Hostname: page-component-848d4c4894-x5gtn Total loading time: 0 Render date: 2024-05-20T00:14:42.566Z Has data issue: false hasContentIssue false

Programmed changes in the adult rat offspring caused by maternal protein restriction during gestation and lactation are attenuated by maternal moderate–low physical training

Published online by Cambridge University Press:  01 May 2012

Marco Fidalgo
Affiliation:
Department of Physical Education and Sports Science, CAV, Federal University of Pernambuco, Pernambuco, Brazil
Filippe Falcão-Tebas
Affiliation:
Department of Nutrition, Federal University of Pernambuco, Pernambuco, Brazil
Adriano Bento-Santos
Affiliation:
Department of Nutrition, Federal University of Pernambuco, Pernambuco, Brazil
Elaine de Oliveira
Affiliation:
Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
José Firmino Nogueira-Neto
Affiliation:
Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Egberto Gaspar de Moura
Affiliation:
Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Patrícia Cristina Lisboa
Affiliation:
Department of Physiological Sciences, Roberto Alcantara Gomes Biology Institute, State University of Rio de Janeiro, Rio de Janeiro, Brazil
Raul Manhães de Castro
Affiliation:
Department of Nutrition, Federal University of Pernambuco, Pernambuco, Brazil
Carol Góis Leandro*
Affiliation:
Department of Physical Education and Sports Science, CAV, Federal University of Pernambuco, Pernambuco, Brazil
*
*Corresponding author: C. G. Leandro, fax +55 81 35233351, E-mail: carolleandro22@gmail.com
Rights & Permissions [Opens in a new window]

Abstract

The effects of maternal moderate–low physical training on postnatal development, glucose homeostasis and leptin concentration in adult offspring subjected to a low-protein diet during the perinatal period were investigated. Male Wistar rats (aged 150 d old) were divided into four groups according to maternal group: untrained (NTp, n 8); trained (Tp, n 8); untrained with a low-protein diet (NT+LPp, n 8); trained with a low-protein diet (T+LPp, n 8). The trained mothers were subjected to a protocol of moderate physical training over a period of 4 weeks (treadmill, 5 d/week, 60 min/d, at 65 % VO2max) before mating. At pregnancy, the intensity and duration of exercise was progressively reduced (50–20 min/d, at 65–30 % VO2max). The low-protein diet groups received an 8 % casein diet, and their peers received a 17 % casein diet during gestation and lactation. The pups' birth weight and somatic growth were recorded weekly up to the 150th day. Fasting blood glucose, cholesterol, serum leptin concentration, glucose and insulin tolerance tests were evaluated. The Tp animals showed no changes in somatic and biochemical parameters, while the NT+LPp group showed a greater abdominal circumference, hyperglycaemia, hypercholesterolaemia, glucose intolerance and lower plasma leptin. In the T+LPp animals, all of those alterations were reversed except for plasma leptin concentration. In conclusion, the effects of a perinatal low-protein diet on growth and development, glucose homeostasis and serum leptin concentration in the offspring were attenuated in pups from trained mothers.

Type
Full Papers
Copyright
Copyright © The Authors 2012

The peri- and preconception periods are now thought to be critical for the long-term effects on fetal development and postnatal growth and may predispose offspring to phenotypic changes and metabolic diseases later in life(Reference Hanson and Gluckman1). Unbalanced nutrient intake during this critical period of development has been associated with subsequent health risks and disease in the offspring, according to epidemiological data and numerous experimental observations(Reference Barker2). This phenomenon has been termed ‘developmental plasticity’(Reference Gluckman and Hanson3). Developmental plasticity is the property of a given genotype to produce different phenotypes in response to distinct environmental conditions(Reference Hanson, Godfrey and Lillycrop4).

The maternal low-protein diet model is one of the most extensively studied models of early growth restriction(Reference Ozanne and Hales5). A low-protein diet (8 % casein) during gestation followed by a normal diet throughout the life course has been associated with growth restriction, slightly elevated systolic blood pressure and increased fasting plasma insulin concentrations compared with control offspring(Reference Ozanne and Hales5). If maternal protein restriction is continued during lactation, there is lasting growth restriction, age-dependent loss of glucose tolerance, insulin resistance, hypertension and hyperleptinaemia, even when the offspring are weaned onto a control diet(Reference Ozanne and Hales5). In adult rats (110 d) subjected to a low-protein diet (10 % casein) during pregnancy and lactation, Zambrano et al. (Reference Zambrano, Bautista and Deas6) observed a lower serum leptin concentration. Leptin is a hormone mainly produced by adipocytes; however, it is also produced by several tissues, including skeletal muscle(Reference Wang, Liu and Hawkins7). The presence of leptin in the muscle could be indicative of adipocyte infiltration, or leptin may have been produced directly by muscle fibres(Reference Trevenzoli, Rodrigues and Oliveira8).

Recently, we demonstrated that controlled moderate- to low-intensity physical training before and during gestation attenuated the impact of the low-protein diet by improving the mothers' resting oxygen consumption and the growth rate of the offspring(Reference Amorim, dos Santos and Hirabara9). In addition, physical exercise training has been associated with a reduced risk of metabolic disease and enhances both cardiorespiratory and metabolic functions. During gestation, the beneficial effects of physical exercise are dependent on the volume of exercise(Reference Amorim, dos Santos and Hirabara9). There are different physiological responses according to the type and frequency of exercise, the physical fitness of the mother, the time point in pregnancy when the exercise is carried out and the duration and intensity of exercise(Reference Clapp, Kim and Burciu10, Reference Clapp11). In 2002, the American College of Obstetricians and Gynecologists published exercise guidelines for pregnancy(12). In these recommendations, it was suggested that after medical approval, 30 min or more of moderate exercise a day on most, if not all, days of the week is recommended for pregnant women. Exercise is considered moderate when oxygen consumption is approximately 50–70 % of VO2max. Regular practice of moderate exercise has been associated with improved cardiorespiratory fitness, increased metabolic rate (reduction in body weight) and increased muscle mitochondrial biogenesis(Reference Leandro, Levada and Hirabara13). At rest, the rate of placental red blood flow increases, and more glucose and oxygen delivery to the placental site are observed in women subjected to a physical training regimen(Reference Clapp11). An epidemiological study found that moderate physical exercise during pregnancy is associated with a 100–150 g increase in birth weight(Reference Hatch, Shu and McLean14).

Little is known about the long-term effects of maternal physical activity on adult offspring subjected to perinatal undernutrition. This is a topic of particular interest as a maternal lifestyle can be considered a therapeutic means of countering the effects of either maternal undernutrition or overnutrition. Thus, in the present study, the effects of a maternal moderate–low protocol of physical training on postnatal development, glucose homeostasis and leptin concentration in adult offspring whose mothers were subjected to a low-protein diet during the perinatal period were investigated. Our hypothesis is that exercise-induced physiological adaptations during gestation, as seen in our previous studies, attenuate or modulate the impact of a perinatal low-protein diet on glucose homeostasis and leptin concentrations in adult offspring.

Materials and methods

The experimental protocol was approved by the Ethical Committee of the Biological Sciences Centre (protocol no. 80 23 076.049077/2010-80), Federal University of Pernambuco, Brazil and followed the Guidelines for the Care and Use of Laboratory Animals(Reference Bayne15).

Animals

Virgin female albino Wistar rats (Rattus norvegicus) aged 60 d were obtained from the Department of Nutrition, Federal University of Pernambuco, Brazil. Female rats were maintained at a room temperature of 23 ± 2°C with a controlled light–dark cycle (dark 09.00–21.00 hours). Standard laboratory chow (52 % carbohydrate, 21 % protein and 4 % lipids; Agribrands-Purina Limited) and water were given ad libitum. The animals were randomly divided into two groups: untrained rats (n 8) and trained rats (n 8). The trained rats were subjected to a training programme of moderate running over a period of 4 weeks (5 d/week and 60 min/d) on a treadmill (EP-131; Insight Equipments) at a controlled intensity based on their VO2max(Reference Amorim, dos Santos and Hirabara9). After the 4-week training period, the rats were mated (two females for one male). The day on which spermatozoa were present in a vaginal smear was designated as the day of conception, day 0 of pregnancy. Pregnant rats were then transferred to individual cages. Half of the rats from each group received a 17 % casein diet, and the other half received an 8 % casein isoenergetic diet (low-protein group, LP)(Reference Reeves, Nielsen and Fahey16)ad libitum. Thus, two more groups were formed, which are as follows: untrained (NT, n 4); trained (T, n 4); untrained with a low-protein diet (NT+LP, n 4); trained with a low-protein diet (T+LP, n 4). The exercise programme was maintained during gestation, with a progressive reduction in duration and intensity until the 19th day of gestation. There was no physical exercise during the suckling period. During lactation, the offspring were kept in litters of six pups, and their mothers continued to be fed with the 8 % casein or 17 % casein diet. Only male offspring from each litter were used. In all subsequent experiments, two male offspring were randomly chosen from each litter. The pups were divided into four groups according to their mothers' assigned group: untrained (NTp, n 8); trained (Tp, n 8); untrained with a low-protein diet (NT+LPp, n 8); trained with a low-protein diet (T+LPp, n 8). At 150 d of age, pups were killed by cardiac exsanguination.

Physical training protocol

Physical training was performed according to Amorim et al. (Reference Amorim, dos Santos and Hirabara9). Briefly, rats ran on a treadmill during the 4 weeks (5 d/week, 60 min/d, at 65 % VO2max) before pregnancy. The protocol was divided into four progressive stages in each session: (1) warm-up (5 min); (2) intermediary (10 min); (3) training (30 min); (4) cool-down (5 min). The percentage of VO2max during the sessions of training before gestation was kept at approximately 55–65 %. During pregnancy, rats ran (5 d/week) at a progressively reduced duration and intensity of effort (first week 50 min/d (approximately 65 % of VO2max), second week 30 min/d (approximately 45 % of VO2max) and third week 20 min/d (approximately 32 % of VO2max)). There was no physical training during the lactation period.

Measurement of food intake and body weight during gestation

During gestation, dams were housed individually, and their daily food consumption was determined by the difference between the amount of food provided at the onset of the light cycle and the amount of food remaining 24 h later(Reference Lopes de Souza, Orozco-Solis and Grit17). Body and food weights were recorded to the nearest 0·01 g. Body weight was recorded daily throughout the experiment by a Marte Scale (AS-1000; Marte Científica) approaching 0·01 g. Percentage of weight gain (%BWG) was calculated by the formula:

$$\begin{eqnarray} \%\,BWG = (body\,weight\,(g)\times 100/weight\,at\,the\,first\,day\,of\,gestation\,(g)) - 100. \end{eqnarray}$$

Blood glucose measurements

Fasting glycaemia levels were evaluated weekly during gestation using blood samples from the tail vein of rats, using a glucometer (Accu Check Advantage and Roche) and the glucose oxidase method. The animals were fasted overnight.

Postnatal developmental patterns of offspring

The body weights of pups were recorded weekly throughout the experiment with a Marte Scale (AS-1000) approaching 0·01 g. Percentage of weight gain (%BWG) was calculated at different intervals of time until 150 d old (birth to 30th, 31st to 90th and 91st to 150th) by the formula:

$$\begin{eqnarray} \%\,BWG\, = \,(body\ weight\,(g)\times 100/weight\quad at\quad the\quad first\quad day\quad in\quad the\quad interval\,\,(g)) - 100. \end{eqnarray}$$

The body lengths of pups were recorded by measuring the external surfaces (nose-to-anus length) using a digital calliper (0·01 mm accuracy). Body weight and body length were used to determine BMI (weight (g)/length (cm)2)(Reference Novelli, Diniz and Galhardi18). Abdominal circumference was recorded by measuring the midpoint between the lower rib margin and the iliac crest in the horizontal plane(Reference Novelli, Diniz and Galhardi18).

Glucose tolerance test and insulin tolerance test of offspring

The glucose tolerance test (GTT) was performed at 145 d and the insulin tolerance test (ITT) at 147 d of age. In both tests, animals were fasted overnight. Blood sample collections were performed by cutting the tip of the tail to remove approximately 10 μl of blood. The first blood sample was collected (time zero) before the injection of glucose. In the GTT, a 50 % glucose solution (Equiplex Pharmaceutical Limited) at a dose of 1 mg/g body weight was administered intraperitoneally. Blood samples were then collected at 15, 30, 45, 60 and 120 min after administration. The area under the glucose curve was obtained by blood glucose values at 0, 30, 60 and 120 min using the trapezoidal method(Reference Le Floch, Escuyer and Baudin19). In the ITT, a solution of insulin (Eli Lilly do Brazil Limited) at a dose of 0·75 mU (34.125 ng)/g body weight was administered intraperitoneally, and additional blood samples were collected at 15, 30, 45, 60 and 120 min. Glucose disappearance constant was calculated from the blood glucose values obtained at 0, 30 and 60 min (Kitt)(Reference Le Floch, Escuyer and Baudin19).

Resting blood glucose, cholesterol and leptin

At 150 d old, animals were anaesthetised with ketamine (0·25 ml/100 g body weight) and xylazine (0·25 ml/100 g body weight). Blood was sampled by cardiac puncture for the quantification of overnight fasting serum glucose, cholesterol and leptin concentrations. Glycaemia and cholesterolaemia were determined in blood samples using a glucometer and a cholesterol meter (Accu Check Advantage and Accutrend GCT; Roche Diagnostics Limited), respectively. Plasma leptin concentration was determined by a RIA kit (Linco Research, Inc.), with an assay sensitivity of 0·5 ng/ml and an intra-assay variation coefficient of 2·9 %. The samples were analysed in one assay.

Western blotting for leptin content analysis

The soleus muscle (right) was weighed and homogenised in an ice-cold lysis buffer (50 mm-HEPES, 1 mm-MgCl2, 10 mm-EDTA, Triton X-100 1 %, pH 6·4) and protease inhibitor cocktail (1 mg/ml aprotinin, leupeptin and phenylmethylsulfonyl fluoride; Sigma-Aldrich, Inc.). Total protein content of the supernatants was determined by the bicinchoninic acid method (Protein Assay Kit; Thermo Fisher Scientific, Inc.). Samples with 10 μg total protein were separated by SDS-PAGE (12 %) and transferred to a nitrocellulose membrane (Hybond P; Amersham Pharmacia Biotech, Inc.). Membranes were blocked for 90 min with 5 % non-fat dry milk in TBS-T (20 mm-Tris, 0·5 m-NaCl and 0·1 % Tween 20). The membranes were then washed three times with T-TBS and incubated overnight with primary antibody anti-leptin (rabbit monoclonal – 1:1000; Sigma Chemical Company). They were then washed and incubated for 1 h with secondary antibody anti-rabbit (goat anti-rabbit conjugated with horseradish peroxidase 1:1000; Santa Cruz Biotechnology, Inc.). After the membranes had been washed three times with T-TBS, antibody binding was visualised using 3,3-diaminobenzidine tetrahydrochloride (10 mg in 15 ml Tris buffer, 0·1 m, pH 7·4). Images were scanned, and the bands were quantified by densitometry using Image J (Cibernetics Media, Inc.).

Statistical analysis

Results are presented as means with their standard errors. Intra-litter analyses were performed and found not to be significant. For statistical analysis, data were analysed by two-way repeated-measures ANOVA, with mothers' diet (NT and NT+LP) and physical training (T and T+LP) as factors. Pearson's correlation coefficient was used to correlate body-weight gain with the number of pups born per mother. Significance was set at P <0·05. Data analysis was performed using the statistical program Graphpad Prism 5® (GraphPad Software, Inc.).

Results

Before mating, there was no difference between the groups in terms of body weight (P>0·05). During gestation, moderate physical training attenuated the effects of a low-protein diet as reflected by the mother diet–training interaction for this analysis (F 6,36= 4·757, P< 0·0001). Actually, the effects of a low-protein diet on BWG during gestation were seen at the third week of gestation in the untrained dams (NT+LP), while in the T+LP dams, these effects were normalised (Fig. 1(a) and (b)). Data were adjusted for the number of pups born to each dam (NT, median 11·0 (minimum–maximum 9–13); T, median 11·5 (minimum–maximum 9–14); NT+LP, median 10·5 (minimum–maximum 8–11); T+LP, median 11·0 (minimum–maximum 9–12)). Pearson's correlation coefficient between the number of pups and the mother's BWG was not significant (r 2 0·27, P= 0·452). Relative daily food intake during gestation was not different between the groups (F 6,36= 0·423, P= 0·85; Fig. 1(c)). Fasting blood glucose only changed in the NT+LP mother group, which displayed greater values compared with the NT+LP group (F 6,36= 2·316, P< 0·05; Fig. 1(d)).

Fig. 1 (a) Body weight (g), (b) percentage of body-weight gain in each third week of gestation, relative to the body mass on the first day of pregnancy, (c) relative daily food intake expressed in g/d during gestation and (d) fasting blood glucose by non-trained (NT, n 4; (a, c) □ and (b, d) ), trained (T, n 4; (a, c) □ and (b, d) ), non-trained+low-protein (NT+LP, n 4; (a, c) and (b, d) ) and trained+low-protein (T+LP, n 4; (a, c) and (b, d) ) dams. Values are means with their standard errors represented by vertical bars. * Mean values were significantly different from the NT group (P< 0·05; two-way ANOVA). † Mean values were significantly different from the NT+LP group (P< 0·05; two-way ANOVA).

Pups from mothers subjected to physical training before and during gestation and/or to a low-protein diet during gestation were evaluated from birth to 150 d. Pups from mothers subjected to physical training showed a less pronounced reduction in body weight, body length and BMI especially in the interval between 60th and 120th day old, with a significant mother diet–physical training interaction (F 4,50= 7·439, P< 0·001; Table 1). In addition, at the 150th day of life, the NT+LPp animals displayed a greater abdominal circumference than the NTp animals that was attenuated in the T+LP pups (Table 1).

Table 1 Body weight, body length, BMI and abdominal circumference of the offspring at 30, 60, 90, 120 and 150 d old (Mean values with their standard errors)

NTp, pups of untrained rats; Tp, pups of trained rats; NT+LPp, pups of untrained rats with a low-protein diet; T+LPp, pups of trained rats with a low-protein diet; %BWG, percentage of body-weight gain.

* Mean values were significantly different from the NTp group (P< 0·05; two-way ANOVA).

Mean values were significantly different from the NT+LPp group (P< 0·05; two-way ANOVA).

During gestation, the dams were subjected to physical training and fed a low-protein diet. During lactation, the dams continued to receive a low-protein diet.

From 145–147 d old, pups were subjected to the GTT and ITT. There were no differences between the groups on the GTT and ITT curves (Fig. 2(a) and (c)); however, areas under the glycaemic curve were greater in the NT+LPp group than in the NTp group (Fig. 2(b)). The rate of disappearance of glucose was lower in the NT+LPp animals (Fig. 2(d)). The effects of the maternal low-protein diet were attenuated in response to physical training (T+LPp) (mother diet–physical training interaction: F 15,245= 20·21, P< 0·001).

Fig. 2 (a) Glucose tolerance test, (b) areas under glycaemic curve, (c) insulin tolerance test and (d) rate of disappearance of glucose ‘Kitt’ of offspring at 145–147 d old from untrained (NTp, n 8; (a, c) ), trained (Tp, n 8; (a, c) ), untrained+low-protein (NT+LPp, n 8; (a, c) ) and trained+low-protein mothers (T+LPp, n 8; (a, c) ). Values are means with their standard errors represented by vertical bars. * Mean values were significantly different from the NTp group (P< 0·05; two-way ANOVA).

At 150 d old, the NT+LPp animals showed greater fasting glycaemia and cholesterolaemia when compared with the NTp group. However, in the T+LPp animals, glycaemia (NTp: mean 91·1 (sem 1·6); Tp: mean 98·0 (sem 2·1); NT+LPp: mean 108·4 (sem 2·0); T+LPp: mean 91·3 (sem 3·8)) and cholesterolaemia (NTp: mean 153·3·1 (sem 0·6); Tp: mean 156·0 (sem 1·4); NT+LPp: mean 172·1 (sem 1·3); T+LPp: mean 164·1 (sem 0·9)) were normalised (F 3,56= 5·145, P= 0·003; Fig. 3).

Fig. 3 Fasting blood glucose and cholesterol of the offspring at 150 d old from untrained (NTp, n 8; □), trained (Tp, n 8; ■), untrained+low-protein (NT+LPp, n 8; ) and trained+low-protein mothers (T+LPp, n 8; ). Values are means with their standard errors represented by vertical bars. * Mean values were significantly different from the NT group (P< 0·05; two-way ANOVA). † Mean values were significantly different from the NT+LP group (P< 0·05; two-way ANOVA).

Leptin content in the soleus muscle was greater (+45 %) in the NT+LPp pups in comparison with the NTp group. Conversely, leptin content was lower in pups from trained mothers (T+LPp − 48 %) in comparison with the NT+LPp pups, with a significant mother diet–physical training interaction (F 3,58= 3·967, P< 0·05). Plasma leptin concentrations were lower in the NT+LPp animals when compared with the NTp group, and physical training was not able to attenuate this effect (NTp: mean 4·4 (sem 1·3); NT+LPp: mean 1·8 (sem 0·1); T+LPp: mean 1·3 (sem 0·1); Fig. 4).

Fig. 4 (a) Leptin in the soleus muscle and (b) plasma leptin of the offspring at 150 d old from untrained (NTp; n 8), trained (Tp; n 8), untrained+low-protein (NT+LPp; n 8) and trained+low-protein mothers (T+LPp; n 8). Values are means with their standard errors represented by vertical bars. * Mean values were significantly different from the NT group (P< 0·05; two-way ANOVA). † Mean values were significantly different from the NT+LP group (P< 0·05; two-way ANOVA).

Discussion

An active maternal lifestyle, including regular physical activity and moderate physical training, improves aerobic fitness and the maternal–fetal physiological reserve and, thus, enhances nutrient and oxygen delivery to the fetus(Reference Clapp11). In the mother, improved cardiovascular function, limited BWG and a reduced risk of gestational diabetes mellitus and gestational hypertension have been observed(Reference Melzer, Schutz and Boulvain20). For the fetus, reduced fat mass, high tolerance to stress and an advanced neurobehavioural maturation have been observed(Reference Melzer, Schutz and Boulvain20). Our previous studies using animal models have shown that maternal physical training on a treadmill (5 d/week, progressive reduction in duration and intensity 50–20 min/d, 65–30 % VO2max) resulted in reduced BWG and high resting oxygen consumption (resting VO2) and attenuated the delayed reflex ontogeny induced by undernutrition(Reference Amorim, dos Santos and Hirabara9, Reference Falcão-Tebas, Bento-Santos and Fidalgo21). The present study has evaluated the long-term effects of a maternal moderate–low physical training protocol on BWG, glucose homeostasis and leptin concentration in adult offspring whose mothers were subjected to a low-protein diet during the perinatal period.

In the present study, although trained mothers demonstrated increased body-weight gain in the second and third weeks of gestation, the food intake did not differ, with the exception of the T+LPp animals. The present results are in agreement with previous studies(Reference Amorim, dos Santos and Hirabara9, Reference Falcão-Tebas, Bento-Santos and Fidalgo21). Because physical training can induce an increase in maternal lean body mass(Reference Leandro, Levada and Hirabara13), it may be inferred that body weight is increased by a mechanism that includes an increase in the synthesis of muscle proteins(Reference Amorim, dos Santos and Hirabara9). In dams, physical training was also able to normalise the effects of a low-protein diet on fasting serum glucose. This differential effect observed in previously active compared with undernourished dams may be related to reductions in the risk of gestational diabetes mellitus, as an exercise programme may improve insulin sensitivity and fasting plasma glucose concentrations of women at risk for gestational diabetes(Reference Oostdam, van Poppel and Eekhoff22).

Low-protein diet offspring remained growth retarded throughout life and maintained a greater abdominal circumference, even when fed the control diet ad libitum from weaning to adult life. The present results are in agreement with previous studies that have indicated that pups from undernourished mothers experienced a reduced postnatal growth trajectory(Reference Amorim, dos Santos and Hirabara9, Reference Desai, Crowther and Ozanne23Reference Ozanne and Hales25). Several hormonal changes, including greater leptin and T3 and lower serum insulin-like growth factor concentrations, are associated with malnutrition during gestation and lactation and can be possible imprinting factors in the growth and development programming of the progeny(Reference Moura, Santos and Lisboa26). Maternal physical training had beneficial effects on the postnatal growth rate in offspring from undernourished mothers. The underlying mechanisms of these effects may be related to metabolic changes, redistribution of blood flow and changes in the production of fetal and placental hormones that control growth(Reference Amorim, dos Santos and Hirabara9). The insulin-like growth factor and their associated binding proteins are thought to be an important mechanism underlying the long-term effects of maternal physical training(Reference Turgut, Kaptanoglu and Emmungil27). Treadmill exercise (20 m/min, 20 min/d, during 19 d) results in an increase in the plasma concentration of growth hormone, insulin-like growth factor I and insulin-like growth factor binding protein-3 in the late period of pregnancy(Reference Turgut, Kaptanoglu and Emmungil27). Nevertheless, these effects are directly dependent on the volume of effort (duration and intensity), and opposite effects are observed in high-intensity exercise during pregnancy(Reference Hopkins, Baldi and Cutfield28). In the present study, the intensity and duration of each session of exercise was controlled in order to keep the effort approximately 65–30 % of VO2max. Thus, positive effects on offspring growth are observed when undernourished dams are subjected to moderate–low physical training.

In the present study, a perinatal low-protein diet induced greater glycaemia and cholesterolaemia, a greater area under the glucose curve and a reduced rate of disappearance of glucose than observed in their pairs. The present results confirm those presented in previous studies(Reference Ozanne and Hales25, Reference Ozanne, Jensen and Tingey29). It is interesting to note that physical training attenuated the deleterious effects of perinatal undernutrition. These observations indicate that maternal physical exercise initiated in early pregnancy induces feto-placental adaptations and can be considered as a therapeutic means of countering the effects of maternal undernutrition, which may provide a useful strategy for enhancing nutrient and oxygen availability to the fetus. The underlying mechanism can be related to epigenetic modulation induced by physical activity that regulates gene expression(Reference Gomez-Pinilla, Zhuang and Feng30). For example, it has been found that physical exercise induces the DNA methylation of brain neurotrophic factors (BDNF-IV), increases the concentration of protein involved in DNA methylation and mRNA, and increases the acetylation of histones(Reference Gomez-Pinilla, Zhuang and Feng30). In the case of these changes occurring during the critical period of fetal development, physical activity assumes an important role in the control of gene transcription in the context of the long-term effects of developmental plasticity. According to advances in the studies of the Developmental Origin of Health and Disease and epigenomic factors, physical activity during gestation opens new therapeutic possibilities for low-cost treatment for disorders associated with perinatal undernutrition.

In the present study, leptin concentrations were evaluated in the plasma and skeletal muscle. We have observed that protein restriction during gestation and lactation induced a reduced plasma leptin concentration at 150 d of age. In contrast, previous studies in which dams were subjected to undernutrition during lactation have indicated that adult offspring developed greater leptinaemia and central leptin resistance with increased total (39 %) and visceral fat mass (2·3 times)(Reference de Oliveira Cravo, Teixeira and Passos31, Reference Bonomo, Lisboa and Pereira32). Different periods early in life to induce undernutrition appear to be crucial in the long-term effects of plasma leptin concentrations. Maternal physical training was not able to normalise the effects of maternal low-protein diet. Thus, a maternal low-protein diet during gestation and lactation seems to affect the synthesis and metabolism of leptin, and also causes the effects on their receptors that persist long after the subsequent normalisation of nutrition(Reference Lisboa, Passos and Dutra33).

Leptin is mainly produced by the adipocyte; however, it is also produced by several other tissues, including skeletal muscle(Reference Wang, Liu and Hawkins7), where its expression may be different from that produced in the adipocyte. In the present study, the NT+LPp animals experienced an increase in the expression of leptin in the skeletal muscle. Leptin in the muscle could be indicative of adipocyte infiltration or may be due to leptin produced directly by the muscle fibres(Reference Miljkovic-Gacic, Gordon and Goodpaster34). The dissociation of leptin serum levels and muscle leptin content in the NT+LPp group may be explained by the fact that leptin serum levels are associated with adipose tissue mass, and the muscle contribution to plasma leptin levels may not be important. The importance of leptin muscle content may be on the control of local glucose homeostasis, as it has already been shown that intramuscular fat is associated with insulin resistance(Reference Miljkovic-Gacic, Gordon and Goodpaster34). With training, this supposed adipocyte infiltration may decrease, thereby normalising leptin intramuscular production.

The primary aim of the present study was to test the hypothesis that moderate physical training before and during gestation attenuates the effects of perinatal low-protein undernutrition. Indeed, the effects of a perinatal low-protein diet on the development, glucose homeostasis and leptin concentrations in the skeletal muscle of the offspring were attenuated in pups from trained mothers.

Acknowledgements

The authors are indebted to Lucia Pires and Edeones França for their technical assistance. This study was supported by the Foundation for Support of Science and Research in Pernambuco State, Brazil (FACEPE), Coordination for the Improvement of Higher Level Personnel (PROCAD-Capes) and the National Council for Research, Brazil (CNPq). The authors' contributions are as follows: M. F. and C. G. L. performed the study design; F. F.-T., A. B.-S., E. O., J. F. N.-N. and M. F. performed the experiments and data collection; M. F., R. M. C. and C. G. L. performed the statistical analyses, interpreted the data and wrote the manuscript; M. F., P. C. L., E. G. M. and C. G. L. were responsible for critical revisions to the manuscript, and all authors approved the final version. The authors declare that there are no conflicts of interest. M. F. and C. G. L. declare that all authors listed are eligible for authorship.

References

1Hanson, MA & Gluckman, PD (2005) Developmental processes and the induction of cardiovascular function: conceptual aspects. J Physiol 565, 2734.CrossRefGoogle ScholarPubMed
2Barker, DJ (2007) The origins of the developmental origins theory. J Intern Med 261, 412417.Google Scholar
3Gluckman, PD & Hanson, MA (2004) Living with the past: evolution, development, and patterns of disease. Science 305, 17331736.Google Scholar
4Hanson, M, Godfrey, KM, Lillycrop, KA, et al. (2011) Developmental plasticity and developmental origins of non-communicable disease: theoretical considerations and epigenetic mechanisms. Prog Biophys Mol Biol 106, 272280.CrossRefGoogle ScholarPubMed
5Ozanne, SE & Hales, CN (2004) Lifespan: catch-up growth and obesity in male mice. Nature 427, 411412.Google Scholar
6Zambrano, E, Bautista, CJ, Deas, M, et al. (2006) A low maternal protein diet during pregnancy and lactation has sex- and window of exposure-specific effects on offspring growth and food intake, glucose metabolism and serum leptin in the rat. J Physiol 571, 221230.Google Scholar
7Wang, J, Liu, R, Hawkins, M, et al. (1998) A nutrient-sensing pathway regulates leptin gene expression in muscle and fat. Nature 393, 684688.Google Scholar
8Trevenzoli, IH, Rodrigues, AL, Oliveira, E, et al. (2010) Leptin treatment during lactation programs leptin synthesis, intermediate metabolism, and liver microsteatosis in adult rats. Horm Metab Res 42, 483490.Google Scholar
9Amorim, MF, dos Santos, JA, Hirabara, SM, et al. (2009) Can physical exercise during gestation attenuate the effects of a maternal perinatal low-protein diet on oxygen consumption in rats? Exp Physiol 94, 906913.Google Scholar
10Clapp, JF 3rd, Kim, H, Burciu, B, et al. (2002) Continuing regular exercise during pregnancy: effect of exercise volume on fetoplacental growth. Am J Obstet Gynecol 186, 142147.CrossRefGoogle ScholarPubMed
11Clapp, JF 3rd (2003) The effects of maternal exercise on fetal oxygenation and feto-placental growth. Eur J Obstet Gynecol Reprod Biol 110, Suppl. 1, S80S85.CrossRefGoogle ScholarPubMed
12ACOG Committee Obstetric Practice (2002) ACOG Committee opinion. Number 267, January 2002: exercise during pregnancy and the postpartum period. Int J Gynaecol Obstet 77, 7981.Google Scholar
13Leandro, CG, Levada, AC, Hirabara, SM, et al. (2007) A program of moderate physical training for Wistar rats based on maximal oxygen consumption. J Strength Cond Res 21, 751756.Google Scholar
14Hatch, MC, Shu, XO, McLean, DE, et al. (1993) Maternal exercise during pregnancy, physical fitness, and fetal growth. Am J Epidemiol 137, 11051114.Google Scholar
15Bayne, K (1996) Revised guide for the care and use of laboratory animals available. American Physiological Society. Physiologist 39, 208–111.Google Scholar
16Reeves, PG, Nielsen, FH & Fahey, GC Jr (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-76A rodent diet. J Nutr 123, 19391951.Google Scholar
17Lopes de Souza, S, Orozco-Solis, R, Grit, I, et al. (2008) Perinatal protein restriction reduces the inhibitory action of serotonin on food intake. Eur J Neurosci 27, 14001408.Google Scholar
18Novelli, EL, Diniz, YS, Galhardi, CM, et al. (2007) Anthropometrical parameters and markers of obesity in rats. Lab Anim 41, 111119.Google Scholar
19Le Floch, JP, Escuyer, P, Baudin, E, et al. (1990) Blood glucose area under the curve. Methodological aspects. Diabetes Care 13, 172175.Google Scholar
20Melzer, K, Schutz, Y, Boulvain, M, et al. (2010) Physical activity and pregnancy: cardiovascular adaptations, recommendations and pregnancy outcomes. Sports Med 40, 493507.Google Scholar
21Falcão-Tebas, F, Bento-Santos, A, Fidalgo, MA, et al. (2012) Maternal low-protein diet-induced delayed reflex ontogeny is attenuated by moderate physical training during gestation in rats. Br J Nutr 107, 372377.CrossRefGoogle ScholarPubMed
22Oostdam, N, van Poppel, MN, Eekhoff, EM, et al. (2009) Design of FitFor2 study: the effects of an exercise program on insulin sensitivity and plasma glucose levels in pregnant women at high risk for gestational diabetes. BMC Pregnancy Childbirth 9, 1.CrossRefGoogle Scholar
23Desai, M, Crowther, NJ, Ozanne, SE, et al. (1995) Adult glucose and lipid metabolism may be programmed during fetal life. Biochem Soc Trans 23, 331335.Google Scholar
24Ozanne, SE & Hales, CN (1999) The long-term consequences of intra-uterine protein malnutrition for glucose metabolism. Proc Nutr Soc 58, 615619.CrossRefGoogle ScholarPubMed
25Ozanne, SE & Hales, CN (2002) Early programming of glucose–insulin metabolism. Trends Endocrinol Metab 13, 368373.Google Scholar
26Moura, EG, Santos, RS, Lisboa, PC, et al. (2008) Thyroid function and body weight programming by neonatal hyperthyroidism in rats – the role of leptin and deiodinase activities. Horm Metab Res 40, 17.Google Scholar
27Turgut, S, Kaptanoglu, B, Emmungil, G, et al. (2006) Increased plasma levels of growth hormone, insulin-like growth factor (IGF)-I and IGF-binding protein 3 in pregnant rats with exercise. Tohoku J Exp Med 208, 7581.Google Scholar
28Hopkins, SA, Baldi, JC, Cutfield, WS, et al. (2010) Exercise training in pregnancy reduces offspring size without changes in maternal insulin sensitivity. J Clin Endocrinol Metab 95, 20802088.Google Scholar
29Ozanne, SE, Jensen, CB, Tingey, KJ, et al. (2005) Low birthweight is associated with specific changes in muscle insulin-signalling protein expression. Diabetologia 48, 547552.Google Scholar
30Gomez-Pinilla, F, Zhuang, Y, Feng, J, et al. (2011) Exercise impacts brain-derived neurotrophic factor plasticity by engaging mechanisms of epigenetic regulation. Eur J Neurosci 33, 383390.Google Scholar
31de Oliveira Cravo, C, Teixeira, CV, Passos, MC, et al. (2002) Leptin treatment during the neonatal period is associated with higher food intake and adult body weight in rats. Horm Metab Res 34, 400405.CrossRefGoogle ScholarPubMed
32Bonomo, IT, Lisboa, PC, Pereira, AR, et al. (2007) Prolactin inhibition in dams during lactation programs for overweight and leptin resistance in adult offspring. J Endocrinol 192, 339344.Google Scholar
33Lisboa, PC, Passos, MC, Dutra, SC, et al. (2006) Leptin and prolactin, but not corticosterone, modulate body weight and thyroid function in protein-malnourished lactating rats. Horm Metab Res 38, 295299.CrossRefGoogle Scholar
34Miljkovic-Gacic, I, Gordon, CL, Goodpaster, BH, et al. (2008) Adipose tissue infiltration in skeletal muscle: age patterns and association with diabetes among men of African ancestry. Am J Clin Nutr 87, 15901595.CrossRefGoogle ScholarPubMed
Figure 0

Fig. 1 (a) Body weight (g), (b) percentage of body-weight gain in each third week of gestation, relative to the body mass on the first day of pregnancy, (c) relative daily food intake expressed in g/d during gestation and (d) fasting blood glucose by non-trained (NT, n 4; (a, c) □ and (b, d) ), trained (T, n 4; (a, c) □ and (b, d) ), non-trained+low-protein (NT+LP, n 4; (a, c) and (b, d) ) and trained+low-protein (T+LP, n 4; (a, c) and (b, d) ) dams. Values are means with their standard errors represented by vertical bars. * Mean values were significantly different from the NT group (P< 0·05; two-way ANOVA). † Mean values were significantly different from the NT+LP group (P< 0·05; two-way ANOVA).

Figure 1

Table 1 Body weight, body length, BMI and abdominal circumference of the offspring at 30, 60, 90, 120 and 150 d old‡ (Mean values with their standard errors)

Figure 2

Fig. 2 (a) Glucose tolerance test, (b) areas under glycaemic curve, (c) insulin tolerance test and (d) rate of disappearance of glucose ‘Kitt’ of offspring at 145–147 d old from untrained (NTp, n 8; (a, c) ), trained (Tp, n 8; (a, c) ), untrained+low-protein (NT+LPp, n 8; (a, c) ) and trained+low-protein mothers (T+LPp, n 8; (a, c) ). Values are means with their standard errors represented by vertical bars. * Mean values were significantly different from the NTp group (P< 0·05; two-way ANOVA).

Figure 3

Fig. 3 Fasting blood glucose and cholesterol of the offspring at 150 d old from untrained (NTp, n 8; □), trained (Tp, n 8; ■), untrained+low-protein (NT+LPp, n 8; ) and trained+low-protein mothers (T+LPp, n 8; ). Values are means with their standard errors represented by vertical bars. * Mean values were significantly different from the NT group (P< 0·05; two-way ANOVA). † Mean values were significantly different from the NT+LP group (P< 0·05; two-way ANOVA).

Figure 4

Fig. 4 (a) Leptin in the soleus muscle and (b) plasma leptin of the offspring at 150 d old from untrained (NTp; n 8), trained (Tp; n 8), untrained+low-protein (NT+LPp; n 8) and trained+low-protein mothers (T+LPp; n 8). Values are means with their standard errors represented by vertical bars. * Mean values were significantly different from the NT group (P< 0·05; two-way ANOVA). † Mean values were significantly different from the NT+LP group (P< 0·05; two-way ANOVA).