Maternal protein and folic acid intake during gestation does not program leptin transcription or serum concentration in rat progeny
© The Author(s) 2011
Received: 23 March 2011
Accepted: 25 June 2011
Published: 7 July 2011
Maternal nutrition during gestation influences the development of the fetus, thereby determining its phenotype, including nutrient metabolism, appetite, and feeding behavior. The control of appetite is a very complex process and can be modulated by orexigenic and anorexigenic mediators such as leptin, which is involved in the regulation of energy homeostasis by controlling food intake and energy expenditure. Leptin transcription and secretion are regulated by numerous factors, nutrition being one of them. The present study was designed to test whether maternal nutrition can permanently affect leptin gene transcription and leptin serum concentration in rat progeny. Moreover, we analyzed whether leptin expression and secretion in response to high-fat postweaning feeding depends on the maternal diet during gestation. Pregnant rats were fed either a normal protein, normal folic acid diet (the AIN-93 diet); a protein-restricted, normal folic acid diet; a protein-restricted, folic acid-supplemented diet; or a normal protein, folic acid-supplemented diet. After weaning, the progeny was fed either the AIN-93 diet or a high-fat diet. Neither maternal nutrition nor the postweaning diet significantly affected Lep transcription. High-fat feeding after weaning was associated with higher serum leptin concentration, but the reaction of an organism to the fat content of the diet was not determined by maternal nutrition during gestation. There was no correlation between Lep mRNA level and serum leptin concentration. Global DNA methylation in adipose tissue was about 30% higher in rats fed postnatally the high-fat diet (P < 0.01). Our study showed that the protein and folic acid content in the maternal diet had no significant programming effect on Lep transcription and serum leptin concentration in the rats.
KeywordsLeptin Fetal programming Protein deficiency Folic acid supplementation High-fat diet Rat
Epidemiological observations and studies using animal models have identified characteristics of fetal programming—a form of developmental plasticity by which organisms adapt to their environment in utero. Maternal nutrition during gestation can exert a long-term effect on the progeny’s health by regulating the development, physiology, and function of the body (Chmurzynska 2010). The mechanisms of developmental programming are not thoroughly known, but it has been shown that epigenetic mechanisms may contribute to this phenomenon. The maternal intake of nutrients involved in one-carbon metabolism (e.g., sulfur amino acids, folic acid, choline, vitamin B6) may dictate the establishment of epigenetic patterns in the progeny and thus permanently alter gene expression (Mathers and McKay 2009; Burdge and Lillycrop 2010; McKay et al. 2010). A mismatch between fetal and postnatal nutritional environment may have maladaptive consequences and is considered to be a disease-promoting factor (Bateson et al. 2004; Gluckman and Hanson 2008).
Studies of rodent models indicate that fetal undernutrition can determine excess weight gain and adiposity in adulthood (Langley-Evans et al. 2005). The pathogenesis of obesity triggered by nutritional stimuli acting during prenatal life is not fully understood, but programming of appetite may be one of the contributing factors (Muhlhausler et al. 2008). It was shown that exposure to a low-protein diet during prenatal life can modify feeding behavior in the rat and stimulate a preference for high-fat foods (Bellinger et al. 2004; Bellinger and Langley-Evans 2005; Bellinger et al. 2006). However, providing a higher level of folate in the maternal diet prevents this effect (Engeham et al. 2010). It has also been reported that supplementation of protein-restricted diets with folic acid prevented the programming effects of undernutrition on DNA methylation patterns and altered metabolic phenotype (Lillycrop et al. 2005; Burdge et al. 2008).
The regulation of appetite is a complex process based on the interaction of peripheral signals with the central nervous system, in which the hypothalamus plays a pivotal role (Hainerová and Lebl 2010). Feeding behavior can be modulated by several hormones, including leptin—a polypeptide secreted by adipocytes which controls energy balance and body weight by decreasing food intake (Davis et al. 2010; Morris and Rui 2009). Since leptin concentration during fetal and neonatal life may alter hypothalamic structures, it has been proposed that leptin has an influence on the programming of appetite (Döotsch et al. 2004; Bouret 2009; reviewed by Stocker and Cawthorne 2008).
We hypothesize that maternal diet may alter the growth and development of the body and thus determine the long-term pattern of leptin secretion, which in turn may lead to changes in feeding behavior. The aim of the present study is therefore to assess the effect of protein and folic acid content in the maternal diet during pregnancy on Lep gene transcription and leptin serum concentration in the rat. The circulating leptin level reflects adiposity and energy balance but is also influenced by dietary fat content, and so we analyze here whether the reaction of an organism to the nutritional environment can be programmed in utero. We therefore test whether Lep expression and secretion in response to a postweaning high-fat diet depends on maternal nutrition during gestation.
Materials and methods
Composition of the prenatal and postnatal diets (g/kg diet)
NP + NF (modified AIN-93)
RP + NF
RP + SF
NP + SF
Total energy* (kcal/100 g diet)
At the end of the experimental period, the animals were euthanized. Blood was collected by cardiac puncture. The abdominal fat was dissected, accurately weighted, and immediately frozen and stored at −75°C for further expression analyses.
The total RNA was extracted from visceral fat tissues using Tripure Isolation Reagent (Roche), according to the manufacturer’s protocol. Approximately 1 μg RNA was taken for cDNA synthesis. Samples of RNA were incubated with a set of random hexamers (Roche, 0.25 μg/μl) and oligodT(15) (Roche, 0.25 μg/μl) at 70°C/10 min. A mixture of dNTP (5.0 mM, Roche), 1 U reverse transcriptase AMV (EURX, Poland), and 20 U Protector RNase Inhibitor (Roche) was added. After two hours of incubation at 37°C, the AMV enzyme was inactivated at 94°C/5 min. The cDNA was then diluted 3× and stored at −20°C.
The real-time PCRs were performed on a Light Cycler 2.0 (Roche) based on the SYBR Green detection system (Roche). 18S ribosomal RNA (18S rRNA) and β-actin (Actb) genes were used to normalize Lep expression. The reaction mixture (total 10 μl) consisted of 3 μl cDNA, 0.5 μl LightCycler Fast Start DNA Master SYBR Green I® kit (Roche), 20 μM MgCl2 (18S rRNA), 30 μM MgCl2 (Lep), or 50 μM MgCl2 (Actb), and 0.3 μM forward and reverse primers. The following primers were used: LEP-F: 5′GCC GGT TCC TGT GGC TTT GGT C, LEP-R: 5′GGC GGA TAC CGA CTG CGT GTG; ACTB-F: 5′CTGGGA CGA TAT GGA GAA GAT TTG, ACTB-R: 5′CAT GGC TGGGGT GTT GAA GG; 18S-F: 5′GAC CGG CGC AAG ACG AAC CAG AGC, 18S-R: 5′ AAT AAC GCC GCCGCA TCG CCA GTC. The PCR cycling conditions included predenaturation at 95°C for 10 min, followed by 45 cycles of denaturation at 95°C for 10 s, annealing at 62°C for 2 s, and elongation at 72°C for 8 s. Relative quantification of the mRNA level was performed in duplicates based on a Second Derivative Maximum Method (Roche). The abundance of Lep gene transcripts was then normalized to a geometric mean of two reference genes (18S rRNA and Actb) (Vandesompele et al. 2002).
Serum leptin concentration was measured using radioimmunological assay (Rat Leptin Radioimmunoassay Kit; Millipore Corporation, Billerica, MA, USA), according to the manufacturer’s protocol. The global DNA methylation shift in the adipose tissue of animals of different maternal dietary groups was determined using Imprint® Methylated DNA Quantification Kit (Sigma–Aldrich), according to the manufacturer’s procedure.
The results are presented as group means with their standard errors. Statistical analysis was performed using STATISTICA. The effect of maternal diet and postweaning diets on Lep expression was assessed by multi-way ANOVA, followed by a post hoc Scheffé’s test. P < 0.05 was accepted as statistically significant. Correlations were evaluated for statistical significance with Pearson’s test.
Results and discussion
Food intake was calculated as group mean calorie daily intake and was normalized for body weight (kcal/day per 100 g body weight), and no differences between maternal dietary groups were found (NP + NF 5.8 ± 0.32; RP + NF 6.06 ± 0.09; RP + SF 6.59 ± 0.27; NP + SF 6.05 ± 0.35 g/day per 100 g body weight). Neither maternal diet during gestation nor postnatal feeding affected body weight in the juvenile rats. Abdominal fat mass was influenced by fat content in the postweaning diet (P < 0.001) and was much higher in the high-fat diet–fed groups (17.2 ± 0.9 g) than in the AIN diet–fed groups (12.3 ± 0.8 g).
In the present study, there was no effect of maternal nutrition during gestation on leptin serum level in the progeny. Moreover, no interaction effect between prenatal and postnatal nutrition on leptin concentrations was found. This is in agreement with the results obtained by Engeham et al. (2010). Although they used slightly different experimental diets, they also observed no alterations in leptin serum concentrations upon prenatal protein restriction or folic acid supplementation. These data might suggest that dietary factors involved in one-carbon metabolism in the maternal diet have no programming effect on leptin expression and secretion. However, other elements of the leptin signaling cascade may be involved in adaptation to the intrauterine environment. Manuel-Apolinar et al. (2010) have shown, for example, that food restriction during gestation is associated with an increase in the hypothalamic expression of the leptin receptor gene. On the other hand, a maternal low-protein diet may change the expression of other central or peripheral mediators of appetite, as was shown by Orozco-Sólis et al. (2009). The prenatally protein-restricted animals exhibited hyperphagia, which was accompanied by enhanced expression of orexigenic peptides, agouti-related protein, and neuropeptide Y, and by decreased hypothalamic levels of the anorexigenic peptide proopiomelanocortin.
Correlation coefficients between serum leptin concentration and body weight, abdominal fat weight and calorie intake in each maternal dietary group
Leptin concentration and body weight
Leptin concentration and abdominal fat
Leptin concentration and calorie intake
NP + NF AIN
NP + NF HF
RP + NF AIN
RP + NF HF
RP + SF AIN
RP + SF HF
NP + SF AIN
NP + SF HF
The global methylation status was determined in adipose tissue, and there were no differences found between the maternal dietary groups. This is in agreement with the observations of Engeham et al. (2010). However, methylation shift was observed depending on the type of postnatal diet, with about 30% higher global methylation level being observed in the HF groups, compared to the AIN groups (P < 0.01). It has been shown that alterations in the methylation status of gene promoters are associated with the dietary fat content. Methylation of the CpGs near the transcription start site of the melanocortin-4 receptor gene decreases after high-fat feeding (Widiker et al. 2010). On the other hand, leptin promoter was methylated more in cafeteria-fed animals (Milagro et al. 2009). This may suggest that diet-induced changes in methylation are locus-specific.
Summarizing, our study has shown that protein and folic acid content in the maternal diet during gestation does not determine Lep mRNA level in adipose tissue and serum leptin concentration in the rat progeny. Moreover, the response to the high-fat diet after weaning is independent of maternal dietary history.
This study was supported by the Polish Ministry of Education and Science, grant number N N312 151034.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
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