Epigenetic modification of fetal baboon hepatic phosphoenolpyruvate carboxykinase following exposure to moderately reduced nutrient availability

Abstract

Decreased maternal nutrient availability during pregnancy induces compensatory fetal metabolic and endocrine responses. Knowledge of cellular changes involved is critical to understanding normal and abnormal development. Several studies in rodents and sheep report increased fetal plasma cortisol and associated increased gluconeogenesis in response to maternal nutrient reduction (MNR) but observations in primates are lacking. We determined MNR effects on fetal liver phosphoenolpyruvate carboxykinase 1 (protein, PEPCK1; gene, PCK1 orthologous/homologous human chromosomal region 20q13.31) at 0.9 gestation (G). Female baboon social groups were fed ad libitum (control, CTR) or 70% CTR (MNR) from 0.16 to 0.9G when fetuses were delivered by caesarean section under general anaesthesia. Plasma cortisol was elevated in fetuses of MNR mothers (P < 0.05). Immunoreactive PEPCK1 protein was located around the liver lobule central vein and was low in CTR fetuses but rose to 63% of adult levels in MNR fetuses. PCK1 mRNA measured by QRT-PCR increased in MNR (2.3-fold; P < 0.05) while the 25% rise in protein by Western blot analysis was not significant. PCK1 promoter methylation analysis using bisulfite sequencing was significantly reduced in six out of nine CpG-dinucleotides evaluated in MNR compared with CTR liver samples. In conclusion, these are the first data from a fetal non-human primate indicating hypomethylation of the PCK1 promoter in the liver following moderate maternal nutrient reduction.

Figure 2. Baboon liver PEPCK1 immunoreactivity

Fraction stained (A) and density of staining (B) in arbitrary units; 0.9 gestation (G) fetuses of ad libitum fed control (CTR) mothers (n= 8); 0.9G fetuses of mothers fed 70% of control mothers (MNR; n= 6); adult CTR pregnant mothers (n= 6) at caesarean section at 0.9G. Data are means ±s.e.m.; *P < 0.05 vs. CTR fetuses.

Figure 1. Baboon liver PEPCK1 immunoreactivity

A, 0.9G fetus of an ad libitum fed mother; B, 0.9G fetus of a mother fed 70% of control mothers; C, negative control – 0.9G fetus of a mother fed 70% of control mothers; D, control maternal liver at 0.9G. Inset: confirmation of PEPCK1 antibody by Western blot (β-actin was the loading control).

Figure 3. Fetal liver PCK1 abundance: mRNA measured by QRT-PCR and protein measured by Western analysis

A, PCK1 mRNA by QRT-PCR shown as relative expression (RQ); B, PEPCK1 protein by Western blot analysis shown as PEPCK1/β-actin ratio at 0.9G. Filled bars indicate samples from fetuses of control (CTR; n= 8) ad libitum fed mothers, open bars from mothers receiving 70% of food eaten by CTR (MNR; n= 6). Data are means ±s.e.m.; *P < 0.05.

Figure 4. Location of CpG islands on the PCK1 gene promoter

A, the genomic sequences from the PCK1 promoter region that is subjected to bisulfite sequencing analysis. Primer sequences used for bisulfite sequencing are underlined. The transcriptional start site located within the TATA is denoted as +1. CpG dinucleotides that are potential targets for DNA methylation are numbered relative to the transcription start site of the PCK1 gene (shown in bold). One CpG residue is excluded from this study because it represents a SNP (single nucleotide polymorphism) in baboons. Human (HSA), baboon (PHA), and rat (RNO) genomic sequences are aligned using ClustalW and nucleotides that are conserved in humans or rats are shaded. The TESS (transcription element search system) was used to identify potential transcription binding sites using a log-likelihood cutoff of 12.5. B, 0.9G fetal baboon liver PCK1 promoter methylation for each CpG site (CTR = 8 vs. MNR = 6). Data are expressed as means ±s.e.m. **P < 0.01; ***P < 0.001.