Research Article: Hepatic DNA methylation and expression profiles under prenatal restricted diet in three generations of female rat fetuses

Date Published: April 16, 2019

Publisher: Public Library of Science

Author(s): Joanna Nowacka-Woszuk, Adrian Grzemski, Magdalena Sliwinska, Agata Chmurzynska, Cristina Óvilo.


The nutritional factors acting during early life can affect the development of the organism. It has been hypothesized that such programmed traits can be inherited by later generations. In this work, we present for the first time the effect of food deprivation in pregnant dams and its consequences for the transcription and DNA methylation profiles in the offspring of the next three generations. We used a 50% reduction in dietary intake during pregnancy in the rat and determined whether this altered the hepatic DNA methylation and transcription levels in female fetuses over three generations. Targeted next-generation sequencing (tNGS) was used in the first generation for 1748 genes associated with six selected biological processes. The selected cytosines were then studied by pyrosequencing in F1–F3. The transcript level of the selected genes was determined by the real-time PCR. The tNGS approach indicated 394 cytosines, in close proximity to 374 genes, with a statistically significant difference in methylation levels between the control and restricted groups. A gene clustering analysis revealed 23 molecular pathways to which the studied genes were assigned. Only seven cytosines were differently methylated to more than 10%, and so these sites were studied next using pyrosequencing. The observation from NGS was confirmed for only one cytosine located near the St6galnac5 gene, though this was with the opposite effect. A difference was also observed for the Usp30 gene, though in proximity to the cytosine selected from NGS. In F3, the differences were observed for the Oxct2b gene. We also found differences in methylation levels between generations for the Grb10 and St6galnac5 genes, but independently of the diet used. The transcript levels of selected genes (Usp30, Grb10, Pld1, St6galnac5, Oxct2b, Khk, and Acsl4) were not altered in F1, while changes were detected for Pld1 and Oxct2b in F2 and F3, respectively. Prenatal food deprivation did not induce broad changes in hepatic DNA methylation of the genes involved in lipid or carbohydrate metabolism, and did not result in alterations in their transcription. Thus, the hypothesis that transgenerational inheritance is induced by dietary restriction was not confirmed.

Partial Text

The use of prenatal restricted diets in rodent models can be considered to be a counterpart of the Dutch Hunger Winter syndrome observed in humans after the Second World War, as malnutrition during pregnancy leads to an effect on the health of the progeny in their adult life [1, 2]. It has already been well documented that suboptimal in utero environments in humans lead to long-term consequences for gene expression, DNA methylation, and phenotype [3]. These consequences of prenatal factors acting on the developing fetus are referred to as fetal programming [4]. In rodent models, nutrient-deficient prenatal diets have been widely studied with different modifications in terms of regimen type, time of exposure, and so on [5]. Our previous study applied a calorie-restricted diet [6], which was found to have greater impact on the lipid profile in the F0 dams than in their progeny. The hepatic expression level of the key lipid metabolism gene (Fasn) was also altered, while its methylation remained unchanged in response to the applied diet. This is in agreement with the earlier study of Lee et al. [7], in which the hepatic Fasn mRNA level also increased in response to the maternal calorie-restricted diet.

We performed targeted bisulfite NGS to determine the methylation level of over 1700 genes mostly involved in energy homeostasis pathways, and we found that over 300 cytosines showed very small though significant differences between the control and restricted groups. The clustering analysis indicated 23 detailed molecular pathways involving these genes. In the majority of these pathways, these genes were not directly functionally related to each other, with the exception of the oxidative phosphorylation process. In this pathway, 22 genes had altered DNA methylation—those encoding enzymes for NADH dehydrogenases, succinate dehydrogenases, ubiquinol-cytochrome c reductases, cytochrome c oxidases, pyrophosphatase, and ATP synthases. The differences were small (± 3%), but it cannot be excluded that such minor differences may affect metabolism, especially when they are involved in the same processes. The oxidative phosphorylation process that takes place in mitochondria is a key pathway where adenosine triphosphate (ATP) is produced as the main source of energy for cell functioning, as well as for the maintenance of metabolic homeostasis [14]. It can thus be speculated that altered methylation of genes involved in this process can change their expression levels, leading to a disruption of energy balance. Since small differences in a majority of genes were observed for other metabolic pathways, we anticipated that the use of the restricted diet during pregnancy did not dramatically alter DNA methylation in the offspring. In a previous study by Ogawa et al. [15], it was found that a prenatally experience calorie-restricted diet induced a wide range of changes in the hepatic methylome in the fetal liver of mice. It should be mentioned that Ogawa et al. used a different experimental approach in which the biological samples from individual animals were pooled and the methylation level was established using methylated DNA immunoprecipitation with microarray detection (Me-Dip-ChiP). They found that multiple genes involved in glucocorticoid signaling, insulin resistance, PPAR targets, cholesterol and fatty acid metabolism pathways, and immune response processes had altered methylation levels in their promoter regions. Of these, the imprinted Grb10 gene (growth factor receptor-bound protein 10), involved in insulin and mTOR signaling pathways, was changed by the restricted maternal diet. This was also seen in our study, where NGS analysis showed reduced methylation in the restricted group. Ogawa et al. [15] also observed methylation differences in the Acsl6 (long-chain-fatty-acid-CoA ligase 6) gene, which is involved in fatty acid metabolism, whereas our NGS study found such differences in Acsl4, which belongs to the same gene family. Another study employing a 50% reduction in the calorie intake of pregnant mice was performed by Ganguly et al. [16]. To determine the effect of the pregnant dams’ diet on the developing fetuses, the authors focused on transplacental glucose transport processes and studied the Glut1 and Glut3 genes. They noted that, in the restricted group, the methylation level in the promoter region of Glut3 was significantly increased. Moreover, this led to enhanced binding of methyl-CpG-binding protein (Mecp2), together with histone deacetylases (Hdac2); consequently, a reduced level of placental transcription of Glut3 gene was observed. This demonstrates that gene expression was altered by changed epigenetic mechanisms induced by the dietary factor. In our earlier study, we did not observe any such correlation for Fasn, a key lipid metabolism gene [6]. It should be mentioned that, in this study, the seven differently methylated cytosines indicated by NGS were also analyzed by pyrosequencing, and only one of them (near the St6galnac5 gene) had such a correlation, though with a very small difference between the studied groups. This shows that the technological approach must be considered when drawing conclusions. Since minor effects on DNA methylation could be expected from the experimental procedures, the targeted methods applied here for mainly energy homeostasis genes should be extended to global DNA methylation analysis in the future. Moreover, we found that the methylation level for some cytosines in Grb10 and St6galnac5 genes in the first generation was lower than in F2 and F3, in both the C and R groups. A similar tendency, though not significant, was observed for the other examined genes. It can thus be speculated that unknown factors acting on both control and restricted groups affected the methylation profile in F1.

In summary, we postulate that use of the calorie-restricted diet during rat pregnancy did not dramatically alter the DNA methylation of the genes involved in energy homeostasis, and transgenerational inheritance was not observed. Moreover, changed CpG methylation was not accompanied by alterations in transcription level. However, the effect of some minor differences in the methylation level of strictly functionally related genes cannot be excluded. We speculate that overall food deficiency activates regulatory processes that optimize the intrauterine environment for the developing fetuses. These processes stimulate the use of stored nutrients and prevent deregulation of DNA methylation. It must be also borne in mind that we did not perform genome-wide methylation analysis, so changes in other groups of genes were possible.




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