Food and Chemical Toxicology 69 (2014) 69–75
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Flaxseed oil during lactation changes milk and body composition in male and female suckling pups rats Deysla Sabino Guarda a, Patricia Cristina Lisboa a, Elaine de Oliveira a, José Firmino Nogueira-Neto b, Egberto Gaspar de Moura a,⇑, Mariana Sarto Figueiredo a a b
Department of Physiological Sciences, Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ 20551-030, Brazil Lipids Laboratory, Medical School, State University of Rio de Janeiro, Rio de Janeiro, RJ 20551-030, Brazil
a r t i c l e
i n f o
Article history: Received 9 November 2013 Accepted 2 April 2014 Available online 12 April 2014 Keywords: Flaxseed oil Lactation Milk Body composition
a b s t r a c t We have reported several changes in neonate or adult offspring after the maternal use of whole flaxseed or its components. However, it is unknown the use of higher oil intake in the neonatal period. Here we evaluated the effects of high maternal intake of flaxseed oil during lactation upon milk and body composition in male and female offspring. Lactating rats were divided into: (1) control (C, n = 10), 7% soybean oil; (2) hyper 19% soybean oil (HS, n = 10); and (3) hyper 17% flaxseed oil + 2% soybean oil (HF, n = 10). Dams and offspring were killed at weaning. HS and HF dams, male and female offspring presented lower body weight during lactation. HF mothers presented lower body and visceral fat masses. HF male offspring presented lower body and subcutaneous fat masses. HS and HF milk presented lower triglycerides (TG) and cholesterol. HF male and female offspring showed lower triglyceridemia and insulinemia, but no changes in glycemia and leptinemia. The higher intake of flaxseed oil during lactation reduced the body weight of mothers and offspring, decreases milk lipids and apparently increases insulin sensitivity in this critical period of life. Those changes may explain the previously reported programming effect of maternal flaxseed intake during lactation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Functional foods are those that beyond basic nutritional functions can produce beneficial health effects and should be safe for consumption without medical supervision. Flaxseed is the seed from the flax plant (Linum usitatissimum L), called linseed, which is a member of the Linaceae family (Oomah, 2001). Flaxseed contains 32–45% of its mass as oil, of which 51–55% is alpha-linolenic acid (ALA, which belongs to omega-3 family) and 15–18% is linoleic acid (omega-6) (Carter, 1993). The ratio of omega-3/omega-6 seems to be an important factor to lipid and glucose homeostasis. Flaxseed and canola oil are rich in omega-3 and lower in omega-6, while soybean oil that is less expensive and because of this, highly used by poor population had a lower omega-3/omega-6 ratio. Beside this, there is a trend of increase the proportion of vegetable oil on the diet (Takahashi and Ide, 2000). Flaxseed presented potential health benefits associated with decrease in the risk of cardiovascular disease (Cunnane et al., ⇑ Corresponding author. Address: Departamento de Ciências Fisiológicas, 5o andar, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Rio de Janeiro, RJ 20550-030, Brazil. Tel./fax: +55 21 28688334. E-mail address: [email protected] (E.G. de Moura). http://dx.doi.org/10.1016/j.fct.2014.04.003 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
1995; Jenkins et al., 1999). Several functional properties of flaxseed have been reported including antioxidant activity and the ability to lower blood glucose, serum total cholesterol, LDL-c and triacylglycerol while increasing serum HDL-c (Prasad, 2008). Based in the recommendation to increase the use of oils rich in omega-3, there is an increase in consumption by the population. However, this could lead to an overconsumption of a hyperlypidic diet, which health risk is not completely evaluated. Recently, it was compared the differences in the effects of canola vs. soybean oil in a normal proportion (7%) or hyperlipidic proportion (19%) (Costa et al., 2012, 2013). It is interesting that in the normal proportion canola oil decreases abdominal fat and increases insulin sensitivity, but in the higher proportion canola oil is worse than soybean oil both in relation to obesity and insulin resistance. Some authors, including our previous data have suggested caution when flaxseed is consumed during pregnancy and lactation (Tou et al., 1998; Troina et al., 2010). Pups whose mothers consumed dietary flaxseed (10%) during gestation and lactation presented lower body weight at birth (Tou et al., 1998). At weaning, the male offspring, whose mothers received whole dietary flaxseed (25%) showed higher body mass but lower body fat mass, lower serum total cholesterol and triglycerides, hyperleptinemia,
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hypoinsulinemia and higher insulin sensitivity, lower serum triiodothyronine (T3) and higher thyrotropin (TSH) (Figueiredo et al., 2009). In another study we reported that the female offspring at weaning showed similar phenotype, presenting lower total fat mass, lower visceral fat mass, lower serum total cholesterol and triglycerides and higher serum leptin, but contrary to the higher body mass observed in the male, the female also presented lower body mass (Troina et al., 2010). Since, the main components of flaxseed is the oil and secoisolariciresinol diglucoside (SDG), we showed in another study, the effects of maternal supplementation with SDG alone or SDG plus 7% flaxseed oil during lactation. In the mother, SDG treatment caused a higher total fat mass, while the oil reduced the fat mass. Also, the oil reduced serum triglycerides and cholesterol in the dams. In the offspring, a lower body fat mass was observed in the oil-treated group both in male and female. Serum triglyceride levels were also lower in both SDG and flaxseed oil + SDG groups, both in male and female (Troina et al., 2012). Based in the previous reports, we hypothesized that the overconsumption of flaxseed oil during lactation, instead to be health promoting, could be detrimental as was reported for canola oil (Costa et al., 2013). Thus in this study we aimed to evaluate the milk composition, body composition of dams and pups as well as serum leptin, glucose homeostasis and lipid profile. To our knowledge, this is the first study to adress the question of the use of hyperlipidic flaxseed diet during lactation over body composition, glucose homeostasis and lipid profile. 2. Materials and methods Three-month-old Wistar rats were maintained in a temperature-controlled room (25 ± 1 °C) with a 12:12 dark-light cycle. Virgin female rats (200–220 g) were mated and each female was placed in an individual cage with free access to water and food until parturition. The use of the animals according our experimental design was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro (CEUA/060/2011). At birth, 30 lactating rats were randomly assigned to each of the following groups: (1) control (C, n = 10), with free access to a diet containing 7% lipid from soybean oil; (2) hyper soybean oil (HS, n = 10), with free access to a diet containing 19% lipid exclusive from soybean oil; and (3) hyper flaxseed oil (HF, n = 10), with free access to a diet containing 19% lipid (2% from soybean oil and 17% from flaxseed oil) Table 1. The soybean oil presented 54% of linoleic acid (n-6) and 8% of alpha-linolenic acid (n-3) showing n-6/n3 as 7:1, and the flaxseed oil presented 16% of linoleic acid (n-6) and 57% of alpha-linolenic acid (n-3) showing n-6/n3 as
Table 1 Composition of 100 g of diet during lactation. Ingredients (%)
Control
a
c d e f g
2.1. Nutritional evaluation Maternal food intake (FI) and body mass (BM) as well as BM of the offspring were daily monitored during lactation. The maternal cumulative FI was calculated through the summation of the daily mother’s food intake and the caloric density of the diets was calculated considering 4 kcal/g of protein, 4 kcal/g of carbohydrate and 9 kcal/g of lipid.
2.2. Body composition Visceral fat mass (VFM) was evaluated weighing the retroperitoneal, mesenteric, and around the uterus and ovaries or testis fat depots. The carcasses 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 were used to determine fat mass content gravimetrically. 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, tubes were weighed and results were expressed as% fat. The subcutaneous fat mass was estimated from the total fat mass minus the visceral fat mass, and the results were expressed as percentages. The total protein concentrations were determined by the Lowry method. Data were expressed as g protein/100 g carcass (Figueiredo et al., 2009).
2.3. Milk composition Milk samples were obtained on 14 and 20 days of lactation. Mothers were separated from their pups for 2 h and were injected with oxytocin (5 UI/ml sc – Eurofarm, São Paulo, SP, Brazil). After 30 min, dams were lightly anesthetized with pentobarbital and milk was extracted manually from the thoracic and abdominal teats. All milk samples were analyzed for lactose, total protein, total cholesterol and triglycerides. Lactose was estimated by a colorimetric method using picric acid. Commercial lactose (Sigma, St. Louis, MO, USA) was used as a standard. Protein was estimated by a colorimetric method using bovine serum albumin (Sigma, St. Louis, MO, USA) as a standard. Total cholesterol and triglycerides were determined by an enzymatic and colorimetric method using a commercial kit (Bioclin, Belo Horizonte, MG, Brazil) (Troina et al., 2010).
Hyper soybean oil Hyper flaxseed oil
Caseina 20.00 20.00 Corn starchb 50.29 43.95 Sucrosec 10.00 10.00 Mineral mixa 3.50 3.50 Vitamin mixa 1.00 1.00 d Soybean oil 7.00 19.00 e Flaxseed oil – – f Fiber 5.00 – Choline bitartratee 0.25 0.25 l-Cystinee 0.30 0.30 Tert-Butylhydroquinoneg 0.0014 0.0014 Macronutrient composition (per 100 g of diet) Protein (g) 19.55 19.53 Carbohydrate (g) 61.14 50.28 Fat (g) 19.49 49.50 Total energy (kJ/100 g) 1486.80 1770.80 b
1:3. In the higher flaxseed oil diet we added 2% of soybean oil to attend the minimum amount of n-6 that has to be offer to growing animals according to the recommendation of the American Institute of Nutrition/AIN 93G (Reeves et al., 1993), since has explained above flaxseed oil present already n-6. The experimental and control diets were offered ad libitum and started at birth, which was defined as day 0 (d0) of lactation, and were ended at weaning (d21). After birth, the litters were adjusted to 4 males and 4 female for each dam. When the number of litter from one mother was not enough, we borrow from other randomly selected mother from the same group that gave birth on the same day. At weaning, the dams and 2 pups (1 male and 1 female) for each dam were killed with a lethal dose of pentobarbital (0.06 g/kg/b.w.); blood was collected by cardiac puncture, and the tissues adiposity compartments and carcass were analyzed.
20.00 43.95 10.00 3.50 1.00 2.00 17.00 – 0.25 0.30 0.0014 19.53 50.28 49.50 1770.80
M Cassab Comercio & Industria LTDA (São Paulo, SP, Brazil). Maisena, Unilever Best Foods Brasil LTDA (Mogi Guaçu, SP, Brazil). União (Rio de Janeiro, RJ, Brazil). Liza Cargil Agricultura LTDA (Mairinque, SP, Brazil). Pragsoluções Biociências LTDA (Jaú, SP, Brazil). Microcel, Blanver LTDA (Cotia, SP, Brazil). Vogler Ingredients (Eastman, USA).
2.4. Biochemical analysis The blood samples were centrifuged and the serum was separated to determine the lipid profile. Total cholesterol, triglycerides (TG) and HDL-c were analyzed using Biosystem Ò commercial test kits. LDL-c and VLDL-c were obtained using Friedewald calculations: LDL-c (mg/dl) = Total cholesterol (TG/5) HDL-c and VLDL-c (mg/dl) = TG/5. Glucose concentration was determined in blood samples from the tail vein of fasting rats using glucose oxidase reagent strips, and read in a reflectance glucosimeter (ACCU-CHEK Advantage; Roche Diagnostics, Mannheim, Germany). The results were expressed as mg/dl (Figueiredo et al., 2009). The insulin resistance index (IRI) was calculated (fasting insulin fasting glucose).
2.5. Hormone analysis Blood samples were centrifuged to obtain serum, which was individually kept at 20 °C until assay. All measurements were performed in one assay. Serum insulin levels were determined by radioimmunoassay (RIA), using a commercial kit (ImmuChem™ 125I, coated tube; ICN Biomedical Inc., Aurora, OH, USA) with an assay sensitivity of 0.1 ng/ml and an intra-assay coefficient of variance of 3.2%. Serum leptin levels were determined by RIA, using a commercial kit (Millipore Corporation, Billerica, MA, USA) with an assay sensitivity of 0.639 ng/ml and intraassay coefficient of variance of 5.7% (Figueiredo et al., 2009).
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D.S. Guarda et al. / Food and Chemical Toxicology 69 (2014) 69–75 2.6. Statistical analysis The data were analyzed by statistical program GraphPad Prism 5 and expressed the data are reported as the means ± SEM. The statistical significance was determined by One-way ANOVA and Newman Keuls as post-test, and considered statistically significant when p < 0.05.
mothers ( 54% and 63%, respectively, P < 0.0001) compared with the control group. At weaning, these effects persist (HS: 59% and 70%, P < 0.0001, HF: 62% and 64%, P < 0.0001). The lactose and protein content in the milk had no changes during lactation (Fig. 4). 3.4. Serum biochemical and hormonal analysis
3. Results 3.1. Nutritional evaluation The HS and HF mothers presented lower body weight from day four of lactation until weaning (approximately 16%, at weaning, P = 0.001) (Fig. 1A). The mothers from HS and HF groups presented transitory changes in food intake during lactation (Fig. 1B). The cumulative food intake were lower (approximately 24%, P = 0.0001) in HS and HF groups when compared with the C mothers, but since the HS and HF diets had 16% higher total energy/mass there was no significant change in food intake caloric density between the groups during lactation. The male (P = 0.01) and female (P = 0.01) pups from HS and HF mothers had lower body weights than control offspring during lactation, as shown in Fig. 2A and B.
We observed lower total cholesterol ( 18%, P = 0.041) and HDLc ( 16%, P = 0.048) in serum of mothers from HS group (Table 2). No changes were observed in others biochemical and hormonal parameters among groups. At 21 days old, male and female offspring from HF group presented lower serum VLDL-c ( 41%, P = 0.05 and 51%, P = 0.025 respectively) and TG ( 41%, P = 0.013 and 50%, P = 0.018 respectively) (Tables 3 and 4). The HF male and female offspring presented hypoinsulinemia ( 23%, P = 0.026 and 27%, P = 0.0003 respectively), but no changes were observed in glycemia. Thus, the HF male and female offspring showed lower IRI ( 29%, P = 0.042 and 43%, P < 0.0001 respectively, p < 0.05) (Tables 3 and 4). There were no changes in leptinemia.
3.2. Body composition
4. Discussion
The mothers from HF group during lactation presented lower body fat mass ( 32%, P = 0.038) and lower visceral fat mass ( 47%, P = 0.034) compared with the control group (Fig. 1C and D). No differences were observed in mother’s body protein mass among the groups. The HF male offspring presented lower body and subcutaneous fat mass ( 20%, P = 0.043 and 20%, P = 0.042, respectively) and no changes in visceral fat mass and body protein mass (Fig. 3). No changes were observed in female body protein and fat mass at weaning.
This study showed for the first time that supplementation of a hyperlipidic flaxseed oil diet during lactation produces lower triglycerides and greater insulin sensitivity than the same proportion of soybean oil diet in male and female offspring. These findings are similar when we used whole flaxseed at the same period (Figueiredo et al., 2009). Those animals developed insulin resistance when adults. Thus, the present finding is important to indicate that flaxseed oil can be responsible for the changes in insulin sensitivity caused by whole flaxseed in the weaning period, which potentially can program the insulin resistance at adulthood. Flaxseed oil was supplemented in the diet because the compound has gained popularity with regard to bioactive compounds in functional foods that promote beneficial effects on health and their therapeutic properties in preventing diseases such as obesity
3.3. Milk composition At 14 days of lactation we observed lower TG and cholesterol in the milk from HS ( 26% and 60%, respectively, P < 0.0001) and HF
(A)
(B)
(D)
Body Fat Mass (%)
10 8
*
6 4 2 0 C
HS
HF
Visceral Fat Mass (%)
(C)
2.5 2.0
*
1.5 1.0 0.5 0.0 C
HS
HF
Fig. 1. Body weight and food intake during lactation and total and visceral body fat mass of dams at weaning. (A) Body weight of dams during lactation that were fed control (j), hyper soybean oil (N) and hyper flaxseed oil (s) diet. (B) Food Intake of mothers during lactation that were fed control (j), hyper soybean oil (N) and hyper flaxseed oil (s) diet. (C) Body fat mass of dams during lactation. (D) Visceral Fat mass during lactation. Values are means for 10 mothers/group during lactation. Mean values were significantly different to those of control animals: p < 0.05.
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Fig. 2. Body weight of male and female pups during lactation from dams that were fed control (j), hyper soybean oil (N) and hyper flaxseed oil (s) diet. (A) Body weight of male pups during lactation. (B) Body weight of female pups during lactation. Values are means for 4 pups male and 4 pups female from each mothers/group during lactation. Mean values were significantly different to those of control animals: p < 0.05.
(B) Subcutaneous fat mass (%)
(A) Body Fat Mass (%)
20 15
*
10 5 0 C
HS
15
* 10
5
0
HF
C
HS
HF
Fig. 3. Body fat composition of male offspring at weaning. (A) Body fat mass of male offspring at weaning. (B) Subcutaneous fat mass of male at weaning. Values are means for 10 pups male from each mothers/ group during lactation. Mean values were significantly different to those of control animals: p < 0.05.
C HS HF
100
50
0 14 days
Triacylglycerol (mg/ml)
Lactose milk content (mg/ml)
(B)
200
100 50
C HS HF
*
#
*
*
0 14 days
30 20 10 0 14 days
(C)
150
C HS HF
40
20 days
20 days
Cholesterol milk content (mg/dl)
Milk protein content (mg/ml)
(A) 150
20 days
(D) 1500
C HS HF
1000
500
*
*
*
*
0 14 days
20 days
* HS vs C # HF vs C and HS Fig. 4. Biochemical composition concentration in milk on days 14 and 20 days of lactation. (A) Protein content. (B) Lactose content. (C) Triglycerides content and (D) cholesterol content in milk on days 14 and 20 days of lactation. Values are means for 10 dams/group during lactation. Mean values were significantly different to those of control animals: p < 0.05.
and dyslipidemia (Oomah, 2001). According to Baranowski et al. (2012), dietary intervention with ALA-rich flaxseed oil in obese Zucker rats reduced adipocyte hypertrophy, protein levels of inflammatory markers MCP-1 and TNF-a, and T-cell infiltration
in adipose tissue. Thus, those results suggest that, due to its ability to improve adipocyte function, ALA-rich flaxseed oil confers health benefits in obesity. However, here we showed that the use during lactation is not without risk.
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D.S. Guarda et al. / Food and Chemical Toxicology 69 (2014) 69–75 Table 2 Biochemical and hormonal parameters from dams at weaning. Control
Glycemia (mg/dl) Insulin (lUI/ml) IRI Leptin (ng/ml) Total Cholesterol (mg/dl) HDL-c (mg/dl) VLDL-c (mg/dl) Triglycerides (mg/dl)
Hyper soybean oil
Hyper flaxseed oil
Mean
SEM
Mean
SEM
Mean
SEM
82.56 40.29 286.00 15.47 54.90 40.20 12.00 59.78
3.78 2.74 13.80 1.46 2.84 1.53 1.06 5.16
78.50 36.87 235.80 15.32 44.80* 33.80* 10.44 51.89
3.41 2.54 12.82 0.90 2.75 1.56 0.97 4.33
84.50 35.79 236.0 14.38 49.89 39.90 12.78 63.22
1.59 2.73 17.66 0.77 2.45 2.59 1.10 5.58
Values are expressed as means and SEM of 10 animals per group. HS vs. C p < 0.05.
*
Table 3 Biochemical and hormonal parameters in male offspring at weaning. Control
Glycemia (mg/dl) Insulin (lUI/ml) IRI Leptin (ng/ml) Total Cholesterol (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL-c (mg/dl) Triglycerides (mg/dl)
Hyper Soybean Oil
Hyper Flaxseed Oil
Mean
SEM
Mean
SEM
Mean
SEM
81.9 19.10 126.1 13.44 105.3 43.5 46.25 30.63 132.9
2.26 1.44 11.34 1.01 6.29 2.68 8.31 5.00 17.13
87.05 17.10 115.4 14.58 105.4 42.20 42.00 24.29 121.9
1.93 0.56 6.58 1.50 3.96 1.00 4.37 2.39 11.83
85.63 14.77* 90.00* 12.71 99.80 42.10 40.90 18.00* 79.38*
3.33 0.78 6.08 0.74 5.44 2.12 3.46 2.59 7.14
Values are expressed as means and SEM of 10 animals per group from each dam. HF vs. C p < 0.05.
*
Table 4 Biochemical and hormonal parameters in female offspring at weaning. Control
Glycemia (mg/dl) Insulin (lUI/ml) IRI Leptin (ng/ml) Total Cholesterol (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL-c (mg/dl) Triglycerides (mg/dl)
Hyper soybean oil
Hyper flaxseed oil
Mean
SEM
Mean
SEM
Mean
SEM
81.90 18.87 147.50 14.54 106.20 45.00 43.75 22.89 98.38
1.62 0.77 9.23 1.40 5.09 2.82 6.89 4.10 15.16
86.05 16.77 109.0# 15.33 96.80 44.80 39.63 15.78 78.22
2.47 0.66 4.94 1.29 7.56 2.90 6.92 2.43 12.02
81.95 13.88* 84.14* 13.47 92.90 42.00 39.60 11.30* 49.56*
3.75 0.75 4.69 0.65 4.51 2.14 3.09 1.60 4.35
Values are expressed as means ± SEM of 10 animals per group from each dam. HF vs. C p < 0.05. # HS vs. C p < 0.05.
*
The diets supplemented with soybean and flaxseed oil reduced the mother’s body weight during lactation, despite a less marked change in food intake, including with no changes in caloric intake, because the hyperlipidic diet is more energetic per gram of food. Since the food efficiency seems to be lower in HS and HF groups, it is possible that the hyperlipidic diet increases the metabolic rate or reduces adipogenesis. If the rats change its mobility, it could increase their metabolic rate. However, there is no study demonstrating changes in rat movement after flaxseed intake or its components. Curiously, rats that received 25% of flaxseed in diet are more manageable and less stressed at the end of treatment (Meneses et al., 2011). In fact, Pérez-Matute et al. (2007) have demonstrated that rats fed with the cafeteria diet and orally treated with omega-3 (1 g/kg) for 5 weeks showed a marginally lower body-weight gain, a decrease in food intake and an increase in leptin production, reduced retroperitoneal adipose tissue weight, which could be secondary to the inhibition of the adipogenic transcription factor PPAR-gamma gene expression, and also to the
increase in adipocyte apoptosis. Thus, it seems that those effects were not due to differences in the n-3/n-6 ratios in the diets, but due only to the higher lipidic proportion on the diet. However, when we administered diets supplemented with 25% of flaxseed, the dams presented no significant changes in body weight and food intake (Figueiredo et al., 2009, 2011, 2012). In another study, the consumption of SDG (lignan) and flaxseed oil + SDG did not alter food intake or body weight of lactating rats (Troina et al., 2012). In these two previous studies, the proportion of oil was normal, around 7%, while in the present study was higher (19%). Thus, hyperlipidic diets even rich in PUFAs seem to reduce body weight and food intake, with a higher effect upon body weight. In the previous study, the combination of SDG to 7% flaxseed oil attenuated the lipogenic effect of SDG (Troina et al., 2012). The HF dams presented lower body fat mass and lower visceral fat mass confirming the effects of n-3 to reduce body fat mass, since HS did not present any change in fat mass. Thus, contrary with
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observed for body mass, the n-3/n-6 ratio seems to be important for the body fat mass regulation. The milk from both HS and HF dams showed lower TG and cholesterol contents on day 14 and 20 of lactation. Troina et al. (2012), observed no differences in lipid content in the milk from the group that received 7% flaxseed oil + SDG at the same two periods of lactation. Also, Yonekubo et al. (1993) showed no significant changes in protein, fat, mineral and total solid contents of the milk between the control and the group that received a diet with 7% fish oil. Thus, it seems that the higher oil proportion is the main cause of lower TG and cholesterol milk content. Priego et al. (2013), showed that maternal dietary source of fat affects milk fat acid composition and circulating fat acid profile, as could be expected, but also body weight gain and thermogenic capacity of offspring during the suckling period. HF and HS male and female offspring had lower body mass. This decrease may be related to the lower lipid content in the milk from the HS and HF mothers. The findings of lower body mass confirm others studies in the female offspring using total flaxseed during pregnancy and/or lactation (Tou et al., 1998; Collins et al., 2003; Troina et al., 2010). When we analyzed the body composition, we observed lower body and subcutaneous fat mass only in male HF offspring and no changes in female offspring at weaning, contrary the reduction in fat mass in female offspring whose mother received total flaxseed (Troina et al., 2010). However, in male rats, Figueiredo et al. (2009) studying the whole flaxseed effects showed also a lower body fat mass, but an increase in body mass. It seems that these differences may be explained by the fact that the oil alone, in the present study, or part of the whole flaxseed has an important effect decreasing fat mass, but other flaxseed component may increase the lean mass, only in the male. Soybean oil has no effect upon the offspring fat mass, suggesting a specific effect of n3-n6 ratio. Essential n-3 fatty acids in flaxseed have been associated with a reduction of cholesterol and TG levels (Morris, 2007). The supplementation of flaxseed oil during lactation, in the present study reduced the serum levels of VLDL-c and TG in male and female offspring at weaning. So, it seems that the n-3 fatty acid present in the maternal diet lower offspring TG and cholesterol, because there is a lower milk transfer or n-3 fatty acid transfer through the milk acts on the pups lipid metabolism. This data corroborates with our previous study that had the diet supplemented with 7% flaxseed oil + SDG during lactation, which observed lower serum TG in male and female offspring at weaning (Troina et al., 2012). Also total flaxseed supplemented to the mothers during lactation caused a similar reduction in offspring TG and cholesterol both in males and female (Figueiredo et al., 2009; Troina et al., 2010). Thus, it seems that the lowering in TG and cholesterol is a flaxseed oil effect, since soybean oil even in this higher proportion (19%) did not changed pups serum TG and cholesterol. The insulinemia was lower in HF male and female offspring at weaning, despite no changes in glycemia. We suggest that in this case, flaxseed oil was able to increase insulin sensitivity. According to Chechi et al. (2010), a spontaneously hypertensive rats (SHR) that received a diet supplemented with 10% of flaxseed oil (10%) for 4 weeks presented lower fasting insulin, lower hepatic TG and cholesterol concentrations and a reduction in hepatic mRNA expression of PPAR-gama, which suggest that the flaxseed oil may activate the PPAR-gama dependent pathway to alter the liver lipid metabolism and to increase insulin sensitivity in SHR rats that become obese with this moderately hyperlipidic diet. Another oil rich in n-3 is the conjugated linolenic acid (CLA) that also improved insulin sensitivity in fa/fa rats. Furthermore, CLA-fed fa/fa rats had decrease perimeter and area of islet cells, suggesting pancreatic preservation by CLA. The authors associate this pancreatic preservation with the reduction in pancreatic insulin production as
indicated by the smaller islet size and lower serum insulin and C-peptide concentrations (Noto et al., 2007). By the contrary, a higher canola oil diet was associated with higher HOMA-IR, but also with a decrease in pancreatic islet size (Costa et al., 2013). Troina et al. (2012) observed that the glycemia and insulinemia were not modified with the consumption of 7% flaxseed oil + SDG during lactation in both male and female offspring. On the other hand, Figueiredo et al. (2009), showed at weaning normal fasting glycemia and hypoinsulinemia in the male offspring from the dams that received whole flaxseed diet during lactation. Also, other flaxseed component, maybe the fibers had a synergistic effect with the oil that allowed that even with a lower oil concentration (around 7%) this effect upon insulin sensitivity occurs. Thus, it seems that not only the presence of a higher n3-n6 ratio is important for improve insulin sensitivity, but also the total amount of n-3, since 19% oil had an effect that was not observed with 7% concentration. The reduction of body fat mass showed in the HF offspring may be one of the main causes to increase the insulin sensitivity in both genders. Figueiredo et al. (2009) showed hyperleptinemia in the male pups from the group that the mothers received whole flaxseed during lactation. It is known that leptin increases insulin sensitivity, despite decrease insulin secretion (Kieffer and Habener, 2000). Unexpectedly, in the present study, no changes were observed in serum leptin in dams and offspring at weaning, even with the lower body fat mass. However, leptin may be produced in other tissues, mainly muscle, as already showed in a preview study of our group, where there was an increase in the amount of leptin in skeletal muscle (Trevenzoli et al., 2010). Thus, we suppose that flaxseed oil could induce leptin production in extra-adipose tissues. Paz-Filho et al. (2009), have demonstrated that when the leptin levels were adjusted for body fat mass they were significantly lower in obese hyperleptinemic patients, and less leptin might be available to the peripheral tissues, which may predispose to lipotoxicity and the development of comorbidities associated with metabolic syndrome, such as liver steatosis, insulin resistance, dyslipidemia and beta cell failure. Thus, in the present study, even normoleptinemic, HF group may have a higher leptin action, because leptin/fat mass ratio is higher. Thus, it is possible that flaxseed oil may have a direct effect on leptin production that through leptin better action improves insulin sensitivity and decreases visceral fat mass. 5. Conclusions According to our findings, the higher intake of flaxseed oil during lactation reduced the body weight of mothers during lactation and changes the milk composition. The reduction of cholesterol and triglycerides in milk in both periods of lactation can be responsible for the reduction of pup’s body weight during lactation and changes in lipid profile in male and female at weaning. The lower offspring fat mass can contribute to the higher insulin sensitivity, without discard an indirect effect of n-3 fatty acids on leptin production that improves insulin sensitivity. The higher insulin sensitivity in critical periods of life may imprint the pup future metabolic, hormonal and neural changes. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
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Acknowledgments The authors are grateful to Mr. Ulisses Siqueira and Miss Monica Moura for their technical assistance. Research was supported by the National Council for Scientific and Technological Development (CNPq), the State of Rio de Janeiro Carlos Chagas Filho Research Foundation (FAPERJ) and Coordination for the Enhancement of Higher Education Personnel (CAPES). D.S.G. was a recipient of a CNPq fellowship. The authors E.G.M. and M.S.F. designed the study and wrote the manuscript. P.C.L. and E.O. revised the manuscript and analyzed the data. D.S.G. and M.S.F. were responsible for animal programming, biochemical and hormonal analysis. J.F.N. performed lipids measurements. All authors contributed to and approved the final manuscript. References Baranowski, M., Enns, J., Blewett, H., Yakandawala, U., Zahradka, P., Taylor, C.G., 2012. Dietary flaxseed oil reduces adipocyte size, adipose monocyte chemoattractant protein-1 levels and T-cell infiltration in obese, insulinresistant rats. Cytokine 59, 382–391. Carter, J., 1993. Flaxseed as a source of alpha linolenic acid. J. Am. Coll. Nutr. 12, 551. Chechi, K., Yasui, N., Ikeda, K., Yamori, Y.K., Cheema, S., 2010. Flax oil-mediated activation of PPAR-c correlates with reduction of hepatic lipid accumulation in obese spontaneously hypertensive/NDmcr-cp rats, a model of the metabolic syndrome. Br. J. Nutr. 104, 1313–1321. Collins, T.F.X., Sprando, R.L., Black, T.N., Olejnik, N., Wiesenfeld, P.W., Babu, U.S., Bryant, M., Flynn, T.J., Ruggles, D.I., 2003. Effects of flaxseed and defatted flaxseed meal on reproduction and development in rats. Food Chem. Toxicol. 41, 819–834. Costa, C.A., Carlos, A.S., Gonzalez, Gde. P., Reis, R.P., Ribeiro, M. dos S., Dos Santos, A. de S., Monteiro, A.M., de Moura, E.G., Nascimento-Saba, C.C., 2012. Diet containing low n-6/n-3 polyunsaturated fatty acids ratio, provided by canola oil, alters body composition and bone quality in young rats. Eur. J. Nutr. 51, 191–198. Costa, C.A., Carlos, A.S., de Sousa Dos Santos, A., de Moura, E.G., Nascimento-Saba, C.C., 2013. High-fat diets containing soybean or canola oil affect differently pancreas function of young male rats. Horm. Metab. Res. 45, 652–654. Cunnane, S.C., Hamedeh, M.J., Liede, A.C., Thompson, L.U., Wolever, T.M., Jenkins, D.J., 1995. Nutritional attributes of traditional flaxseed in healthy young adults. Am. J. Clin. Nutr. 61, 62–68. Figueiredo, M.S., de Moura, E.G., Lisboa, P.C., Troina, A.A., Trevenzoli, I.H., Oliveira, E., Boaventura, G.T., da Fonseca Passos, M.C., 2009. Flaxseed supplementation of rats during lactation changes the adiposity and glucose homeostasis of their offspring. Life Sci. 85, 365–371. Figueiredo, M.S., de Moura, E.G., Lisboa, P.C., Troina, A.A., Trevenzoli, I.H., Oliveira, E., Boaventura, G.T., da Fonseca Passos, M.C., 2011. Maternal flaxseed diet during lactation programs thyroid hormones metabolism and action in the male adult offspring in rats. Horm. Metab. Res. 43, 410–416. Figueiredo, M.S., Passos, M.C.F., Trevenzoli, I.H., Troina, A.A., Carlos, A.S., Alves Nascimento-Saba, C.C., Fraga, M.C., Manhães, A.C., de Oliveira, E., Lisboa, P.C., de Moura, E.G., 2012. Adipocyte morphology and leptin signaling in rat offspring from mothers supplemented with flaxseed during lactation. Nutrition 28, 307– 315.
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Jenkins, D.J.A., Kendall, C.W.W., Vidgen, E., Agarwal, S., Rao, A.V., Rosenberg, R.S., Diamandis, E.P., Novokmet, R., Mehling, C.C., Perera, T., Griffin, L.C., Cunnane, S.C., 1999. Health aspects of partially defatted flaxseed, including effects on serum lipids, oxidative measures, and ex vivo androgen and progestin activity: a controlled crossover trial. Am. J. Clin. Nutr. 69, 395–402. Kieffer, T.J., Habener, J.F., 2000. The adipoinsular axis effects of leptin on pancreatic beta-cells. Am. J. Physiol. Endocrinol. Metab. 278, E1–E14. Meneses, J.A., Junqueira Lopes, C.A., Coca Velarde, L.G., Boaventura, G.T., 2011. Behavioral analysis of Wistar rats fed with a flaxseed based diet added to an environmental enrichment. Nutr. Hosp. 26, 716–721. Morris, D.H., 2007. Focus: Flax – A health and nutrition primer. Flax Council of Canada. (accessed May 2009). Noto, A., Zahradka, P., Ryz, N.R., Yurkova, N., Xie, X., Taylor, C.G., 2007. Dietary conjugated linoleic acid preserves pancreatic function and reduces inflammatory markers in obese, insulin-resistant rats. Metabol. Clin. Exp. 56, 142–151. Oomah, B.D., 2001. Flaxseed as a functional food source. J. Sci. Food Agric. 81, 889– 894. Paz-Filho, G.J., Volaco, A., Suplicy, H.L., Radominski, R.B., Boguszewski, C.L., 2009. Decrease in leptin production by the adipose tissue in obesity associated with severe metabolic syndrome. Arq. Bras. Endocrinol. Metabol. 53, 1088–1095. Pérez-Matute, P., Pérez-Echarri, N., Martínez, J.A., Marti, A., Moreno-Aliaga, M.J., 2007. Eicosapentaenoic acid actions on adiposity and insulin resistance in control and high-fat-fed rats: role of apoptosis, adiponectin and tumour necrosis factor-alpha. Br. J. Nutr. 97, 389–398. Prasad, K., 2008. Regression of hypercholesterolemic atherosclerosis in rabbits by secoisolariciresinol diglucoside isolated from flaxseed. Atherosclerosis 197, 34– 42. Priego, T., Sánchez, J., García, A.P., Palou, A., Picó, C., 2013. Maternal dietary fat affects milk fatty acid profile and impacts on weight gain and thermogenic capacity of suckling rats. Lipids 48, 481–495. Reeves, P.G., Nielsen, F.H., Fahey Jr., G.C., 1993. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76 rodent diet. J. Nutr. 123, 1939– 1951. Takahashi, Y., Ide, T., 2000. Dietary n-3 fatty acids affect mRNA level of brown adipose tissue uncoupling protein 1, and white adipose tissue leptin and glucose transporter 4 in the rat. Br. J. Nutr. 84, 175–184. Tou, J.C.L., Chen, J., Thompson, L.U., 1998. Flaxseed and its lignan precursor, secoisolariciresinol diglycoside, affect pregnancy outcome and reproductive development in rats. J. Nutr. 128, 861–868. Trevenzoli, I.H., Rodrigues, A.L., Oliveira, E., Thole, A.A., Carvalho, L., Figueiredo, M.S., Toste, F.P., Neto, J.F., Passos, M.C., Lisboa, P.C., Moura, E.G., 2010. Leptin treatment during lactation programs leptin synthesis, intermediate metabolism, and liver microsteatosis in adult rats. Horm. Metab. Res. 42, 483–490. Troina, A.A., Figueiredo, M.S., Moura, E.G., Boaventura, G.T., Soares, L.L., Cardozo, L.F., Oliveira, E., Lisboa, P.C., Passos, M.A., Passos, M.C., 2010. Maternal flaxseed diet during lactation alters milk composition and programs the offspring body composition, lipid profile and sexual function. Food Chem. Toxicol. 48, 697–703. Troina, A.A., Figueiredo, M.S., Passos, M.C., Reis, A.M., Oliveira, E., Lisboa, P.C., Moura, E.G., 2012. Flaxseed bioactive compounds change milk, hormonal and biochemical parameters of dams and offspring during lactation. Food Chem. Toxicol. 50, 2388–2396. Yonekubo, A., Honda, S., Okano, M., Takahashi, K., Yamamoto, Y., 1993. Dietary fish oil alters rat milk composition and liver and brain fatty acid composition of fetal and neonatal rats. J. Nutr. 123, 1703–1708.
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Flaxseed oil during lactation changes milk and body composition in male and female suckling pups rats Deysla Sabino Guarda a, Patricia Cristina Lisboa a, Elaine de Oliveira a, José Firmino Nogueira-Neto b, Egberto Gaspar de Moura a,⇑, Mariana Sarto Figueiredo a a b
Department of Physiological Sciences, Laboratory of Endocrine Physiology, Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, RJ 20551-030, Brazil Lipids Laboratory, Medical School, State University of Rio de Janeiro, Rio de Janeiro, RJ 20551-030, Brazil
a r t i c l e
i n f o
Article history: Received 9 November 2013 Accepted 2 April 2014 Available online 12 April 2014 Keywords: Flaxseed oil Lactation Milk Body composition
a b s t r a c t We have reported several changes in neonate or adult offspring after the maternal use of whole flaxseed or its components. However, it is unknown the use of higher oil intake in the neonatal period. Here we evaluated the effects of high maternal intake of flaxseed oil during lactation upon milk and body composition in male and female offspring. Lactating rats were divided into: (1) control (C, n = 10), 7% soybean oil; (2) hyper 19% soybean oil (HS, n = 10); and (3) hyper 17% flaxseed oil + 2% soybean oil (HF, n = 10). Dams and offspring were killed at weaning. HS and HF dams, male and female offspring presented lower body weight during lactation. HF mothers presented lower body and visceral fat masses. HF male offspring presented lower body and subcutaneous fat masses. HS and HF milk presented lower triglycerides (TG) and cholesterol. HF male and female offspring showed lower triglyceridemia and insulinemia, but no changes in glycemia and leptinemia. The higher intake of flaxseed oil during lactation reduced the body weight of mothers and offspring, decreases milk lipids and apparently increases insulin sensitivity in this critical period of life. Those changes may explain the previously reported programming effect of maternal flaxseed intake during lactation. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Functional foods are those that beyond basic nutritional functions can produce beneficial health effects and should be safe for consumption without medical supervision. Flaxseed is the seed from the flax plant (Linum usitatissimum L), called linseed, which is a member of the Linaceae family (Oomah, 2001). Flaxseed contains 32–45% of its mass as oil, of which 51–55% is alpha-linolenic acid (ALA, which belongs to omega-3 family) and 15–18% is linoleic acid (omega-6) (Carter, 1993). The ratio of omega-3/omega-6 seems to be an important factor to lipid and glucose homeostasis. Flaxseed and canola oil are rich in omega-3 and lower in omega-6, while soybean oil that is less expensive and because of this, highly used by poor population had a lower omega-3/omega-6 ratio. Beside this, there is a trend of increase the proportion of vegetable oil on the diet (Takahashi and Ide, 2000). Flaxseed presented potential health benefits associated with decrease in the risk of cardiovascular disease (Cunnane et al., ⇑ Corresponding author. Address: Departamento de Ciências Fisiológicas, 5o andar, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Rio de Janeiro, RJ 20550-030, Brazil. Tel./fax: +55 21 28688334. E-mail address: [email protected] (E.G. de Moura). http://dx.doi.org/10.1016/j.fct.2014.04.003 0278-6915/Ó 2014 Elsevier Ltd. All rights reserved.
1995; Jenkins et al., 1999). Several functional properties of flaxseed have been reported including antioxidant activity and the ability to lower blood glucose, serum total cholesterol, LDL-c and triacylglycerol while increasing serum HDL-c (Prasad, 2008). Based in the recommendation to increase the use of oils rich in omega-3, there is an increase in consumption by the population. However, this could lead to an overconsumption of a hyperlypidic diet, which health risk is not completely evaluated. Recently, it was compared the differences in the effects of canola vs. soybean oil in a normal proportion (7%) or hyperlipidic proportion (19%) (Costa et al., 2012, 2013). It is interesting that in the normal proportion canola oil decreases abdominal fat and increases insulin sensitivity, but in the higher proportion canola oil is worse than soybean oil both in relation to obesity and insulin resistance. Some authors, including our previous data have suggested caution when flaxseed is consumed during pregnancy and lactation (Tou et al., 1998; Troina et al., 2010). Pups whose mothers consumed dietary flaxseed (10%) during gestation and lactation presented lower body weight at birth (Tou et al., 1998). At weaning, the male offspring, whose mothers received whole dietary flaxseed (25%) showed higher body mass but lower body fat mass, lower serum total cholesterol and triglycerides, hyperleptinemia,
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hypoinsulinemia and higher insulin sensitivity, lower serum triiodothyronine (T3) and higher thyrotropin (TSH) (Figueiredo et al., 2009). In another study we reported that the female offspring at weaning showed similar phenotype, presenting lower total fat mass, lower visceral fat mass, lower serum total cholesterol and triglycerides and higher serum leptin, but contrary to the higher body mass observed in the male, the female also presented lower body mass (Troina et al., 2010). Since, the main components of flaxseed is the oil and secoisolariciresinol diglucoside (SDG), we showed in another study, the effects of maternal supplementation with SDG alone or SDG plus 7% flaxseed oil during lactation. In the mother, SDG treatment caused a higher total fat mass, while the oil reduced the fat mass. Also, the oil reduced serum triglycerides and cholesterol in the dams. In the offspring, a lower body fat mass was observed in the oil-treated group both in male and female. Serum triglyceride levels were also lower in both SDG and flaxseed oil + SDG groups, both in male and female (Troina et al., 2012). Based in the previous reports, we hypothesized that the overconsumption of flaxseed oil during lactation, instead to be health promoting, could be detrimental as was reported for canola oil (Costa et al., 2013). Thus in this study we aimed to evaluate the milk composition, body composition of dams and pups as well as serum leptin, glucose homeostasis and lipid profile. To our knowledge, this is the first study to adress the question of the use of hyperlipidic flaxseed diet during lactation over body composition, glucose homeostasis and lipid profile. 2. Materials and methods Three-month-old Wistar rats were maintained in a temperature-controlled room (25 ± 1 °C) with a 12:12 dark-light cycle. Virgin female rats (200–220 g) were mated and each female was placed in an individual cage with free access to water and food until parturition. The use of the animals according our experimental design was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro (CEUA/060/2011). At birth, 30 lactating rats were randomly assigned to each of the following groups: (1) control (C, n = 10), with free access to a diet containing 7% lipid from soybean oil; (2) hyper soybean oil (HS, n = 10), with free access to a diet containing 19% lipid exclusive from soybean oil; and (3) hyper flaxseed oil (HF, n = 10), with free access to a diet containing 19% lipid (2% from soybean oil and 17% from flaxseed oil) Table 1. The soybean oil presented 54% of linoleic acid (n-6) and 8% of alpha-linolenic acid (n-3) showing n-6/n3 as 7:1, and the flaxseed oil presented 16% of linoleic acid (n-6) and 57% of alpha-linolenic acid (n-3) showing n-6/n3 as
Table 1 Composition of 100 g of diet during lactation. Ingredients (%)
Control
a
c d e f g
2.1. Nutritional evaluation Maternal food intake (FI) and body mass (BM) as well as BM of the offspring were daily monitored during lactation. The maternal cumulative FI was calculated through the summation of the daily mother’s food intake and the caloric density of the diets was calculated considering 4 kcal/g of protein, 4 kcal/g of carbohydrate and 9 kcal/g of lipid.
2.2. Body composition Visceral fat mass (VFM) was evaluated weighing the retroperitoneal, mesenteric, and around the uterus and ovaries or testis fat depots. The carcasses 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 were used to determine fat mass content gravimetrically. 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, tubes were weighed and results were expressed as% fat. The subcutaneous fat mass was estimated from the total fat mass minus the visceral fat mass, and the results were expressed as percentages. The total protein concentrations were determined by the Lowry method. Data were expressed as g protein/100 g carcass (Figueiredo et al., 2009).
2.3. Milk composition Milk samples were obtained on 14 and 20 days of lactation. Mothers were separated from their pups for 2 h and were injected with oxytocin (5 UI/ml sc – Eurofarm, São Paulo, SP, Brazil). After 30 min, dams were lightly anesthetized with pentobarbital and milk was extracted manually from the thoracic and abdominal teats. All milk samples were analyzed for lactose, total protein, total cholesterol and triglycerides. Lactose was estimated by a colorimetric method using picric acid. Commercial lactose (Sigma, St. Louis, MO, USA) was used as a standard. Protein was estimated by a colorimetric method using bovine serum albumin (Sigma, St. Louis, MO, USA) as a standard. Total cholesterol and triglycerides were determined by an enzymatic and colorimetric method using a commercial kit (Bioclin, Belo Horizonte, MG, Brazil) (Troina et al., 2010).
Hyper soybean oil Hyper flaxseed oil
Caseina 20.00 20.00 Corn starchb 50.29 43.95 Sucrosec 10.00 10.00 Mineral mixa 3.50 3.50 Vitamin mixa 1.00 1.00 d Soybean oil 7.00 19.00 e Flaxseed oil – – f Fiber 5.00 – Choline bitartratee 0.25 0.25 l-Cystinee 0.30 0.30 Tert-Butylhydroquinoneg 0.0014 0.0014 Macronutrient composition (per 100 g of diet) Protein (g) 19.55 19.53 Carbohydrate (g) 61.14 50.28 Fat (g) 19.49 49.50 Total energy (kJ/100 g) 1486.80 1770.80 b
1:3. In the higher flaxseed oil diet we added 2% of soybean oil to attend the minimum amount of n-6 that has to be offer to growing animals according to the recommendation of the American Institute of Nutrition/AIN 93G (Reeves et al., 1993), since has explained above flaxseed oil present already n-6. The experimental and control diets were offered ad libitum and started at birth, which was defined as day 0 (d0) of lactation, and were ended at weaning (d21). After birth, the litters were adjusted to 4 males and 4 female for each dam. When the number of litter from one mother was not enough, we borrow from other randomly selected mother from the same group that gave birth on the same day. At weaning, the dams and 2 pups (1 male and 1 female) for each dam were killed with a lethal dose of pentobarbital (0.06 g/kg/b.w.); blood was collected by cardiac puncture, and the tissues adiposity compartments and carcass were analyzed.
20.00 43.95 10.00 3.50 1.00 2.00 17.00 – 0.25 0.30 0.0014 19.53 50.28 49.50 1770.80
M Cassab Comercio & Industria LTDA (São Paulo, SP, Brazil). Maisena, Unilever Best Foods Brasil LTDA (Mogi Guaçu, SP, Brazil). União (Rio de Janeiro, RJ, Brazil). Liza Cargil Agricultura LTDA (Mairinque, SP, Brazil). Pragsoluções Biociências LTDA (Jaú, SP, Brazil). Microcel, Blanver LTDA (Cotia, SP, Brazil). Vogler Ingredients (Eastman, USA).
2.4. Biochemical analysis The blood samples were centrifuged and the serum was separated to determine the lipid profile. Total cholesterol, triglycerides (TG) and HDL-c were analyzed using Biosystem Ò commercial test kits. LDL-c and VLDL-c were obtained using Friedewald calculations: LDL-c (mg/dl) = Total cholesterol (TG/5) HDL-c and VLDL-c (mg/dl) = TG/5. Glucose concentration was determined in blood samples from the tail vein of fasting rats using glucose oxidase reagent strips, and read in a reflectance glucosimeter (ACCU-CHEK Advantage; Roche Diagnostics, Mannheim, Germany). The results were expressed as mg/dl (Figueiredo et al., 2009). The insulin resistance index (IRI) was calculated (fasting insulin fasting glucose).
2.5. Hormone analysis Blood samples were centrifuged to obtain serum, which was individually kept at 20 °C until assay. All measurements were performed in one assay. Serum insulin levels were determined by radioimmunoassay (RIA), using a commercial kit (ImmuChem™ 125I, coated tube; ICN Biomedical Inc., Aurora, OH, USA) with an assay sensitivity of 0.1 ng/ml and an intra-assay coefficient of variance of 3.2%. Serum leptin levels were determined by RIA, using a commercial kit (Millipore Corporation, Billerica, MA, USA) with an assay sensitivity of 0.639 ng/ml and intraassay coefficient of variance of 5.7% (Figueiredo et al., 2009).
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D.S. Guarda et al. / Food and Chemical Toxicology 69 (2014) 69–75 2.6. Statistical analysis The data were analyzed by statistical program GraphPad Prism 5 and expressed the data are reported as the means ± SEM. The statistical significance was determined by One-way ANOVA and Newman Keuls as post-test, and considered statistically significant when p < 0.05.
mothers ( 54% and 63%, respectively, P < 0.0001) compared with the control group. At weaning, these effects persist (HS: 59% and 70%, P < 0.0001, HF: 62% and 64%, P < 0.0001). The lactose and protein content in the milk had no changes during lactation (Fig. 4). 3.4. Serum biochemical and hormonal analysis
3. Results 3.1. Nutritional evaluation The HS and HF mothers presented lower body weight from day four of lactation until weaning (approximately 16%, at weaning, P = 0.001) (Fig. 1A). The mothers from HS and HF groups presented transitory changes in food intake during lactation (Fig. 1B). The cumulative food intake were lower (approximately 24%, P = 0.0001) in HS and HF groups when compared with the C mothers, but since the HS and HF diets had 16% higher total energy/mass there was no significant change in food intake caloric density between the groups during lactation. The male (P = 0.01) and female (P = 0.01) pups from HS and HF mothers had lower body weights than control offspring during lactation, as shown in Fig. 2A and B.
We observed lower total cholesterol ( 18%, P = 0.041) and HDLc ( 16%, P = 0.048) in serum of mothers from HS group (Table 2). No changes were observed in others biochemical and hormonal parameters among groups. At 21 days old, male and female offspring from HF group presented lower serum VLDL-c ( 41%, P = 0.05 and 51%, P = 0.025 respectively) and TG ( 41%, P = 0.013 and 50%, P = 0.018 respectively) (Tables 3 and 4). The HF male and female offspring presented hypoinsulinemia ( 23%, P = 0.026 and 27%, P = 0.0003 respectively), but no changes were observed in glycemia. Thus, the HF male and female offspring showed lower IRI ( 29%, P = 0.042 and 43%, P < 0.0001 respectively, p < 0.05) (Tables 3 and 4). There were no changes in leptinemia.
3.2. Body composition
4. Discussion
The mothers from HF group during lactation presented lower body fat mass ( 32%, P = 0.038) and lower visceral fat mass ( 47%, P = 0.034) compared with the control group (Fig. 1C and D). No differences were observed in mother’s body protein mass among the groups. The HF male offspring presented lower body and subcutaneous fat mass ( 20%, P = 0.043 and 20%, P = 0.042, respectively) and no changes in visceral fat mass and body protein mass (Fig. 3). No changes were observed in female body protein and fat mass at weaning.
This study showed for the first time that supplementation of a hyperlipidic flaxseed oil diet during lactation produces lower triglycerides and greater insulin sensitivity than the same proportion of soybean oil diet in male and female offspring. These findings are similar when we used whole flaxseed at the same period (Figueiredo et al., 2009). Those animals developed insulin resistance when adults. Thus, the present finding is important to indicate that flaxseed oil can be responsible for the changes in insulin sensitivity caused by whole flaxseed in the weaning period, which potentially can program the insulin resistance at adulthood. Flaxseed oil was supplemented in the diet because the compound has gained popularity with regard to bioactive compounds in functional foods that promote beneficial effects on health and their therapeutic properties in preventing diseases such as obesity
3.3. Milk composition At 14 days of lactation we observed lower TG and cholesterol in the milk from HS ( 26% and 60%, respectively, P < 0.0001) and HF
(A)
(B)
(D)
Body Fat Mass (%)
10 8
*
6 4 2 0 C
HS
HF
Visceral Fat Mass (%)
(C)
2.5 2.0
*
1.5 1.0 0.5 0.0 C
HS
HF
Fig. 1. Body weight and food intake during lactation and total and visceral body fat mass of dams at weaning. (A) Body weight of dams during lactation that were fed control (j), hyper soybean oil (N) and hyper flaxseed oil (s) diet. (B) Food Intake of mothers during lactation that were fed control (j), hyper soybean oil (N) and hyper flaxseed oil (s) diet. (C) Body fat mass of dams during lactation. (D) Visceral Fat mass during lactation. Values are means for 10 mothers/group during lactation. Mean values were significantly different to those of control animals: p < 0.05.
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Fig. 2. Body weight of male and female pups during lactation from dams that were fed control (j), hyper soybean oil (N) and hyper flaxseed oil (s) diet. (A) Body weight of male pups during lactation. (B) Body weight of female pups during lactation. Values are means for 4 pups male and 4 pups female from each mothers/group during lactation. Mean values were significantly different to those of control animals: p < 0.05.
(B) Subcutaneous fat mass (%)
(A) Body Fat Mass (%)
20 15
*
10 5 0 C
HS
15
* 10
5
0
HF
C
HS
HF
Fig. 3. Body fat composition of male offspring at weaning. (A) Body fat mass of male offspring at weaning. (B) Subcutaneous fat mass of male at weaning. Values are means for 10 pups male from each mothers/ group during lactation. Mean values were significantly different to those of control animals: p < 0.05.
C HS HF
100
50
0 14 days
Triacylglycerol (mg/ml)
Lactose milk content (mg/ml)
(B)
200
100 50
C HS HF
*
#
*
*
0 14 days
30 20 10 0 14 days
(C)
150
C HS HF
40
20 days
20 days
Cholesterol milk content (mg/dl)
Milk protein content (mg/ml)
(A) 150
20 days
(D) 1500
C HS HF
1000
500
*
*
*
*
0 14 days
20 days
* HS vs C # HF vs C and HS Fig. 4. Biochemical composition concentration in milk on days 14 and 20 days of lactation. (A) Protein content. (B) Lactose content. (C) Triglycerides content and (D) cholesterol content in milk on days 14 and 20 days of lactation. Values are means for 10 dams/group during lactation. Mean values were significantly different to those of control animals: p < 0.05.
and dyslipidemia (Oomah, 2001). According to Baranowski et al. (2012), dietary intervention with ALA-rich flaxseed oil in obese Zucker rats reduced adipocyte hypertrophy, protein levels of inflammatory markers MCP-1 and TNF-a, and T-cell infiltration
in adipose tissue. Thus, those results suggest that, due to its ability to improve adipocyte function, ALA-rich flaxseed oil confers health benefits in obesity. However, here we showed that the use during lactation is not without risk.
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D.S. Guarda et al. / Food and Chemical Toxicology 69 (2014) 69–75 Table 2 Biochemical and hormonal parameters from dams at weaning. Control
Glycemia (mg/dl) Insulin (lUI/ml) IRI Leptin (ng/ml) Total Cholesterol (mg/dl) HDL-c (mg/dl) VLDL-c (mg/dl) Triglycerides (mg/dl)
Hyper soybean oil
Hyper flaxseed oil
Mean
SEM
Mean
SEM
Mean
SEM
82.56 40.29 286.00 15.47 54.90 40.20 12.00 59.78
3.78 2.74 13.80 1.46 2.84 1.53 1.06 5.16
78.50 36.87 235.80 15.32 44.80* 33.80* 10.44 51.89
3.41 2.54 12.82 0.90 2.75 1.56 0.97 4.33
84.50 35.79 236.0 14.38 49.89 39.90 12.78 63.22
1.59 2.73 17.66 0.77 2.45 2.59 1.10 5.58
Values are expressed as means and SEM of 10 animals per group. HS vs. C p < 0.05.
*
Table 3 Biochemical and hormonal parameters in male offspring at weaning. Control
Glycemia (mg/dl) Insulin (lUI/ml) IRI Leptin (ng/ml) Total Cholesterol (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL-c (mg/dl) Triglycerides (mg/dl)
Hyper Soybean Oil
Hyper Flaxseed Oil
Mean
SEM
Mean
SEM
Mean
SEM
81.9 19.10 126.1 13.44 105.3 43.5 46.25 30.63 132.9
2.26 1.44 11.34 1.01 6.29 2.68 8.31 5.00 17.13
87.05 17.10 115.4 14.58 105.4 42.20 42.00 24.29 121.9
1.93 0.56 6.58 1.50 3.96 1.00 4.37 2.39 11.83
85.63 14.77* 90.00* 12.71 99.80 42.10 40.90 18.00* 79.38*
3.33 0.78 6.08 0.74 5.44 2.12 3.46 2.59 7.14
Values are expressed as means and SEM of 10 animals per group from each dam. HF vs. C p < 0.05.
*
Table 4 Biochemical and hormonal parameters in female offspring at weaning. Control
Glycemia (mg/dl) Insulin (lUI/ml) IRI Leptin (ng/ml) Total Cholesterol (mg/dl) HDL-c (mg/dl) LDL-c (mg/dl) VLDL-c (mg/dl) Triglycerides (mg/dl)
Hyper soybean oil
Hyper flaxseed oil
Mean
SEM
Mean
SEM
Mean
SEM
81.90 18.87 147.50 14.54 106.20 45.00 43.75 22.89 98.38
1.62 0.77 9.23 1.40 5.09 2.82 6.89 4.10 15.16
86.05 16.77 109.0# 15.33 96.80 44.80 39.63 15.78 78.22
2.47 0.66 4.94 1.29 7.56 2.90 6.92 2.43 12.02
81.95 13.88* 84.14* 13.47 92.90 42.00 39.60 11.30* 49.56*
3.75 0.75 4.69 0.65 4.51 2.14 3.09 1.60 4.35
Values are expressed as means ± SEM of 10 animals per group from each dam. HF vs. C p < 0.05. # HS vs. C p < 0.05.
*
The diets supplemented with soybean and flaxseed oil reduced the mother’s body weight during lactation, despite a less marked change in food intake, including with no changes in caloric intake, because the hyperlipidic diet is more energetic per gram of food. Since the food efficiency seems to be lower in HS and HF groups, it is possible that the hyperlipidic diet increases the metabolic rate or reduces adipogenesis. If the rats change its mobility, it could increase their metabolic rate. However, there is no study demonstrating changes in rat movement after flaxseed intake or its components. Curiously, rats that received 25% of flaxseed in diet are more manageable and less stressed at the end of treatment (Meneses et al., 2011). In fact, Pérez-Matute et al. (2007) have demonstrated that rats fed with the cafeteria diet and orally treated with omega-3 (1 g/kg) for 5 weeks showed a marginally lower body-weight gain, a decrease in food intake and an increase in leptin production, reduced retroperitoneal adipose tissue weight, which could be secondary to the inhibition of the adipogenic transcription factor PPAR-gamma gene expression, and also to the
increase in adipocyte apoptosis. Thus, it seems that those effects were not due to differences in the n-3/n-6 ratios in the diets, but due only to the higher lipidic proportion on the diet. However, when we administered diets supplemented with 25% of flaxseed, the dams presented no significant changes in body weight and food intake (Figueiredo et al., 2009, 2011, 2012). In another study, the consumption of SDG (lignan) and flaxseed oil + SDG did not alter food intake or body weight of lactating rats (Troina et al., 2012). In these two previous studies, the proportion of oil was normal, around 7%, while in the present study was higher (19%). Thus, hyperlipidic diets even rich in PUFAs seem to reduce body weight and food intake, with a higher effect upon body weight. In the previous study, the combination of SDG to 7% flaxseed oil attenuated the lipogenic effect of SDG (Troina et al., 2012). The HF dams presented lower body fat mass and lower visceral fat mass confirming the effects of n-3 to reduce body fat mass, since HS did not present any change in fat mass. Thus, contrary with
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observed for body mass, the n-3/n-6 ratio seems to be important for the body fat mass regulation. The milk from both HS and HF dams showed lower TG and cholesterol contents on day 14 and 20 of lactation. Troina et al. (2012), observed no differences in lipid content in the milk from the group that received 7% flaxseed oil + SDG at the same two periods of lactation. Also, Yonekubo et al. (1993) showed no significant changes in protein, fat, mineral and total solid contents of the milk between the control and the group that received a diet with 7% fish oil. Thus, it seems that the higher oil proportion is the main cause of lower TG and cholesterol milk content. Priego et al. (2013), showed that maternal dietary source of fat affects milk fat acid composition and circulating fat acid profile, as could be expected, but also body weight gain and thermogenic capacity of offspring during the suckling period. HF and HS male and female offspring had lower body mass. This decrease may be related to the lower lipid content in the milk from the HS and HF mothers. The findings of lower body mass confirm others studies in the female offspring using total flaxseed during pregnancy and/or lactation (Tou et al., 1998; Collins et al., 2003; Troina et al., 2010). When we analyzed the body composition, we observed lower body and subcutaneous fat mass only in male HF offspring and no changes in female offspring at weaning, contrary the reduction in fat mass in female offspring whose mother received total flaxseed (Troina et al., 2010). However, in male rats, Figueiredo et al. (2009) studying the whole flaxseed effects showed also a lower body fat mass, but an increase in body mass. It seems that these differences may be explained by the fact that the oil alone, in the present study, or part of the whole flaxseed has an important effect decreasing fat mass, but other flaxseed component may increase the lean mass, only in the male. Soybean oil has no effect upon the offspring fat mass, suggesting a specific effect of n3-n6 ratio. Essential n-3 fatty acids in flaxseed have been associated with a reduction of cholesterol and TG levels (Morris, 2007). The supplementation of flaxseed oil during lactation, in the present study reduced the serum levels of VLDL-c and TG in male and female offspring at weaning. So, it seems that the n-3 fatty acid present in the maternal diet lower offspring TG and cholesterol, because there is a lower milk transfer or n-3 fatty acid transfer through the milk acts on the pups lipid metabolism. This data corroborates with our previous study that had the diet supplemented with 7% flaxseed oil + SDG during lactation, which observed lower serum TG in male and female offspring at weaning (Troina et al., 2012). Also total flaxseed supplemented to the mothers during lactation caused a similar reduction in offspring TG and cholesterol both in males and female (Figueiredo et al., 2009; Troina et al., 2010). Thus, it seems that the lowering in TG and cholesterol is a flaxseed oil effect, since soybean oil even in this higher proportion (19%) did not changed pups serum TG and cholesterol. The insulinemia was lower in HF male and female offspring at weaning, despite no changes in glycemia. We suggest that in this case, flaxseed oil was able to increase insulin sensitivity. According to Chechi et al. (2010), a spontaneously hypertensive rats (SHR) that received a diet supplemented with 10% of flaxseed oil (10%) for 4 weeks presented lower fasting insulin, lower hepatic TG and cholesterol concentrations and a reduction in hepatic mRNA expression of PPAR-gama, which suggest that the flaxseed oil may activate the PPAR-gama dependent pathway to alter the liver lipid metabolism and to increase insulin sensitivity in SHR rats that become obese with this moderately hyperlipidic diet. Another oil rich in n-3 is the conjugated linolenic acid (CLA) that also improved insulin sensitivity in fa/fa rats. Furthermore, CLA-fed fa/fa rats had decrease perimeter and area of islet cells, suggesting pancreatic preservation by CLA. The authors associate this pancreatic preservation with the reduction in pancreatic insulin production as
indicated by the smaller islet size and lower serum insulin and C-peptide concentrations (Noto et al., 2007). By the contrary, a higher canola oil diet was associated with higher HOMA-IR, but also with a decrease in pancreatic islet size (Costa et al., 2013). Troina et al. (2012) observed that the glycemia and insulinemia were not modified with the consumption of 7% flaxseed oil + SDG during lactation in both male and female offspring. On the other hand, Figueiredo et al. (2009), showed at weaning normal fasting glycemia and hypoinsulinemia in the male offspring from the dams that received whole flaxseed diet during lactation. Also, other flaxseed component, maybe the fibers had a synergistic effect with the oil that allowed that even with a lower oil concentration (around 7%) this effect upon insulin sensitivity occurs. Thus, it seems that not only the presence of a higher n3-n6 ratio is important for improve insulin sensitivity, but also the total amount of n-3, since 19% oil had an effect that was not observed with 7% concentration. The reduction of body fat mass showed in the HF offspring may be one of the main causes to increase the insulin sensitivity in both genders. Figueiredo et al. (2009) showed hyperleptinemia in the male pups from the group that the mothers received whole flaxseed during lactation. It is known that leptin increases insulin sensitivity, despite decrease insulin secretion (Kieffer and Habener, 2000). Unexpectedly, in the present study, no changes were observed in serum leptin in dams and offspring at weaning, even with the lower body fat mass. However, leptin may be produced in other tissues, mainly muscle, as already showed in a preview study of our group, where there was an increase in the amount of leptin in skeletal muscle (Trevenzoli et al., 2010). Thus, we suppose that flaxseed oil could induce leptin production in extra-adipose tissues. Paz-Filho et al. (2009), have demonstrated that when the leptin levels were adjusted for body fat mass they were significantly lower in obese hyperleptinemic patients, and less leptin might be available to the peripheral tissues, which may predispose to lipotoxicity and the development of comorbidities associated with metabolic syndrome, such as liver steatosis, insulin resistance, dyslipidemia and beta cell failure. Thus, in the present study, even normoleptinemic, HF group may have a higher leptin action, because leptin/fat mass ratio is higher. Thus, it is possible that flaxseed oil may have a direct effect on leptin production that through leptin better action improves insulin sensitivity and decreases visceral fat mass. 5. Conclusions According to our findings, the higher intake of flaxseed oil during lactation reduced the body weight of mothers during lactation and changes the milk composition. The reduction of cholesterol and triglycerides in milk in both periods of lactation can be responsible for the reduction of pup’s body weight during lactation and changes in lipid profile in male and female at weaning. The lower offspring fat mass can contribute to the higher insulin sensitivity, without discard an indirect effect of n-3 fatty acids on leptin production that improves insulin sensitivity. The higher insulin sensitivity in critical periods of life may imprint the pup future metabolic, hormonal and neural changes. Conflict of Interest The authors declare that there are no conflicts of interest. Transparency Document The Transparency document associated with this article can be found in the online version.
D.S. Guarda et al. / Food and Chemical Toxicology 69 (2014) 69–75
Acknowledgments The authors are grateful to Mr. Ulisses Siqueira and Miss Monica Moura for their technical assistance. Research was supported by the National Council for Scientific and Technological Development (CNPq), the State of Rio de Janeiro Carlos Chagas Filho Research Foundation (FAPERJ) and Coordination for the Enhancement of Higher Education Personnel (CAPES). D.S.G. was a recipient of a CNPq fellowship. The authors E.G.M. and M.S.F. designed the study and wrote the manuscript. P.C.L. and E.O. revised the manuscript and analyzed the data. D.S.G. and M.S.F. were responsible for animal programming, biochemical and hormonal analysis. J.F.N. performed lipids measurements. All authors contributed to and approved the final manuscript. References Baranowski, M., Enns, J., Blewett, H., Yakandawala, U., Zahradka, P., Taylor, C.G., 2012. Dietary flaxseed oil reduces adipocyte size, adipose monocyte chemoattractant protein-1 levels and T-cell infiltration in obese, insulinresistant rats. Cytokine 59, 382–391. Carter, J., 1993. Flaxseed as a source of alpha linolenic acid. J. Am. Coll. Nutr. 12, 551. Chechi, K., Yasui, N., Ikeda, K., Yamori, Y.K., Cheema, S., 2010. Flax oil-mediated activation of PPAR-c correlates with reduction of hepatic lipid accumulation in obese spontaneously hypertensive/NDmcr-cp rats, a model of the metabolic syndrome. Br. J. Nutr. 104, 1313–1321. Collins, T.F.X., Sprando, R.L., Black, T.N., Olejnik, N., Wiesenfeld, P.W., Babu, U.S., Bryant, M., Flynn, T.J., Ruggles, D.I., 2003. Effects of flaxseed and defatted flaxseed meal on reproduction and development in rats. Food Chem. Toxicol. 41, 819–834. Costa, C.A., Carlos, A.S., Gonzalez, Gde. P., Reis, R.P., Ribeiro, M. dos S., Dos Santos, A. de S., Monteiro, A.M., de Moura, E.G., Nascimento-Saba, C.C., 2012. Diet containing low n-6/n-3 polyunsaturated fatty acids ratio, provided by canola oil, alters body composition and bone quality in young rats. Eur. J. Nutr. 51, 191–198. Costa, C.A., Carlos, A.S., de Sousa Dos Santos, A., de Moura, E.G., Nascimento-Saba, C.C., 2013. High-fat diets containing soybean or canola oil affect differently pancreas function of young male rats. Horm. Metab. Res. 45, 652–654. Cunnane, S.C., Hamedeh, M.J., Liede, A.C., Thompson, L.U., Wolever, T.M., Jenkins, D.J., 1995. Nutritional attributes of traditional flaxseed in healthy young adults. Am. J. Clin. Nutr. 61, 62–68. Figueiredo, M.S., de Moura, E.G., Lisboa, P.C., Troina, A.A., Trevenzoli, I.H., Oliveira, E., Boaventura, G.T., da Fonseca Passos, M.C., 2009. Flaxseed supplementation of rats during lactation changes the adiposity and glucose homeostasis of their offspring. Life Sci. 85, 365–371. Figueiredo, M.S., de Moura, E.G., Lisboa, P.C., Troina, A.A., Trevenzoli, I.H., Oliveira, E., Boaventura, G.T., da Fonseca Passos, M.C., 2011. Maternal flaxseed diet during lactation programs thyroid hormones metabolism and action in the male adult offspring in rats. Horm. Metab. Res. 43, 410–416. Figueiredo, M.S., Passos, M.C.F., Trevenzoli, I.H., Troina, A.A., Carlos, A.S., Alves Nascimento-Saba, C.C., Fraga, M.C., Manhães, A.C., de Oliveira, E., Lisboa, P.C., de Moura, E.G., 2012. Adipocyte morphology and leptin signaling in rat offspring from mothers supplemented with flaxseed during lactation. Nutrition 28, 307– 315.
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