Adipocyte morphology and leptin signaling in rat offspring from mothers supplemented with flaxseed during lactation

Nutrition 28 (2012) 307–315

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Basic nutritional investigation

Adipocyte morphology and leptin signaling in rat offspring from mothers supplemented with flaxseed during lactation Mariana Sarto Figueiredo Ph.D. a, Magna Cottini da Fonseca Passos Ph.D. a, Isis Hara Trevenzoli Ph.D. b, Aline Andrade Troina Ph.D. a, Aluana Santana Carlos M.S. c, ~es Ph.D. d, Celly Cristina Alves Nascimento-Saba Ph.D. c, Mabel Carneiro Fraga Ph.D. a, d, Alex C. Manha a a a Elaine de Oliveira Ph.D. , Patrıcia Cristina Lisboa Ph.D. , Egberto Gaspar de Moura Ph.D. , * a

Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil Laboratory of Molecular Endocrinology, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil c Laboratory of Morphofunctional Analysis, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil d Laboratory of Neurophysiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 March 2011 Accepted 4 July 2011

Objective: We have recently shown that maternal flaxseed supplementation during lactation induces insulin resistance in adult offspring. Here, we studied the effects of maternal dietary flaxseed during lactation on adipocyte morphology and leptin signaling in the hypothalamicpituitary-thyroid axis as well as on behavioral traits in the adult progeny. Methods: Lactating rats were fed a control (C) diet or a diet with 25% flaxseed (F). After weaning, pups received a standard diet until postnatal day (PN) 180. Male offspring were killed at PN21 and 180. Data were considered significant at P < 0.05. Results: Weaned F rats presented a lower total and subcutaneous fat mass and higher subcutaneous adipocyte area (þ48%), but at adulthood they presented higher subcutaneous and visceral adipocyte areas (þ40% and 1.9-fold increase, respectively), with no change in body fat mass. At PN21, F pups had hyperleptinemia (þ69%), lower T3 (33%), higher TSH (2.1-fold increase), higher pituitary leptin receptor (Ob-R, þ11%), signal transducer and activator of transcription 3 (STAT3, þ21%), and phosphorylated-STAT3 (p-STAT3, þ77%) protein content. Adult F offspring only showed lower T4 (28%) and higher thyroid Ob-R (þ52%) expression. Maternal flaxseed intake during lactation did not result in behavioral changes in the adult offspring. Conclusions: Maternal flaxseed supplementation decreases offspring adiposity and increases pituitary leptin signaling at weaning, but it induces hypertrophic adipocytes and higher thyroid leptin receptor in adulthood. The present data suggest that extensive use of flaxseed during lactation is undesirable. Ó 2012 Elsevier Inc. All rights reserved.

Keywords: Adipose tissue Flaxseed Lactation Leptin signaling and programming

Introduction Adverse nutritional or hormonal environments during early life may change some physiological parameters at adulthood. Barker [1] reported low birth weight with diabetes, obesity, and hypertension in adult humans. This association has been named metabolic programming and has been confirmed by several epidemiologic and experimental studies [2,3]. Our group has shown that neonatal imprinting factors during lactation, such as malnutrition [4–6], overnutrition [7], thyroid hormones [8], * Corresponding author. Tel.: (þ55.21) 25876434; fax: (þ55.21) 25876129. E-mail address: [email protected] (E. G. de Moura). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.07.002

leptin [9,10], and hypoprolactinemia [11,12], program changes in adiposity, leptin signaling, thyroid function, glucose homeostasis, and lipid profile. Leptin is a hormone secreted by white adipose tissue that crosses the blood-brain barrier by a saturable transport system and that affects feeding behavior, thermogenesis, and neuroendocrine status by acting through the hypothalamic leptin receptor (Ob-R) and the Janus tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) intracellular pathway [13,14]. The long form (Ob-Rb) and the short form of the leptin receptor (Ob-Ra) are the most studied isoforms and Ob-Rb is fully capable of activating intracellular signaling [15]. Leptin binding to Ob-Rb initiates tyrosine phosphorylation by JAK2.

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Phosphorylated Ob-Rb recruits STAT3, which is activated through phosphorylation by JAK2 [16]. Activated STAT3 dimerizes and translocates to the nucleus to stimulate gene transcription [17]. The JAK2–STAT3 pathway stimulates the transcription of the suppressor of cytokine signaling 3 (SOCS3), which is a negative regulator of leptin signaling [16,18]. Flaxseed (Linum usitatissimum L.) has been a focus of interest in the field of functional food because of its potential health benefits, such as the improvement of lipid profile, glycemia, and cardiovascular function [19–23]. Flaxseed is rich in protein, dietary fat, and fiber fat; approximately 37% of its mass consists of oil, of which 50% is a-linolenic acid, which is an (n-3) fatty acid [18:3(n-3), alpha-linolenic acid] [24]. Flaxseed is one of the richest sources of the plant lignan secoisolariciresinol diglucoside, which is present in a much higher proportion in the seed than in other tissues and is responsible for the observed phytoestrogenic, anticarcinogenic, and antiatherogenic effects [25–27]. Although flaxseed has several potential advantages when consumed throughout life, it may cause adverse effects when it is consumed in critical periods of life, such as gestation and lactation [28,29]. We have shown that maternal flaxseed in the diet during lactation in rats is associated with higher total and visceral fat mass, serum triglycerides, and cholesterol in adult female offspring [30], whereas, in male offspring, there is no change in body adiposity, but hypoadiponectinemia and hyperinsulinemia do occur, suggesting insulin resistance [31]. These effects seem to result from changes observed in the milk composition, such as higher leptin and 17b-estradiol contents [30]. Several studies have investigated the associations between leptin and thyroid hormones. Leptin seems to act on the thyroid system mainly by stimulating Thyrotropin releasing hormone (TRH) secretion [32], but it also acts at the pituitary level [33,34] and thyroid level [35–36] Substitute for [35–36], and it influences thyroid hormone metabolism [37–39] Substitute for [37–39]. In rodents, Ob-R has also been identified in the thyroid and pituitary glands [35,40,41]. Both TRH and leptin have antidepressant effects [42], and one clinical study has demonstrated the beneficial effects of flaxseed oil in the diet on bipolar disorder in children and adolescents [43]. Because flaxseed may influence adipogenesis and leptin production, and because leptin has profound effects on the hypothalamic-pituitary-thyroid (HPT) axis and antidepressant effects [42], our aim was to evaluate the short- and long-term consequences of maternal flaxseed in the diet during lactation on adipose tissue morphology, leptin signaling in the HPT, and behavioral disturbances in rats. Materials and methods Animal care and diets The use of the animals according to our experimental design was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro (protocol 230/2008), which based its analysis on the principles adopted and promulgated by the Brazilian law that concerns the rearing and use of animals in teaching and research activities in Brazil [44]. Three-month-old Wistar rats were maintained in a room with controlled temperature (25  1 C) and dark–light cycle (lights on from 7:00 a.m. to 7:00 p.m.). Virgin rats (200–220 g) were mated, and each female was placed in an individual cage with free access to water and food until the birth of the offspring. At birth, 16 lactating rats were randomly assigned to each of the following groups: control group (C, n ¼ 8), with free access to a diet containing 20% protein, 60.2% carbohydrate, and 19.8% lipid composing the energy value, with 5% of cellulose fiber; and flaxseed group (F, n ¼ 8), with free access to a diet containing 18.9% protein (13.9% casein and 5% flaxseed), 59.3% carbohydrate, and 21.8% lipid exclusively from flaxseed composing the energy value, with 5% of fiber

exclusively from flaxseed. Birth was defined as day 0 of lactation and weaning was at day 21. The lactating rats started to receive the experimental diet (control or flaxseed) on day 0 of lactation until day 21 of lactation. At birth, all of the litters were randomly adjusted to six male pups for each mother to maximize lactation performance [45]. After weaning, all pups received a standard laboratory diet (Nuvilab; Nuvital Nutriente S/A, PR, Brazil) containing 22% protein and 66% carbohydrate until 180 d of age. Two pups of each mother (16 pups) were chosen and killed at 21 d of age, and another two pups from each mother (16 pups) were killed at 180 d of age with a lethal dose of pentobarbital (0.06g/kg/b.w.) for collection of blood by cardiac puncture and collection of tissues. The diets offered to lactating rats (Table 1) were prepared according to the American Institute of Nutrition (AIN 93G) recommendation for rodent diets [46]. The flaxseed diet contained 8.9% versus 7.6% lipid and 54.3% versus 52.1% carbohydrate in comparison with the control diet, but these differences were not significant. The flaxseed was ground in a blender before being added to the diet. The flaxseed diet contained 25% flaxseed, which completely provided the recommended oil and fiber content [46]. The ingredients of the experimental diets were carefully weighed and homogenized with boiling water to gelatinize the starch in an industrial food mixer (Hobart). The resulting dough was formed into pellets and dried in a Fabbe-Primar ventilated oven at 60 C for 24 h, labeled, and stored in a refrigerator until use. The body mass and food intake of the offspring were monitored every 4 d from weaning until they were 180 d old. Behavioral studies From PN150 to PN170, 40 C and 40 F adult offspring were submitted to the behavioral tests. The following tests were used: 1) elevated plus-maze (EPM), 2) hole board arena (HB), and 3) radial arm water maze (RAWM). Full descriptions of the testing equipment and protocols can be found elsewhere [47,48]. All testing sessions were performed between 1:00 and 6:00 p.m. in a sound-attenuated room. Behavioral analyses were performed using video images of the tests. Anxiety-like behavior was assessed at PN150 in the EPM. Animals were allowed 10 min to explore the maze. The number of entries and total time spent in the open and closed arms were recorded. The number of open-arm entries (Entries OA) and the time spent in the open arms (Time OA) were used as anxiety indices. Novelty-seeking behavior was assessed at PN157 in the HB. Animals were given 5 min to explore the arena. The number of nose pokes (head dips) was used as a measure of exploratory activity. Memory and learning were assessed at PN165 in the RAWM. Animals were tested for five consecutive days, four trials per day. Animals were given 2 min per trial to explore the maze and find the hidden platform that was positioned at the end of one of the arms. The latency to find the hidden platform was used to assess memory/learning performance.

Table 1 Composition of 100 g of diet used in the experimental during lactation phase Ingredients

Control %

Casein* 22.70 d Flaxseedy 50.30 Corn starchz 10.00 Sucrosex Mineral Mix AIN93G* 3.50 Vitamin Mix AIN93G* 1.00 k 7.00 Soybean oil { 5.00 Cellulose (Fiber) Choline Bitartrate* 0.25 L-Cystine* 0.30 0.0014 Tert-Butylhydroquinone# Macronutrient composition (100g/diet) Protein 17.40 Carbohydrate 52.10 Fat 7.60 Total energy (kJ/ 100 g) 1451.20 * y z x { k #

Flaxseed kJ 284.51 d 701.45 167.36 12.89 16.40 263.59 d d 5.02 d

%

kJ 16.00 25.00 44.00 10.00 3.50 1.00

d d 0.25 0.30 0.0014 17.30 54.30 8.90 1532.44

~o Paulo, SP, Brasil). M Cassab Comercio & Industria LTDA (Sa Arma Zen Produtos Naturais LTDA (Rio de Janeiro, RJ, Brasil). Maisena, Unilever Best Foods Brasil LTDA (Mogi Guac¸u, SP, Brasil). ~o (Rio de Janeiro, RJ, Brasil). Unia Microcel, Blanver LTDA (Cotia, SP, Brasil). Liza Cargil Agricultura LTDA (Mairinque, SP, Brasil). Vogler Ingredients (Eastman, USA).

200.31 513.38 617.10 167.36 12.89 16.40 d d d 5.00 d

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Total, visceral, and subcutaneous fat masses

Statistical analysisdbiochemistry

The total and visceral fat masses were determined at 21 and 180 d by carcass analysis [5,9] substitute for [5,9]. The carcasses of the C and F groups were weighed, autoclaved for 1 h, and homogenized in distilled water (1:1). Samples of the homogenate were stored at 4 C for analysis. Three grams of homogenate was used to determine the fat mass content gravimetrically [49]. Samples were hydrolyzed in a shaking water bath at 70 C for 2 h with 30% KOH and ethanol. Total fatty acids and non-esterified cholesterol were removed using three successive washings with petroleum ether. After drying overnight in a vacuum, the tubes were weighed, and the results are expressed as g fat/100 g carcass. The visceral (mesenteric, epididymal, and retroperitoneal) fat mass was excised and weighed to evaluate central adiposity [50]. The subcutaneous fat mass was estimated from the total fat mass minus the visceral fat mass, and the results are expressed as percentages (%).

Data are reported as the means  SEM. The statistical significance was determined by two-way ANOVA to analyze body weight and food intake evolution. The other experimental data were analyzed by Student’s unpaired t test. Differences were considered significant at P < 0.05.

Adipose tissue morphology Visceral and subcutaneous (inguinal) adipose tissue samples of the C and F groups at 21 and 180 d were fixed in buffered formaldehyde for 72 h, dehydrated, cleared, and then paraffin-embedded so that the plane of each section corresponded with the that of the wider surface. Five-micrometer-thick sections at the same level were obtained and stained with hematoxylin-eosin to assess morphology. Tissue sections were observed with an Olympus BX40 light microscope using a 10 objective, and digital images were captured with an Optronics CCD video camera system. The calibration was checked regularly using a slide micrometer. At least 100 adipocyte profiles per animal were randomly selected and captured. The area, perimeter, and number of adipocytes (cells/100 mm2) were determined using the ImageJ 1.34s program (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA) [51,52].

Serum hormone concentrations Offspring blood samples were obtained at 21 and 180 d and centrifuged (1000  g, 4 C, 20 min) to obtain the serum, which was individually kept at 20 C until assaying. All measurements were performed in one assay. Leptin levels were measured using a murine leptin–specific radioimmunoassay (RIA) kit (Linco Research, St. Charles, MO, USA), which measures both rat and mouse leptin with an assay sensitivity of 0.5 ng/mL and an intraassay coefficient of variation of 2.4%. Total serum T3 (TT3) and free T4 (FT4) levels were measured by RIA using commercial kits (Coat-A-Coat; DPC, Los Angeles, CA, USA). The coefficient of intraassay variation for the TT3 was 7.5% and that for FT4 was 3.9%. Serum TSH was measured by specific RIA using a kit for rat TSH supplied by the NIDDK (Bethesda, MD, USA). Data are expressed in terms of the reference preparation provided (RP-3). The coefficient of intraassay variation was 2.3%, and the assay sensitivity was 0.18 ng/mL.

Western blot analysis The amounts of leptin signaling pathway proteins (Ob-R, JAK2, STAT3, pSTAT3) in the hypothalamus, pituitary, and thyroid were evaluated by Western blot as described before [41,52] substitute for [41,52]. Briefly, to obtain cell extracts, tissues were homogenized in ice-cold lysis buffer (50 mM HEPES, 1 mM MgCl2, 10 mM EDTA, Triton X-100 1%, pH 6.4) containing the following protease inhibitors: 10 mg/mL aprotinin, 10 mg/mL leupeptin, 2 mg/mL pepstatin, and 1 mM PMSF (Sigma-Aldrich, St. Louis, MO, USA). Actin was used as an internal control. The total protein content in the homogenates was determined by the BCA protein kit assay (Rockford, IL, USA), and cell lysates were denatured in sample buffer (50 mM Tris-HCl, pH 6.8, 1% SDS, 5% 2-mercaptoethanol, 10% glycerol, 0.001% bromophenol blue) and heated at 95 C for 5 min. Samples (30 mg total protein) were separated by 10% SDS-PAGE and transferred to polyvinylidene membranes (PVDF, Hybond-P; Amersham Pharmacia Biotech, Piscataway, NJ, USA). Molecular weight markers (Amersham Biosciences, Uppsala, Sweden) were run in parallel. Membranes were blocked with 5% nonfat milk in Tween-TBS (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.1% Tween-20). The primary antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) used were anti-OB-R (1:500), anti-JAK2 (1:500), anti-STAT3 (1:500), anti-p-STAT3 (1:500), and antiactin (1:500). The membranes were then washed three times with Tween-TBS (0.1%), followed by incubation for 1 h with the appropriate secondary antibody conjugated to biotin (Santa Cruz Biotechnology). Then, the membranes were incubated with streptavidin-conjugated horseradish peroxidase (Caltag Laboratories, Burlingame, CA, USA). All Western blots were allowed to react with horseradish peroxidase substrate (ECL-plus; Amersham Pharmacia Biotech) and then were exposed to x-ray film for 10 s to 1 min. Images were obtained, and the bands were quantified by densitometry using ImageJ and normalized against the bands obtained for actin.

Statistical analysisdbehavior Data are reported as the means  SEM. The Kolmogorov–Smirnov one-sample test (K–S) was used to assess the normality of the distribution of each variable. Significance was assumed for P < 0.05. Differences between the C and F groups in the Entries OA and Time OA measures were analyzed by means of multivariate analyses of variance (mANOVA). TREATMENT (C or F) was used as the betweensubject factor. Differences between the C and F groups in the number of nose pokes were analyzed by means of the repeated-measures ANOVA (rANOVA). Differences between the C and F groups in the latency to find the hidden platform in the RAWM were analyzed by rANOVA. DAY and TRIAL were the within-subject factors. TREATMENT (C or F) was used as the between-subject factor.

Results After weaning, pups from the F mothers had higher body weights at some points throughout their lives, with increases of approximately 10 to 15% (P < 0.05), and their food intake was higher (approximately þ10%, P < 0.05), which confirms previous findings [31]. F pups presented a lower total fat mass at 21 d of age (17.8%, P < 0.05), but at 180 d of age, no changes were observed (Fig. 1A). No changes were observed in the visceral fat mass at 21 d (F ¼ 1.06  0.11 versus C ¼ 1.25  0.05 g) or 180 d (F ¼ 12.20  1.03 versus C ¼ 11.61  0.72 g). At 21 d, the F group presented a lower subcutaneous fat mass (approximately 23%, P < 0.05), and no changes were observed at 180 d old (Fig. 1B). The F group presented a higher subcutaneous adipocyte area at 21 (þ48%, P < 0.05) and 180 d of age (þ40%, P < 0.05), as shown in Figure 2B. No changes were observed in visceral adipocyte area in weaned F pups, but at adulthood, this group showed a higher visceral adipocyte area (1.9-fold increase, P < 0.05; Fig. 2A). As expected, the number of adipocytes per area was lower when the area of the adipocytes was higher (Fig. 2C and 2D). Figure 3 shows representative micrographs of the adipocytes. Leptinemia was higher in the 21-d-old F pups (F ¼ 1.83  0.9 versus C ¼ 1.09  0.16 ng/mL; þ69%, P < 0.05), but no change was observed in adult F rats (F ¼ 1.84  0.24 versus C ¼ 1.60  0.16 ng/mL). At 21 d, F offspring showed lower serum TT3 (F ¼ 75.1  5.2 versus C ¼ 113.1  5.2 ng/dL; P < 0.05), no change in serum FT4, and higher serum TSH (F ¼ 0.72  0.03 versus C ¼ 0.35  0.07 ng/mL; P < 0.05). At 180 d, the F group presented normal levels of TT3 and TSH and lower FT4 (F ¼ 0.66  0.05 versus C ¼ 0.92  0.01 ng/dL; P < 0.05). The levels of leptin signaling pathway proteins (Ob-R, JAK2, STAT3, and p-STAT3) in the hypothalamus of C and F groups were similar at weaning and adulthood. In the pituitary gland at 21 d, weaned F pups had a higher Ob-R content (þ11%, P < 0.05; Fig. 4A) and consequently higher STAT3 and pSTAT3 (þ21% and þ77%, respectively, P < 0.05; Fig. 4C and 4D). In contrast, no changes were detected at adulthood in the pituitary gland. Concerning the leptin pathway in the thyroid gland, no change was observed at 21 d, but Ob-R expression was higher in F offspring than in C offspring (þ52%, P < 0.05; Fig. 5A) at 180 d. The maternal flaxseed-containing diet during lactation had no effect on behavioral traits in adult progeny. No difference was observed between groups in 1) anxiety-like behavior in the EPM, i.e., Entries OA (F ¼ 2.03  0.389 versus C ¼ 2.44  0.41) or Time OA (F ¼ 15794  3673 versus C ¼ 23508  5318 ms); 2) noveltyseeking behavior in the HB, i.e., the number of nose pokes (F ¼ 5.56  0.47 versus C ¼ 6.3  0.4); or 3) memory/learning in the

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A Total fat mass (%)

15

10

C F

*

5

0 21 days

180 days

B

Subcutaneous fat mass (%)

310

15

10

C F

*

5

0 21 days

180 days

Fig. 1. Total and subcutaneous fat mass of the offspring. (A) Total fat mass and (B) subcutaneous fat mass of offspring at PN21 and PN180 whose mothers were fed a control (black bar) or a flaxseed diet (white bar) during lactation. Values are means for eight animals per group with standard errors represented by vertical bars. *Mean values were significantly different from that of the control group (P < 0.05).

RAWM, i.e., the latency to find the hidden platform (F ¼ 548.41  26.49 versus C ¼ 589.74  30.86 s). Discussion In the present study, we observed that maternal flaxseed in the diet during lactation programmed changes in the leptin signaling pathway and in adipocyte morphology. Flaxseed was given in the diet because flaxseed is usually ingested by humans in their food as part of a healthy lifestyle to provide fiber and alpha-linolenic acid [53], especially during pregnancy, when the fiber intake will increase from 25 g/d to 28 g/ d, according to the United States Food and Nutrition Board [54]. The maternal flaxseed diet that we used during lactation had flaxseed as the exclusive source of oil and fiber and represented a dose of 25% flaxseed in the diet. This dose was based on some experimental studies during gestation and/or lactation that used 20-40% flaxseed [29]. Our experimental animals received roughly one quarter of their diet as flaxseed, i.e., around 8.8% of the oil, 5% of the protein, 5% of the carbohydrate, and 5% of the fiber exclusively from flaxseed. For women, who eat around 800 g of food per day, this would represent 200 g of flaxseed and would be equivalent to about nine spoonfuls per day. This amount is easily

reached if the women eat three spoonfuls with each main meal. Consumption of 200 g/d of flaxseed would represent 20% of the recommended daily fiber intake (25 g/d) for women, and this is the amount that we used in our experimental study that showed negative health affects in the pups when they become adults [30,31]. A maternal flaxseed-containing diet during lactation results in small differences in body weight and food intake in the offspring. We have demonstrated that F pups present a lower body fat mass and have no change in visceral fat mass at weaning [31]. In the present study, we observed a higher adipocyte area in the subcutaneous tissue but a normal visceral adipocyte area. Flaxseed oil may activate peroxisome proliferator–activated receptor gamma, which is a nuclear receptor that is highly expressed in adipose tissue and has the ability to activate lipogenic genes and adipocyte differentiation [55,56]. This effect seems to be more pronounced in subcutaneous adipocytes. Also, subcutaneous leptin production seems to be higher than in visceral adipocytes [57]. The hyperleptinemia at weaning in the F group may have been caused by the higher leptin concentration in the milk [30,31]. Thus, at this age, hyperleptinemia may act to decrease insulin secretion and increase insulin sensitivity, which has been previously observed in these animals [31,58].

Fig. 2. Visceral and subcutaneous adipocytes area and number of the offspring at 21 and 180 d old. (A) Visceral adiposity area, (B) subcutaneous adiposity area, (C) visceral, and (D) subcutaneous adiposity number of offspring whose mothers were fed a control (black bar) or a flaxseed diet (white bar) during lactation. Values are means for eight animals per group, with standard errors represented by vertical bars. *Mean values were significantly different from that of the control group (P < 0.05).

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Fig. 3. Morphology of visceral and subcutaneous adipose tissues in rats. Representative photomicrography of visceral (visc) and subcutaneous (sub) adipocytes of control (n ¼ 8) and flaxseed (n ¼ 8) offspring at PN21 and PN180.

Estrogen receptors have been found in adipocytes [59], and the administration of estrogen stimulates the secretion of leptin by adipose tissue in vitro [60]. Flaxseed has high concentrations of phytoestrogens, whose consumption in periods such as pregnancy and/or lactation may result in estrogen changes in the

offspring. In our previous study, we observed a higher 17-bestradiol concentration in the serum and milk of F dams at the end of lactation and in the serum of female and male offspring at 21 d [30,31]. Therefore, these elevations could be attributed to the presence of phytoestrogens (e.g., secoisolariciresinol

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Fig. 4. Leptin signaling pathway in the pituitary of the offspring at PN21. The expression of proteins Ob-R (A), JAK2 (B), STAT3 (C), and p-STAT3 (D) in pituitary from animals at PN21 whose mothers were fed a control (black bar) or a flaxseed diet (white bar) during lactation was done by Western blotting and expressed in arbitrary units. Actin was loaded as a control and data were normalized for actin densitometry. Representative bands are shown (E). Values are means for eight animals per group, with standard errors represented by vertical bars. *Mean values were significantly different from that of the control group (P < 0.05).

diglucoside) in the flaxseed diet, which suggests that high estradiol levels could have been one cause of hyperleptinemia at weaning. At adulthood, F offspring had normal total and visceral fat mass and leptinemia but higher adipocyte areas in subcutaneous and visceral adipose tissues. At this age, normoleptinemia seems to be more related to the fat mass than to the area of adipocytes. We have previously shown in a model of leptin programming that leptin expression in the adipocyte is higher with normal adiposity [61], which suggests a dissociation between leptin production and fat mass. The adipose tissue distribution in obese subjects is an important factor for cardiovascular risk [62,63]. Our data show a greater increase in visceral than in subcutaneous adipocyte area in adult F offspring, which suggests a higher cardiovascular risk in these animals. At 21 d of age, F pups showed no changes in the leptin signaling pathway in the hypothalamus compared with the C group. However, they presented hyperleptinemia at this age, suggesting relative brain insensitivity to leptin as confirmed by a relatively unchanged food intake. Thyroid leptin signaling was not affected, which reinforces the hypothesis of leptin resistance. On the contrary, in the pituitary gland, we observed higher Ob-R,

STAT3, and pSTAT3 expression, which suggests that leptin is acting at this gland. Thus, these tissues had different sensibilities to leptin during development, which cannot be explained at the moment. Leptin regulates pituitary function to stimulate TSH production [33,34]. Thus, in weaned F pups, the higher TSH detected could have been caused, at least in part, by the higher leptin pituitary stimulation at this age. This higher TSH was not able to increase thyroid hormone production, which suggests a primary thyroid dysfunction that only normalized serum T4, probably via hyperleptinemia. Lower deiodination of T4 in the periphery can also explain the lower T3 with normal T4. The possible failure in thyroid function seemed to worsen with age, because at 180 d of age, T4 was significantly lower. The HPT axis seemed also to be impaired because TSH, which was expected to be increased, was actually normal. At 21 d, TSH was still increased, probably because of increased leptin action on the pituitary gland, which was no longer observed at 180 d because those animals presented normoleptinemia and normal leptin signaling in the pituitary gland. However, we observed higher Ob-R expression in the thyroid gland in the F group. This finding has also been observed in other programming models, such as postnatal early overnutrition by litter size reduction [7] and

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Fig. 5. Leptin signaling pathway in the thyroid of the offspring at PN180. The expression of proteins Ob-R (A), JAK2 (B), STAT3 (C), and p-STAT3 (D) in thyroid from animals at PN180 whose mothers were fed a control (black bar) or a flaxseed diet (white bar) during lactation was done by Western blotting and expressed in arbitrary units. Actin was loaded as a control and data were normalized for actin densitometry. Representative bands are shown (E). Values are means for eight animals per group, with standard errors represented by vertical bars. *Mean values were significantly different from that of the control group (P < 0.05).

postnatal nicotine exposure [41], which also show lower serum T4 at adulthood. Serum TSH and T3 levels differ among the three models, suggesting that T4 could directly or indirectly regulate thyroid OB-R expression. The finding that the studied proteins of the leptin signaling pathway were unchanged cannot be explained at present. However, in the nicotine exposure model, higher OB-R is associated with lower p-STAT3, suggesting that mediators other than leptin can modulate those proteins. Regarding the behavioral traits, our results seem to be in line with those of Yu et al. [64], who showed that the adult offspring of dams that were fed flax oil during pregnancy and lactation did not present alterations in memory/learning in a water maze task. In the present study, we further demonstrate that anxiety-like behavior and novelty-seeking behavior were not affected by the flaxseed diet, suggesting the diet does not present significant adverse behavioral effects. Conclusion A flaxseed diet during lactation is associated with lower adiposity but, paradoxically, higher subcutaneous adipocyte area

at weaning, suggesting an impairment of adipogenesis. It is possible that this change alters leptin production and signaling in the pituitary gland, resulting in higher Ob-R, STAT3, and p-STAT3 protein content. At adulthood, F offspring normalized their adiposity but still presented a higher subcutaneous adipocyte area and also visceral adipocyte area, which could be related to insulin resistance [31]. The only change in leptin signaling at adulthood was the increased Ob-R content in the thyroid. Thus, our experimental data suggest that during this period, women should limit their intake of flaxseed or other phytoestrogen-containing substances to prevent altered adipogenesis and leptin action in their progeny. Acknowledgments This research was supported by National Council for Scientific and Technological Development (Conselho Nacional de DesengicodCNPq), Coordination for volvimento Cientıfico e Tecnolo ~o the Enhancement of Higher Education Personnel (Coordenac¸a de Aperfeic¸oamento de Pessoal de Nıvel SuperiordCAPES), and State of Rio de Janeiro Carlos Chagas Filho Research Foundation

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~o Carlos Chagas Filho de Amparo a  Pesquisa do Estado (Fundac¸a do Rio de JaneirodFAPERJ). E.O. was recipient of the Capes Fellowship; M.S.F., I.H.T., and A.S.C. were recipients of the FAPERJ Fellowship; and A.A.T. was a recipient of a CNPq fellowship.

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