Introduction
Early postnatal life is the period of maximal physical development,Reference Hochberg 1 where the mother’s milk is the primary nutrition source. Changes in milk nutrients pose risk to future metabolic disturbances.Reference Golding, Rogers and Emmett 2 Thereby, interests have focused on the influence of lipid consumption during breastfeeding on child growth and its effects on obesity development and osteoporotic fracture origins.Reference Cooper, Westlake and Harvey 3
Adipose tissue and osteoblasts come from a common progenitor – the mesenchymal stem cell.Reference Green, Wong and Weiler 4 The effects of fat tissue on bone health are far from clear: investigators have suggested that fat mass may or may not be associated with bone mass, directly via mechanical loading and indirectly via hormonal production by adipocytes.Reference Costa, Carlos and Santos 5 , Reference Costa, Carlos and Gonzalez 6 Clinical and experimental studies have reported on the importance of α-linolenic acid (ALA, 18:3n-3)-rich diets for adiposity accumulation prevention and promotion of bone formation. Conversely, linoleic acid (LA, 18:2n-6) contributes to bone reabsorption and adipocyte hypertrophy.Reference Kruger, Coetzee, Haag and Weiler 7 , Reference Ruxton, Reed, Simpson and Millington 8
In this context, the flaxseed (Linum usitatissimum) has been described as an excellent ALA source, presenting an average of 30% lipids in its composition, with 51–55% corresponding to ALA.Reference Leite, Vicente and Suzuki 9 , Reference Pacheco, Delaprame and Boaventura 10 Furthermore, because of the presence of dietary fiber, high-quality proteins, antioxidants and minerals, a number of which offer synergistic health benefits and are part of basic nutrition, flaxseed is included in the following categories: functional foods and bioactive food.Reference Goyal, Sharma, Upadhyay, Gill and Sihag 11 , Reference Ribeiro, da Costa, Pereira, Boaventura and Chagas 12 In previous experimental models, our group evidenced protective effects of diet containing 25 out of 100 g flaxseed flour on glycemia, cardiovascular risk and lipid profile reduction.Reference Daleprane, Batista and Pacheco 13 – Reference Cardozo, Soares and Brant 15 Nevertheless, little data are available on flaxseed flour effects on the relationship between adipose tissue and bone development during early life stages.
Thus, this study was designed to evaluate whether maternal flaxseed use during lactation period has effects on body adiposity and skeletal structure of male rat pups at weaning.
Materials and methods
The protocol used for dealing with experimental animals was approved by the Ethics Committee on Animal Research of the Federal Fluminense University, Niteroi-RJ, Brazil (protocol 209/2012). All the procedures were performed in accordance with the Brazilian Society of Laboratory Animal Science and the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised in 1996) provisions.
Wistar rats were maintained in a temperature-controlled (23±1°C) and humidity-controlled (60±10%) room, with artificial dark–light cycle (lights on from 7 am to 7 pm). Virgin female rats were caged with male rats (3 months old, respectively). After mating, each female was placed in an individual cage with free access to water and standard laboratory food (Nuvilab®, Paraná, Brazil).
Within 24 h of birth, excess pups were removed, and only six male pups were retained per dam, which maximized lactation performance.Reference Fishbeck and Rasmussen 16 During the lactation period, pups were randomly assigned to the following groups: control (C, n=12), whose dams were fed control diet containing 20 out of 100 g casein; experimental (FF, n=12), whose mothers were fed diet containing 25 out of 100 g flaxseed flour. During 21 days of lactation, free access to water and diets was not evaluated, owing to difficulties in controlling pup food intake, especially in the 14–21-day period. Diets were manufactured and stored as pellets at 4°C in agreement with American Institute of Nutrition (AIN-93G) recommendations for rodent diets.Reference Reeves 17 The amount of flaxseed flour included – 25 out of 100 g – aimed to meet the entire recommended fiber intake, and it was not necessary to add oil as flaxseed seed comprises a source of this component (Table 1).
Composition of experimental diets
C, control diet; FF, flaxseed flour (experimental group); AIN, American Institute of Nutrition.
FF experimental diet containing 25 out of 100 g flaxseed flour. Mineral and vitamin mix; l-cystine; choline bitartrate: PragSoluções®; casein; cornstarch; cellulose: FARMOS®; soybean: Lisa®; and sucrose: União®. Flaxseed flour: ArmaZen® with 17% protein, 45% carbohydrate and 26% fat. Formulated on AIN-93G recommendations for rodent diets.
At 21 days of age, the pups were weaned, and after 2 h of fasting body mass and length (cm, measured as the distance between nose tip and tail tip)Reference Costa, Carlos and Santos 5 , Reference Costa, Carlos and Gonzalez 6 were evaluated. They were then anesthetized with Thiopentax (Sodium thiopental, 0.1 mg out of 100 g) and subjected to dual-energy X-ray absorptiometry (DXA)Reference Lukaski, Hall, Marchello and Siders 18 using a Lunar DXA 200368 GE instrument (Lunar, with specific software encore 2008 version 12.20; GE Healthcare, Madison, WI, USA). The evaluation was carried out in a blinded manner, as the DXA technician did not know the experimental protocol. Total lean mass (g), total fat mass (% and g), trunk fat mass (g) and bone analysis [bone mineral density (BMD, g/cm2); bone mineral content (BMC, g/cm2); total bone area (cm2)] were measured for each rat.Reference Costa, Carlos and Gonzalez 6 , Reference Ribeiro, da Silva and Pereira 19
Blood was collected by cardiac puncture following DXA procedures. Samples were centrifuged, and the serum samples were stored at −80°C for later analysis. Concentrations of osteoprotegerin (OPG), osteocalcin and leptin (ng/ml, respectively) were measured using multiplex assay kits (Millipore, Billerica, MA, USA). Concentrations of cholesterol, high-density lipoprotein (HDL)-cholesterol and triglycerides (mg/dl, respectively) were measured by colorimetric method (Bioclin BS-120; Bioclin, Belo Horizonte, MG, Brazil).
The determination of fatty acid composition by gas chromatography was carried out using serum samples. Derivatization of lipid extract was performed according to AOAC Official Methods 996.06 with some modifications. 20 Aliquots of 0.2 ml from each serum sample were added to a screw-cap test tube and 5 mg pyrogallic acid, 0.025 ml standard (5 mg/ml tritridecanoin C13:0 in chloroform), 0.1 ml ethanol, 0.5 ml HCl 8.3 M and a number of glass beads were added. The tubes were placed in a water bath at 75°C for 40 min and then cooled at room temperature. Subsequently, 1 ml ethylic ether and 1 ml petroleum ether were added, and the samples were centrifuged at 10,000 rpm for 5 min. The top phase was transferred to another tube and ether was evaporated under N2 in a water bath (below 40°C). Methylation was performed by adding 1 ml BF3 (7% in methanol) and 0.5 ml toluene and subsequent boiling at 100°C for 45 min. After cooling at room temperature, 2.5 ml water, 1 ml hexane and ~0.5 g Na2SO4 anhydrous were added. The tubes were left to rest to allow phase separation, and then the top phase was transferred to a vial containing anhydrous Na2SO4 and evaporated under N2. Before injection into the chromatograph, 0.1 ml hexane was added to each sample. Samples were analyzed using gas chromatography on a GC 17A (Shimadzu) equipped with a flame-ionization detector, automatic injector AOC-20 and a Workstation Class GC10. Fatty acid separation was achieved using a fused-silica column SP-2560 (bis-cyanopropyl polysiloxane) (100 m×0.25 mm×0.2 µm; Supelco, Bellefonte, PA, USA). The column temperature was programmed as follows: 140°C for 5 min; heating at 4°C/min up to 240°C; and 240°C for 30 min. The injector and detector were maintained at 250°C, and helium was used as the carrier gas at a flow rate of 1 ml/min. The split ratio was 1/10. Two microliters of derivatized lipid extract were injected, and the fatty acid methyl ester peaks were identified by comparison of retention times of fatty acid methyl ester standards and the chromatograms viewed using the Ce 1h-05 methods. 21
Intra-abdominal adipose tissue samples were dissected and weighed (g). For morphological analyses, retroperitoneal fat samples were collected and fixed in buffered formaldehyde (mesenteric and epididymal fat were not analyzed because of procedural difficulties). Tissues were embedded in paraffin, cut into 5-µm sections and stained with hematoxylin–eosin. For morphometric analyses, profiles with at least 100 adipocytes were randomly selected and captured for each animal. Sectional adipocyte area (µm2) was determined based on digital images acquired (TIFF format, 36 bit color, 1360×1024 pixels) with an Optronics CCD video camera system and Olympus BX40 light microscope, analyzed using the U.S. National Institutes of Health IMAGE-J software (http://rsbweb.nih.gov/ij/).Reference Costa, Carlos and Santos 5 , Reference Costa, Carlos and Gonzalez 6
The right femur was collected and cleaned of soft tissue, and was preserved in −20°C for later analysis. Bone dimension – distance between the epiphysis (mm, distance between great trochanter and lateral condyle) and middle-point diaphysis width (mm) – was measured using calipers with a readability of 0.01 mm. After drying overnight, the femur was weighted (g) using an analytical balance (Sartorius TE214S; Sartorius, Chicago, IL, USA).Reference Ribeiro, da Costa, Pereira, Boaventura and Chagas 12
Femur biomechanical properties were measured using the three-point bending test by means of a universal test machine (Instron model 4444; Instron, Canton, MA, USA), with a load cell of 100 kgf capacity. The extremities of the bone were supported on two rollers with 3-mm diameter and a distance of 21.70 mm. The load was applied to each bone’s central region.Reference Trebacz and Zduneck 22 At the beginning of the test, a 10 N pre-load was applied in the posterior–anterior direction (perpendicular to longitudinal axis) to stabilize the femur. After 1-min accommodation and stabilization period, force was applied likewise, with constant velocity of 0.5 cm/min up to fracture instant. As a result of the force applied on the femur, Instron software (series IX) generated a load strain graph; in this graph, the main biomechanical properties were obtained: maximal load (higher load withstood by the femur, kN), maximal deformation (mm), break load (the load that fractured the bone, kN), break deformation (mm), resilience (J), tenacity (J) and stiffness (N/mm).
Statistical analyses were performed using GraphPad Prism statistical package (version 5.0, 2007 San Diego, CA, USA). The results were analyzed using Student’s t-test. All results are expressed as means±s.e.m. with significance level of P<0.05.
Results
The body mass of pups was similar between groups at birth. However, pups whose dams were treated with diet containing flaxseed flour showed higher body mass and length (+8 and +6%, P<0.05, respectively) at 21 days of age. Body composition analyzed by DXA showed total lean mass, total fat mass, trunk fat mass and BMD to be similar between groups. BMC (+42%, P<0.05) and bone area (+33%, P<0.05) were higher in the FF group (Table 2).
Body mass and length and dual-energy X-ray absorptiometry analysis at 21 days of age
C, control group; FF, flaxseed flour (experimental group); BMD, bone mineral density; BMC, bone mineral content.
Control group (n=12) and experimental group (n=12), whose dams were treated with control diet or 25 out of 100 g flaxseed diet, during lactation period, respectively. Values are means with their standard errors of mean.
*Significantly different from the control group (Student’s t-test, P<0.05).
Regarding serum analyses, the FF group showed higher concentrations of OPG (P<0.05), osteocalcin (P<0.05), HDL-cholesterol (+8%, P<0.05) and lower concentrations of cholesterol (−12%, P<0.05). Triglyceride and leptin concentrations were similar between groups. Concentrations of stearic, ALA, eicosapentaenoic (EPA) and docosapentaenoic acids were higher (P<0.05), whereas arachidonic acid (AA) concentration was lower (P<0.05) in the FF group (Table 3).
Serum hormonal, biochemical and fatty acids analysis at 21 days of age
C, control group; FF, flaxseed flour (experimental group); HDL, high-density lipoproteins.
Control group (n=12) and experimental group (n=12), whose dams were treated with control diet or 25 out of 100 g flaxseed diet, during lactation period, respectively. Values are means with their standard errors of mean.
*Significantly different from the control group (Student’s t-test, P<0.05).
Intra-abdominal fat mass was similar between groups. However, the FF group showed lower adipocytes area (−40%, P<0.05, FF: 1471±175 v. C: 2486±252 µm2) (Fig. 1).
Intra-abdominal fat mass (a) and adipocyte morphometry (b, sectional area) at 21 days of age. Control group (C, n=12), whose dams were treated with control diet, and experimental group [flaxseed flour (FF), n=12], whose dams were treated with diet containing 25 out of 100 g flaxseed flour, during lactation, respectively. *Significantly different from control group (Student’s t-test, P<0.05). Photomicrographs of adipose tissue (original magnification 200×). (c) Control group, usual adipocyte aspect and (d) experimental group, lower adipocyte area.
Femur measures showed higher mass (+27%, P<0.05), distance between the epiphysis (+4%, P<0.05) and diaphysis middle-point width (+11%, P<0.05) in the FF group. Biomechanical characteristics such as maximal deformation, break deformation and tenacity were similar between groups. Meanwhile, maximal load (+44%, P<0.05), break load (+71%, P<0.05), resilience (+40%, P<0.05) and stiffness (+33%, P<0.05) were higher in the FF group (Table 4).
Mass, distance between the epiphysis, middle-point diaphysis width and biomechanical analysis of the right femur at 21 days of age
C, control group; FF, flaxseed flour (experimental group).
Control group (n=12) and experimental group (n=12), whose dams were treated with control diet or 25 out of 100 g flaxseed diet, during lactation period, respectively. Values are means with their standard errors of mean.
*Significantly different from the control group (Student’s t-test, P<0.05).
Discussion
A limitation of the present study lies in the fact that we were not able to evaluate flaxseed flour effects on gender. Nevertheless, we have observed that male pups whose dams were treated with diet containing flaxseed flour during the lactation period display higher BMC, bone area, bone quality and lower intra-abdominal adipocytes area at weaning. Probably, fatty acid profile in serum was a determining factor to outcomes regarding adiposity and bone structure.
Tinoco et al.Reference Tinoco, Sichieri, Setta, Moura and Carmo 23 observed that ALA content in milk was associated with higher weight and length gain in premature infants. Although the present study has not assessed fatty acid composition in breast milk, we have observed that experimental diet contributes to higher body mass and pup length at weaning, because flaxseed is one of the richest plant ALA sources.Reference Daleprane, Batista and Pacheco 13 – Reference Cardozo, Soares and Brant 15
The experimental and control diets provided the same energy percentage from fat, which justifies the similar body and intra-abdominal fat mass in both groups. However, as reported by McCullough et al.,Reference McCullough, Edel and Bassett 24 we observed that flaxseed intake significantly increased plasma and adipose ALA levels. Flaxseed flour, when compared with soybean oil (present in control diet), displays higher ALA levels and lower LA levels as well as LA/ALA ratio.Reference Gebauer, Psota, Harris and Kris-Etherton 25 ALA is converted to EPA and docosahexaenoic (DHA) acid, which induces fatty acid oxidation genes through peroxisome proliferator-activated receptor alpha (PPARα), suppresses lipogenic genes through sterol regulatory element-binding protein (SREBP-1c) and decreases adipocyte size. However, LA is converted into AA, inducing mature adipocytes formation and hypertrophy.Reference Costa, Carlos and Santos 5 , Reference Costa, Carlos and Gonzalez 6 , Reference Massiera, Saint-Marc and Seydoux 26 , Reference Hsu and Huang 27 These pathways help explain higher ALA, EPA and docosapentaenoic (DHA precursor) acid levels, lower AA in serum and lower intra-abdominal adipocyte area in the FF group.
In addition, the FF group showed higher serum stearic acid concentrations. Flaxseed is source of this saturated fatty acid.Reference Pellizzon, Billheimer, Bloedon, Szapary and Rader 28 Dietschy et al.Reference Dietschy, Woollett and Spady 29 and Pearson et al.Reference Pearson 30 have suggested that stearic acid bears neutral or even reduces cholesterol. Regarding HDL-cholesterol, some studies have not shown any improvement;Reference Ribeiro, da Costa, Pereira, Boaventura and Chagas 12 , Reference Ribeiro, da Silva and Pereira 19 however, Daleprane et al. Reference Daleprane, Batista and Pacheco 13 and Pacheco et al. Reference Pacheco, Delaprame and Boaventura 10 have observed higher HDL profile after flaxseed intake. Experimental studies have reported that cholesterol interferes directly in osteoblast differentiation, by decreasing bone formation and increasing osteoclast bone resorption.Reference Go, Song, Park, Park and Choi 31 , Reference You, Sheng and Tang 32 Meanwhile, Jeong et al.Reference Jeong, Cho and Kim 33 have found a positive correlation between HDL-cholesterol and BMD, favoring bone formation. In this study, low cholesterol and high HDL-cholesterol levels may have had some relation with bone parameters, determined in the FF group at weaning, thus requiring further study.
Flaxseed flour diet, during the lactation period, contributed to higher BMC and bone area in the FF group at weaning. Diets containing high ALA levels are associated with receptor activator of nuclear factor kappa beta ligand (RANKL) down-regulation, lower osteoclast maturation and bone resorption. Moreover, such diets preserve bone mass by increasing the expressions of OPG and osteocalcin (as observed in the present study), enhancing pre-osteoblasts differentiation into mature osteoblasts and bone formation.Reference Kruger, Coetzee, Haag and Weiler 7 , Reference Ruxton, Reed, Simpson and Millington 8 , Reference Corwin 34
DXA is considered a useful reference method for body composition determination, having been successfully employed in whole-body and regional bone studies in rats.Reference Tsujio, Mizorogi and Kitamura 35 In order to complement bone analyses, we observed higher distance between epiphysis, width of diaphysis and femur mass in the FF group. Furthermore, we submitted the femur to three-point bending test in order to assess bone strength. There are only a few studies about bone quality in rats at weaning. Ward et al.Reference Ward, Yuan, Cheung and Thompson 36 have observed that male rats treated with 10% flaxseed diet during lactation showed bone strength similar to our control group. In the present study, 25% flaxseed flour diet was associated with higher femur dimension and biomechanical strength improvement, which suggests resistance to fracture.
In short, our data evidence that flaxseed flour intake during the lactation period promotes adipocyte hypertrophy down-regulation and contributes to the bone quality of pups at weaning. Nevertheless, further studies are necessary to clarify flaxseed flour role in early stages of life and its effects on obesity and osteoporotic fracture prevention during adult life stage.
Acknowledgments
The authors are thankful to the Laboratory of Nutrition and Functional Assessment, College of Nutrition, Federal Fluminense University for technical assistance and use of DXA equipment; to Vitória Cristina Gomes da Silva for English editing; to Coordination for the Enhancement of Higher Education Personnel; and to National Counsel of Technological and Scientific Development.
Financial Support
This work was supported by The State of Rio de Janeiro Carlos Chagas Filho Research Foundation (number process 103373/2012 and 477763/2011).
Conflicts of Interest
None.
Ethical Standards
The protocol used to deal with experimental animals was approved by Ethics Committee on Animal Research of Fluminense Federal University, Niteroi-RJ, Brazil (protocol 597/2014). All procedures are in accordance with the provisions of Brazilian Society of Science and Laboratory’s Animals and the Guide for the Care and Use of Laboratory Animals.
