section fields. Results are means ± SEM, n = 5. *Different from control, P < 0.01.

Paraffin-embedded AT sections immunostained with Ki67 (A) and AT prison cell proliferation (B) in scAT and vAT from controls (C) or rats treated with GT for 6 mo. Note that Ki67 immunostaining is present mainly in the stromal fraction. Scale bar = 40 μm. For quantification, Ki67-labeled nuclei, besides as total DAPI-stained nuclei, were counted in different randomly selected section fields. Results are means ± SEM, n = five. *Different from command, P < 0.01.

Associations betwixt effect variables.

As expected, aromatase expression in scAT and vAT was positively correlated with 17β-estradiol and inversely correlated with testosterone plasma concentrations (Table three). Similarly, 17β-estradiol concentration varied inversely with the plasma concentration of testosterone. Furthermore, with increasing aromatase expression, the per centum of apoptotic and proliferating cells within each corresponding AT location increased. The same relationship was observed betwixt 17β-estradiol plasma concentration and apoptosis in vAT, likewise as proliferation in scAT and vAT. Accordingly, the inverse human relationship was observed for testosterone. The percentages of apoptotic cells in scAT and vAT were directly proportional to the percentages of proliferating cells inside the same depot. scAT adipocyte size was positively correlated with body weight gain. In both AT locations, the 17β-estradiol plasma concentration was inversely correlated with adipocyte diameter. In vAT, aromatase expression and adipocyte size were inversely associated. The plasma testosterone concentration was positively associated with adipocyte diameter in scAT and with body weight gain. In scAT, adipocyte size was inversely correlated with the percentage of proliferating cells.

Tabular array 3

Correlation coefficients between event variables measured in GT and command rats

Torso weight gain sc/v AT, respectively 3
Consequence 17β-Estradiol Testosterone Aromatase Apoptosis Proliferation
thousand pmol/L arbitrary units %
17β-Estradiol, pmol/L −0.61 1
Testosterone, pmol/L 0.76 2 * −0.89 1 *
scAT
    Aromatase, capricious units −0.50 1 0.68 1 * −0.71 1 *
    Adipocyte size, μm 0.71 2 * −0.72 ane * 0.81 2 * −0.57 2 −0.43 2 −0.73 2 *
    Apoptosis, % −0.13 2 0.42 one −0.61 2 0.75 2 *
    Proliferation, % −0.18 2 0.82 one * −0.64 two * 0.66 2 * 0.60 two *
vAT
    Aromatase, capricious units −0.38 1 0.74 one * −0.66 1 *
    Adipocyte size, μm 0.18 2 −0.65 1 * 0.37 two −0.74 2 * −0.50 ii −0.47 two
    Apoptosis, % −0.55 2 0.78 ane * −0.76 two * 0.seventy ii *
    Proliferation, % −0.56 1 0.ninety i * −0.73 i * 0.68 1 * 0.77 1 *
Body weight gain sc/v AT, respectively 3
Event 17β-Estradiol Testosterone Aromatase Apoptosis Proliferation
m pmol/50 arbitrary units %
17β-Estradiol, pmol/50 −0.61 1
Testosterone, pmol/L 0.76 2 * −0.89 1 *
scAT
    Aromatase, arbitrary units −0.50 1 0.68 1 * −0.71 one *
    Adipocyte size, μm 0.71 ii * −0.72 1 * 0.81 2 * −0.57 two −0.43 2 −0.73 2 *
    Apoptosis, % −0.13 two 0.42 i −0.61 2 0.75 two *
    Proliferation, % −0.xviii two 0.82 1 * −0.64 2 * 0.66 2 * 0.60 2 *
vAT
    Aromatase, capricious units −0.38 1 0.74 1 * −0.66 1 *
    Adipocyte size, μm 0.18 2 −0.65 1 * 0.37 ii −0.74 2 * −0.fifty 2 −0.47 two
    Apoptosis, % −0.55 2 0.78 one * −0.76 2 * 0.70 two *
    Proliferation, % −0.56 1 0.90 i * −0.73 ane * 0.68 ane * 0.77 1 *

ane

Spearman correlation. *P < 0.05.

2

Pearson correlation.

3

AT measurements were compared within the same tissue (sc, five).

TABLE three

Correlation coefficients between issue variables measured in GT and control rats

Body weight gain sc/v AT, respectively 3
Consequence 17β-Estradiol Testosterone Aromatase Apoptosis Proliferation
g pmol/L arbitrary units %
17β-Estradiol, pmol/L −0.61 1
Testosterone, pmol/L 0.76 two * −0.89 1 *
scAT
    Aromatase, capricious units −0.50 1 0.68 1 * −0.71 i *
    Adipocyte size, μm 0.71 two * −0.72 i * 0.81 2 * −0.57 two −0.43 2 −0.73 two *
    Apoptosis, % −0.13 2 0.42 1 −0.61 two 0.75 2 *
    Proliferation, % −0.18 2 0.82 1 * −0.64 2 * 0.66 2 * 0.60 2 *
vAT
    Aromatase, capricious units −0.38 1 0.74 1 * −0.66 1 *
    Adipocyte size, μm 0.18 2 −0.65 1 * 0.37 2 −0.74 2 * −0.l 2 −0.47 2
    Apoptosis, % −0.55 2 0.78 1 * −0.76 2 * 0.70 ii *
    Proliferation, % −0.56 one 0.ninety 1 * −0.73 1 * 0.68 i * 0.77 1 *
Trunk weight gain sc/v AT, respectively 3
Event 17β-Estradiol Testosterone Aromatase Apoptosis Proliferation
one thousand pmol/50 arbitrary units %
17β-Estradiol, pmol/L −0.61 1
Testosterone, pmol/L 0.76 ii * −0.89 ane *
scAT
    Aromatase, arbitrary units −0.50 1 0.68 1 * −0.71 1 *
    Adipocyte size, μm 0.71 ii * −0.72 1 * 0.81 ii * −0.57 2 −0.43 2 −0.73 2 *
    Apoptosis, % −0.thirteen 2 0.42 1 −0.61 2 0.75 2 *
    Proliferation, % −0.xviii 2 0.82 1 * −0.64 ii * 0.66 2 * 0.60 2 *
vAT
    Aromatase, arbitrary units −0.38 1 0.74 1 * −0.66 1 *
    Adipocyte size, μm 0.eighteen 2 −0.65 1 * 0.37 2 −0.74 2 * −0.fifty two −0.47 ii
    Apoptosis, % −0.55 2 0.78 1 * −0.76 2 * 0.seventy 2 *
    Proliferation, % −0.56 ane 0.90 1 * −0.73 1 * 0.68 1 * 0.77 i *

one

Spearman correlation. *P < 0.05.

2

Pearson correlation.

three

AT measurements were compared within the same tissue (sc, v).

Give-and-take

GT is a circuitous beverage composed of amino acids, carbohydrates, minerals, vitamins, and xanthines such as caffeine and theophylline. It is also one of the richest food sources of polyphenols, mainly the flavan-3-ols or catechins, including EGCG (∼59% of the total of catechins), (–)-epigallocatechin, EC-three-gallate, and EC (26). Subsequently oral administration of tea, catechins can be found in the plasma and widely distributed in rat tissues (26). From modulation of nutrient intake and energy expenditure, to antiadipogenic and apoptosis-promoting activities, several relevant effects have been described for tea in the past few years; catechins, present in great amounts in GT, take been shown to be involved in nearly of the actions described (27–29). Caffeine, ranging from 0.51–0.86 mmol/L (26), may too account for some of the effects attributed to GT, as it increases thermogenesis and acts synergistically with other tea components such as the catechins (30). It must exist stressed, yet, that some studies did not support a GT outcome on energetic metabolism (31,32).

As shown in the present written report, prolonged GT intake by adult male person Wistar rats resulted in a significantly lower weight gain compared with controls. In association with lower weight gain, increased aromatase expression was observed in both scAT and vAT of GT-treated rats. Furthermore, this increase in aromatase expression reflected upon higher plasma concentration of 17β-estradiol. We used adult rats to avert the variations in torso weight due to changes in lean mass that are associated with growth in younger rats.

Although decreased body weight gain has already been reported both in humans and animals afterwards GT treatment (27,33,34), increased estrogen product has not even so been related to GT chapters to regulate body composition. Previous studies reported that GT catechins were able to inhibit aromatase activeness in the placenta (19,20). The beverage, when placed in contact with the same cells, reduced aromatase activeness by 50%. Notwithstanding, those studies investigated astute effects. Information technology seems probable that chronic inhibition of aromatase activity could result in increased aromatase expression as a feedback mechanism (35–37). Furthermore, catechins may also interfere with estrogen signaling through interaction with estrogen receptor (ER)α or ERβ (38–40), although the actions reported are not consistent between prison cell lines, mayhap due to the presence of distinct costimulators or corepressors, making it difficult to predict the effects on the AT. Xanthines could also exist involved in the upshot of GT upon aromatase by increasing cellular military camp, a known inducer of aromatase expression through the AT aromatase promoter I.3 (vii).

Increased aromatase expression reflected upon plasma 17β-estradiol as this hormone, undetectable in controls, reached measurable concentrations in GT-drinking rats. Indeed, in that location was a pregnant positive association between plasma 17β-estradiol and aromatase expression in scAT and vAT. In parallel, testosterone concentration significantly decreased in the same rats. Plasma concentrations of estradiol and testosterone were negatively associated. This decrease in circulating testosterone may be at least in part due to its aromatization to 17β-estradiol or estrone (7). It has been proposed that the relative amount of estrogens and androgens or of their receptors (10,14) will determine the response of the AT to sex steroids. Furthermore, those ratios are thought to be closely related to the sexual dimorphism in body fat distribution and to the differential chance of men and pre- and postmenopausal women to develop metabolic diseases (41).

Estrogens accept profound effects on free energy metabolism equally demonstrated by the amount of fat aggregating in estrogen insufficiency models (nine,10), with reversion of the upshot by estrogen treatment. On the other hand, estrogen therapy ameliorates the plasma lipid profile in healthy males, although no outcome has been reported on torso weight (42). Mice lacking ERα develop the same metabolic abnormalities equally aromatase knockout mice, showing that ERα mediates the protective actions of estrogens (14). These alterations were absent in ERβ knockouts (fourteen), although recently information technology has been reported that mice defective ERβ, when fed a loftier-fatty diet, had improved insulin sensitivity and glucose tolerance compared with wild-blazon mice (43).

As first described by Vague (44), the 2 chief AT depots display major differences in the metabolic consequences they induce. Unfavorable cardiovascular profiles and the take a chance of developing type 2 diabetes are greater in individuals whose AT is located within the abdominal cavity than in those having mainly sc fatty (2), although there is nevertheless no verbal knowledge of the reasons for these differences. Sex activity steroids contribute to make up one's mind the location of fat distribution and the ability of different AT depots to produce and respond to these hormones varies tremendously (45). Confirming previous findings (8), we found the expression of aromatase in scAT was higher than that in vAT. The differential aromatase expression may contribute to the metabolic differences encountered between the aggregating of fat in the 2 locations. In fact, ii key proteins involved in lipid deposition, lipoprotein lipase and leptin, are transcriptionally regulated past 17β-estradiol, existence downwardly- and upregulated, respectively, by this hormone (46,47). Interestingly, with GT intake, vAT expression of aromatase rose to levels comparable to those found in scAT from controls.

scAT is the chief AT depot responsible for estrogen production. Considering plasma 17β-estradiol was undetectable in command rats, vAT would be under little influence of this hormone due to its low endogenous capacity to produce estrogens. On the other mitt, in GT-treated rats, both scAT and vAT may exist influenced past estrogens and the increase in plasma concentration of 17β-estradiol may likewise indicate that distant tissues or organs may be afflicted. Nutrient intake was not influenced, as rats from both groups ingested similar amounts of nutrient. In fact, in models of estrogen insufficiency, increases in body fat mass are not associated with hyperphagia (48). Estrogens have, however, been shown to increment voluntary energy expenditure (48) and leptin and decrease insulin sensitivities in the hypothalamus (49).

Either induced past the elevation of 17β-estradiol product or directly past the presence of GT components, east.g. catechins or xanthines, other interesting effects were plant on the AT. Outset, measurement of adipocyte diameter revealed a decrease in adipocyte size in both AT depots following GT treatment. Information technology has been pointed out that obesity and its metabolic consequences result from both increased number of adipose cells (hyperplasia) and adipocyte size (hypertrophy) (50). Notwithstanding, adipocyte size seems more critical for predicting morbidities related to AT excess. Inflammatory events originating in AT play a key role in obesity-associated diseases, the infiltration of macrophages in the AT existence a central event (iv). Cinty et al. (51) have shown that macrophages in the AT surround dead adipocytes. We have previously demonstrated that, for simple physical reasons, larger adipocytes are more prone to rupture than small-scale ones (52), peculiarly when they are contained in the abdominal crenel where sudden variations of pressure level oft take place (53,54). The inflammatory events related with large adipocytes are farther supported by the recent written report that adiponectin release from adipocytes is inversely correlated with adipocyte size in obese patients (50). Conversely, tumor necrosis factor-α, interleukin-6, and loftier sensitivity C-reactive protein circulating concentrations were positively associated with the same variable (l). Thus, past reducing adipocyte size, chronic GT handling might have a cracking affect on obesity-driven inflammation. Both estrogens and GT catechins have backdrop that can account for reduced adipocyte size, some of which are like. Ane example is the stimulation of catecholamine-induced lipolysis toward increased β-oxidation of fatty acids (33,55). Information technology was very interesting to find that plasma 17β-estradiol concentrations were inversely correlated with adipocyte size in scAT and vAT. Adipocyte size was as well positively correlated to body weight.

Together with smaller adipocyte size, we found increased apoptosis in vAT and increased proliferation in both AT depots, suggesting that AT is undergoing remodeling. Epidemiological studies have shown that usual GT drinkers have lower waist circumferences (34), which is in agreement with the observation of a high apoptotic rate found in the vAT of GT-treated rats. Modification of the AT profile through selective apoptosis of mature adipocytes in the vAT would avert the rupture of large lipid-laden adipocytes and the generation of an inflammatory response. Furthermore, the presence of higher apoptosis in vAT was positively associated with 17β-estradiol and inversely correlated with testosterone plasma concentrations, in conformity with the sexual dimorphism of vAT accumulation.

AT remodeling extended besides to cell proliferation. Preadipocytes and mature adipocytes are the major jail cell types that compose AT, but vascular endothelial and polish musculus cells are also documented (14). Proliferation was identified mainly in the AT stromal fraction where preadipocytes and blood vessels are predominant. Although we do not know which cell types were Ki67 immunostained, proliferation of either type would reveal greater chapters for lipid trafficking between mature and maturing adipocytes and between locations of AT degradation. Both aromatase expression and plasma 17β-estradiol concentrations had a strong positive correlation with the pct of proliferating cells in scAT and vAT. Thus, it is very likely that 17β-estradiol production in the AT may be responsible for the changes observed in proliferation either in cells from the adipose lineage or others. For example, endothelial, smoothen muscle, and mesenchymal cells may be induced to proliferate in the presence of 17β-estradiol (56). Previous studies reported that GT catechins, particularly EGCG, have antimitogenic effects on adipose cells (29). This apparent contradiction may have arisen from the fact that the antimitogenic action was reported in vitro in 3T3-L1 preadipocytes. In that experimental setting, the effect of GT or GT catechins may not be mediated through estrogens, because these cells either lack or have residue aromatase expression. Furthermore, as mentioned previously, several other types of cells compose AT and many of them are known targets for estrogens actions. Also worth noting is the inverse correlation between plasma testosterone and the percentage of proliferating cells. This, along with the known antiadipogenic effect of testosterone (41), implicates this hormone in the reduction of AT plasticity in response to free energy backlog that predisposes men to metabolic disorders.

The fact that total AT or body composition was not measured is an important limitation of this study. In addition, determination of metabolic parameters and baseline and time course measurements of hormones would strengthen the conclusions. Even so, relevant alterations were observed later on GT treatment, which deserve attending and further study.

In decision, GT intake induced profound remodeling of AT. The causal role of the GT-induced increase in aromatase expression and circulating concentration of 17β-estradiol needs to exist explored, although the strong correlations of 17β-estradiol and aromatase measurements with adipocyte size, apoptosis, and proliferation are in favor of the existence of a causal relationship. We also suggest that the presence of a higher precursor adipose cell pool together with the proadipogenic result of estrogens and attenuation of the antiadipogenic issue of testosterone would facilitate differentiation and the distribution of fat in an increased number of cells, resulting in reduced jail cell size. This could then contribute to the redistribution of body AT and decrease obesity-related inflammation.

The excellent technical support of Dr. Conceição Gonçalves, Luísa Vasques, and Abílio Ferreira are gratefully best-selling. The authors besides thank Prof. Victor de Freitas, from the Faculty of Sciences of the University of Porto, and Prof. Paula Guedes, from the Kinesthesia of Pharmacy of the University of Porto, for the measurements of tea catechins and caffeine, respectively. The authors are thankful for the valuable advice of Dr. Milton Severo on statistical analyses.

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Abbreviations

  • AT

  • DAPI

    4′,half-dozen-diamidine-2′-phenylindole dihydrochloride

  • EC

  • EGCG

    (–)-epigallocatechin-iii-gallate

  • ER

  • GT

  • sc

  • v

Footnotes

1

Supported past Fundação para a Ciência east a Tecnologia (POCTI, Feder, Programa Comunitário de Apoio, U38, U121/94, SFRH/BD/12622/2003 and SFRH/BD/19497/2004).

© 2008 American Club for Nutrition

© 2008 American Lodge for Nutrition