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. 2010 Feb 22;588(Pt 8):1349–1359. doi: 10.1113/jphysiol.2009.184168

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

Mark J Nijland 1,3, Kozoh Mitsuya 1, Cun Li 1,3, Stephen Ford 4, Thomas J McDonald 1,3, Peter W Nathanielsz 1,3, Laura A Cox 2,3
PMCID: PMC2872738  PMID: 20176628

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.

Introduction

Reduced maternal nutrition in pregnancy results in impaired fetal nutrient availability and subsequent adaptive changes in both placental and fetal metabolic and endocrine function. Most studies of maternal nutrient reduction have been conducted in either rodents, which are altricial species, or sheep, which possess a specialized epithelio-chorial placenta that is different from both rodents and primates (Armitage et al. 2004). It is important that similar studies are conducted in primates, which are monotocous, precocial species in which the preparations for delivery both under normal conditions and when exposed to the challenge of reduced nutrient availability are likely to differ from rodents and sheep. We previously demonstrated that feeding pregnant baboons 70% of the global ad libitum diet fed to controls impairs placental development (Schlabritz-Loutsevitch et al. 2007) and has marked effects on fetal renal gene expression and renal structure (Cox et al. 2006; Nijland et al. 2007) as well as producing major changes in the fetal hepatic and placental IGF systems (Li et al. 2007; Schlabritz-Loutsevitch et al. 2007). Thus, this moderate level of maternal nutrient reduction is clearly sensed by the fetal baboon leading to adaptive changes in fetal growth and placental metabolism.

The fetus is completely dependent on its mother for nutrients, but as gestation progresses the fetus develops the ability to respond to decreased nutrient availability by increasing gluconeogenesis (Fowden et al. 1993). Our previous studies show that fetal baboon liver glycogen increases in the face of moderately decreased maternal nutrient intake (Li et al. 2007). Similar changes have been described in fetal sheep in response to maternal hypoglycaemia (Rozance et al. 2007). Phosphoenolpyruvate carboxykinase (PEPCK) is the key rate-limiting enzyme regulating hepatic gluconeogenesis and changes in this enzyme in response to decreased fetal nutrient availability have been extensively investigated in rodents and sheep (Warnes et al. 1977; Fowden et al. 1993; Narkewicz et al. 1993; Kwong et al. 2007; Rozance et al. 2007). Fetal plasma cortisol concentrations show a spontaneous rise in late gestation which has been linked to preparations for postnatal life including such systems as the maturation of the fetal lung (Liggins, 1969), the cardiovascular system (Unno et al. 1999) and the thyroid axis (Thomas et al. 1978). Fetal cortisol has also been shown to stimulate production of fetal liver PEPCK in sheep (Fowden et al. 1993).

PEPCK is the rate-limiting enzyme in gluconeogenesis and exists in two forms in the fetal liver, a cytosolic form PEPCK1 and mitochondrial form PEPCK2. Evaluation of the activities of the two PEPCK isoforms in fetal sheep showed a gradual rise in the cytosolic form over the last third of gestation with a steeper increase after 130 days gestation in the absence of a rise in the mitochondrial form (Warnes et al. 1977) suggesting that the cytosolic form is increased in preparation for delivery and that the mitochondrial form has more of a constitutive function, agreeing with the observations on the human PCK2 gene (Modaressi et al. 1998). When ewes, and hence their fetuses, are maintained chronically hypoglycaemic by continuous infusion of insulin from 80 days of gestation for 6 weeks, activity of hepatic cytosolic PEPCK1 tripled in the absence of any change in mitochondrial PEPCK2 activity (Narkewicz et al. 1993). We have focused on mRNA changes in PCK1, including the potential role of altered promoter methylation in the increase in PEPCK1 protein abundance.

For these reasons presented above, and based on our initial observation of increased glycogen content, we hypothesized that decreased nutrient availability to the fetus resulting from a moderate degree of global reduction in maternal nutrient intake would increase PCK1 mRNA and protein abundance in the fetal baboon liver near term. Because of its discriminatory power to both localize protein to specific cell types in complex tissues we first used immunohistochemistry (IHC) to quantify both distribution and amount of hepatic PEPCK1 protein in fetuses of ad libitum feeding control pregnant baboons and fetuses of baboons eating 70% of the food consumed by the controls. Since hepatic PEPCK1 protein was significantly elevated in fetuses of the undernourished mothers, we repeated protein quantification by Western analysis. We then measured PCK1 mRNA by QRT-PCR to determine if differences in protein expression were due to transcript abundance. Because we observed increased PCK1 transcript abundance in MNR fetal livers and studies in rodents have shown that exposure to reduced nutrient availability during development results in altered methylation of hepatic genes involved in metabolism (Wilson et al. 2002) we further hypothesized that moderate reduction in maternal nutrition would increase abundance of fetal hepatic PCK1 as a result of decreased methylation of its promoter. Finally we investigated whether the PEPCK1 increase would be accompanied by an increase in fetal plasma cortisol providing a potential mechanism linking the fetal metabolic stress induced by decreased nutrient availability to the observed protein changes.

Methods

Animal care and maintenance

A total of 13 baboon (Papio species) pregnancies were studied at the Southwest National Primate Research Center (SNPRC) at the Southwest Foundation for Biomedical Research (SFBR). All animals were housed in outdoor, purpose-built metal and concrete gang cages, each containing 10–16 females and one male. Details of housing, given below, and environmental enrichment including perches, suspended drums, manipulable nylon bones, rubber toys and plastic balls, have been published elsewhere (Schlabritz-Loutsevitch et al. 2004b). All procedures were approved by the University of Texas Health Science Center and SFBR Institutional Animal Care and Use Committees and performed in facilities approved by the Centre for Disease Control (CDC) and the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).

System for controlling and recording individual feeding

The procedures for monitoring non-pregnant morphometric homogeneity prior to pregnancy of the mothers of the fetuses studied have been published previously (Nijland et al. 2007). The animals were individually fed either between 07.00 h and 09.00 h or between 11.00 h and 13.00 h as described in detail elsewhere (Schlabritz-Loutsevitch et al. 2004b). At feeding time, all baboons exited their group cage and passed along a chute and into individual feeding cages. The weight of each baboon was obtained as she crossed an electronic scale system (GSE 665; GSE Scale Systems, MI, USA). The weight recorded was the mean of 50 individual measurements over 3 s. If the standard deviation of the weight measurement was greater than 0.01 of the mean weight, the weight was automatically discarded and the weighing procedure begun again. Water was continuously available in each feeding cage (Lixit, Napa, CA, USA) and the animals fed Purina Monkey Diet 5038 (Purina, St Louis, MO, USA). At the start of the feeding period, ad libitum-fed control baboons (CTR) were given 60 biscuits in their individual cage feeding tray. At the end of the 2 h feeding period, the baboons were returned to the group cage. The biscuits remaining in the tray, on the floor of the cage, and in the pan beneath the cage were counted. Nutrient restricted animals (MNR) were fed 70% of the CTR diet eaten by contemporaneous controls on a per kilogram basis. Food consumption of animals and their weights and health status were recorded daily.

Study design

Healthy female baboons of similar body weights (10–15 kg) were placed into two group cages. Each cage contained a social group of 10–16 randomly assigned non-pregnant females with a vasectomized male. At the end of the acclimation period to the feeding cages (30 days), a fertile male was substituted into each breeding cage. Pregnancy was dated initially by timing of ovulation and changes in sex skin colour and confirmed at 30 days of gestation (dG; term, ∼180 days) by ultrasonography. The MNR group mothers received 70% of the average daily amount of feed eaten, on a weight adjusted basis, by the CTR group at the same gestational age. Over a period of 6 months, eight CTR females and six MNR females became pregnant. Data from these 14 pregnancies, which resulted in five female and three male CTR fetuses and three female and three male MNR fetuses, are presented in this study. In addition liver samples were obtained from six of the CTR mothers at caesarean section when they were in the fasted state and under general anaesthesia.

Caesarean sections

Caesarean sections were performed at 165 days gestation (0.9 gestation (G)) using standard techniques that have been previously described in detail (Schlabritz-Loutsevitch et al. 2004a). All baboons were pre-medicated with ketamine hydrochloride (10 mg kg−1, i.m.). After intubation, isoflurane (2%) was used to maintain a surgical plane of anaesthesia throughout surgery and umbilical cord blood sampling. Following hysterotomy, the umbilical cord was identified and elevated to the surgical opening to enable the fetus to be exsanguinated while still under general anaesthesia. This method is approved by the American Veterinary Medical Association. We have demonstrated fewer tissue artifacts following exsanguination than following administration of euthanasia solutions, which produce marked histological changes, especially in the liver (Grieves et al. 2008). The placenta and fetus were removed from the uterus and immediately submitted for morphometric measurements including fetal and liver weight, complete pathological evaluation, and tissue sampling. Post-operative maternal analgesia was provided (buprenorphine hydrochloride, 0.015 mg kg−1 day−1) for 3 days (Buprenex Injectable, Reckitt Benckiser Health Care Ltd, Hull, UK) as previously described in detail (Schlabritz-Loutsevitch et al. 2004a).

Immunohistochemistry

Paraffin tissue sections from the central lobe of fetal liver were deparaffinized in xylene, rehydrated in descending grades of alcohol (100%, 70% and 45%) to water, immersed in citrate buffer (0.01 m citrate buffer, pH 6.0) and heated to boiling for 10–15 min for antibody retrieval. After cooling for 15 min, the sections were rinsed in potassium phosphate-buffered saline (KPBS; 0.04 m K2HPO4, 0.01 m KH2PO4, 0.154 m NaCl, pH 7.4; 7 times, 6 min each) and for 10 min in a solution of 1.5% H2O2/methanol and then for 5 min in KPBS. Sections were placed in diluted (10%) normal serum for 20 min and covered with primary antibody overnight at 4°C. The primary antibody for PCK-1 was used at final dilution of 1:3000 (cat. ab28455; Abcam, Cambridge, MA, USA) using the standard IHC peroxidase technique with Elite ABC kits (cat. PK-6100, Vector Laboratories, Burlingame, CA, USA) using 0.02% 3,3′-diaminobenzidine tetrahydrochloride (DAB) with 2.5% nickel sulfate as the substrate. In the absence of availability of the antigen negative controls were run with omission of the primary antibody. Three sections at 150 μm intervals were used from each animal. The specificity of the signal was confirmed by Western blot analysis showing a single band at 69 kDa.

Quantification of immunohistochemistry

Images were acquired with a SPOT cooled camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) at a magnification of 40×. For quantification, six 8-bit greyscale pictures per section from each animal were obtained at evenly spaced intervals around the central vein and analysed with Simple PCI imaging software (C Imaging, Compix Inc., Cranberry Township, PA, USA). Photomicrographs were analysed for fraction = (area immunostained/region of interest) × 100%, with minimum threshold set at 1, maximum thresholds set at: red 200, green 200, blue 255 for all images.

Western analysis

Frozen right lobe liver pieces were homogenized in lysis buffer (150 mm NaCl, 1% NP-40, 50 mm, 0.1% SDS, Tris-HCl, pH 8.0) containing protease and phosphatase inhibitors. The homogenates were centrifuged at 4°C for 10 min at 17,000 g. The supernatant proteins were estimated by Lowry assay. Protein samples (40–80 μg) were diluted 1:4 with 5× sample buffer (62.5 mm Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 350 mm DTT) and then diluted with lysis buffer to a total volume of 30 μl. The samples were fractionated on 4–20% Tris-HCl gels and electroblotted for 1 h onto 0.2 μm PVDF membranes. The membranes were immunoblotted with the same primary antibody used for IHC (1:1500 dilution, PEPCK1 (ab28455; Abcam); 1:1000 dilution, β-actin (sc-47778; Santa Cruz Biotechnology, Santa Cruz, CA, USA)) overnight at 4°C and a secondary horseradish peroxidase-conjugated antibody (1:5000 dilution, anti-rabbit sc-2004; 1:5000 dilution, anti-mouse sc-2005) for 90 min at room temperature. Specific proteins were visualized using an enhanced chemiluminescence kit (GE Healthcare, Piscataway, NJ), and immunoblot band intensities were determined using a Phosphoimager (Storm 840, GE Healthcare, Piscataway, NJ). β-Actin was used as a loading control. Protein specificity was confirmed based on detection of a single product of the expected size on the Western blot.

RNA isolation from tissue

RNA was isolated from the right lobe of the fetal liver, using TrIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. The RNA precipitate was washed with 1 ml of 75% ethanol and centrifuged at 4°C at 7500 g for 5 min. The RNA was resuspended in 100 μl diethylpyrocarbonate (DEPC)-treated water and stored at −80°C. RNA quantity and purity were determined spectrophotometrically.

Quantitative reverse transcription polymerase chain reaction (QRT-PCR) quantification of target gene abundance

To measure PCK1 mRNA levels, we used Assays-on-Demand (cat. no.: PCK1, Hs01552565_g1; Applied Biosystems, Foster City, CA, USA). To confirm gene specificity, after quantification using the TaqMan 7900 (Applied Biosystems), amplification of a single product of the correct size was validated by size-fractionating the QRT-PCR products on agarose gels. Although no baboon-specific primers or probes are available, we have successfully used the Assays-on-Demand system for more than 60 different baboon genes using human probe sets. We quantified mRNA according to manufacturer's instructions. In brief, total RNA (50 ng) was reverse transcribed in a 100 μl reaction using a High-Capacity cDNA Archive Kit (Applied Biosystems). Complimentary DNA synthesis was followed by real time PCR using gene specific primers provided by the manufacturer, TaqMan Universal PCR master mix (Applied Biosystems) and the target cDNA. The 18s rRNA (Applied Biosystems Hs99999901_s1) and MRPL48 (Applied Biosystems Hs00740658_m1) were quantified as endogenous controls using the human Assays-on-Demand probe set. All samples were assayed in triplicate.

For relative quantification of gene expression, the comparative threshold cycle (Ct) method was employed (see User Bulletin 2 for ABI Prism 7700 Sequence Detection Systems). The value obtained for Ct represents the PCR cycle at which an increase in reporter fluorescence above a background signal can first be detected (10 times the standard deviation of the baseline). Using this approach, the endogenous control Ct values were subtracted from the gene of interest Ct values to derive a ΔCt value. The ΔCt values were then converted to the relative quotient of expression (RQ value) where an increase in the RQ value represents an increase in mRNA expression.

Promoter DNA methylation analysis

The multiple well-defined cis-regulatory elements that are essential for transcriptional activation of the PEPCK gene have been identified in the promoter region (Hall, 2007; Chakravarty, 2005) and promoter methylation is one of the primary mechanisms by which transcription factor binding is modulated (Watt, 1988; Suzuki, 2008). In addition, a liver-specific control region has also been identified in the 5′-flanking region encompassing the TATA box (Beale, 2004). Hamadryas baboon genomic sequence data (whole genome shotgun reads) from the NCBI Trace Archives were treated with caution after observing a number of inaccurate sequence entries in reference to the fully characterized human PEPCK gene. Our methylation study was therefore focused on the CpG-rich region spanning the transcriptional start site of PEPCK. Genomic DNA (500 ng) from baboon tissues was sodium bisulfite treated and purified with the EpiTect Bisulfite kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Following bisulfite conversion, we performed nested PCR amplification using 1/200 of the first PCR product in the second PCR. The baboon-specific PCR primer sequences for amplification of specific targets in bisulfite-treated DNA were deduced from the NCBI Trace Archive databases (http://www.ncbi.nlm.nih.gov/traces/) and were as follows:

  • Forward (external),5′-TAATTGGAAGTTGTGTTAAAATTTATTA-3′

  • Reverse (external),5′-ACCAAATAATTATTACTCTCAAAAAAAA-3′

  • Forward (internal),5′-TGGTTTAAAGTATAATTGATTTTGGTTA-3′

  • Reverse (internal), 5′-TACAAAACTCTACTTACCTTTCTTCTCTTTAA-3′.

The PCR products were cloned into the pCR4 vector using the TOPO TA Cloning Kit for Sequencing (Invitrogen). Prior to sequencing, plasmids forming individual ampicillin-resistant clones were amplified using the PlasmidAmp Kit (Qiagen) according to the manufacturer's protocol. All sequencing was carried out using 1.0 μl of each amplification product and the BigDye Terminator v3.1 Cycle Sequencing Kits (Applied Biosystems). Sequencing reactions were treated with the BigDye XTerminator Purification Kit (Applied Biosystems) and resolved using the ABI 3730 genetic analyser (Applied Biosystems). Sequencher v4.8 (Gene Cordes, Ann Arbor, MI) and BioEdit v7.0.9 (Ibis Therapeutics, Carlsbad, CA) were used to assemble and align DNA sequences and they were groomed manually. Clones were picked randomly, and completeness of bisulfite conversion was confirmed before scoring. The CpG sites sequenced as cytosine or thymine residues were scored as methylated or unmethylated, respectively, using the BiQ analyzer (Bock et al. 2005) (http://biq-analyzer.bioinf.mpi-sb.mpg.de/) and QUMA (Kumaki et al. 2008) (http://quma.cdb.riken.jp/) programs. Nine sequences with >95% non-CpG C conversion were scored for each DNA sample. DNA methylation differences in mean values were analysed by either unpaired t test for CTR and MNR samples or two-way ANOVA with Bonferroni's post hoc test for male and female groups separately.

Analysis of plasma cortisol

Fetal plasma cortisol was measured using a chemiluminescence immunoassay system (Immulite 1000, Siemens Healthcare Diagnostics, Los Angeles, CA, USA). Within assay coefficient of variation was 4.9% and between assay coefficient of variation was 7.9%.

Statistical analysis

Hormone values were log-transformed before statistical analysis. Differences between treatment groups were evaluated using Student's unpaired t test. All data are expressed as means ±s.e.m. Parameters measured in mothers and CTR and MNR animals were compared using ANOVA with Dunnett's post hoc test. All data are presented as means ±s.e.m. Significance was set at P < 0.05 unless otherwise stated. Where the P value approached 0.05, the actual value is presented. Unless otherwise specified, n= 7 CTR and n= 6 MNR.

Results

Maternal and fetal morphometric measurements

Random allocation of non-pregnant females resulted in similar distribution of maternal weight in the two groups pre-pregnancy (Table 1). Control ad libitum fed mothers gained 11.4% of body weight during pregnancy while MNR mothers lost 5.6% of their pre-pregnancy weight. There was no difference in the duration of gestation in the two groups. At term MNR fetuses had a lower BMI. Although weight of MNR fetuses was numerically less it was not significantly reduced. Placental weight was significantly reduced in the MNR pregnancies. Apart from a trend for fetal heart weight to be decreased (P < 0.1) there were no significant differences between groups in fetal organ weights.

Table 1.

Maternal and fetal morphometrics

CTR (n= 7) MNR (n= 6)
Maternal morphometrics
Pre-conception weight (kg) 15.00 ± 0.82 14.93 ± 0.39
Caesarean section weight (kg) 16.63 ± 0.69 14.11 ± 0.77*
Change during pregnancy (%) 11.43 ± 2.58 −5.63 ± 3.89*
dG at caesarean section 165.86 ± 1.06 166.67 ± 0.80
Fetal morphometrics
Fetal weight (g) 744.43 ± 31.59 668.5 ± 33.41
Length (cm) 36.36 ± 0.76 37.5 ± 1.02
BMI (kg m−2) 5.64 ± 0.20 4.77 ± 0.20*
Placenta weight (g) 177.29 ± 7.89 145.00 ± 7.23*
Placental efficiency 4.23 ± 0.18 4.66 ± 0.31
Adrenals (g) 0.26 ± 0.02 0.24 ± 0.02
Brain (g) 77.17 ± 3.07 77.69 ± 2.49
Heart (g) 4.43 ± 0.37 3.52 ± 0.08†
Kidneys (g) 3.45 ± 0.24 3.56 ± 0.27
Liver (g) 20.48 ± 0.44 17.98 ± 1.36
Lung (g) 17.79 ± 0.86 17.94 ± 1.28
Pancreas (g) 0.54 ± 0.05 0.74 ± 0.30
Spleen (g) 1.43 ± 0.13 1.10 ± 0.03
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Control mothers (CTR; n= 7) were fed ad libitum. Mothers receiving reduced nutrition (MNR; n= 6) ate 70% of the food consumed by CTR. Data are means ±s.e.m. *P < 0.05; †P < 0.10 vs. CTR.

Fetal liver PEPCK protein evaluated by IHC

Immunoreactive PEPCK1 was concentrated around the central vein of the individual liver lobules in all fetal and adult liver tissue studied (Fig. 1A, B and D). PEPCK1 protein was low in fetuses of CTR mothers. MNR significantly increased both the area stained for PEPCK1, which increased 670%, and the density of staining, which increased by 250%, in the fetal liver (Figs 1 and 2). The effect of MNR did not differ with fetal sex (data not shown). As result of exposure to reduced nutrition the level of PEPCK1 in MNR fetuses had risen to approximately 63% of adult levels and was not significantly lower than in the pregnant adult.

Figure 1. Baboon liver PEPCK1 immunoreactivity.

Figure 1

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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 2. Baboon liver PEPCK1 immunoreactivity.

Figure 2

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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.

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

Figure 3A shows that mRNA for PCK1 was increased in the livers of fetuses of the mothers receiving reduced nutrition (2.3-fold; P < 0.05). While PEPCK1 protein measured by Western analysis was numerically higher in the MNR group this did not reach significance (Fig. 3B).

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

Figure 3

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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.

Fetal plasma cortisol

Fetal plasma cortisol was 53% higher in fetuses of MNR mothers (268 ± 2.7 ng ml−1; n= 4, P= 0.03) when compared with fetuses of CTR mothers (175 ± 19.5 ng ml−1n= 5).

Methylation

We analysed methylation of the CpG-rich region spanning the transcriptional start site of PCK1. Comparison of all genomic DNA in this region from CTR and MNR 0.9G livers showed a significant reduction by 28 ± 2.3% (P < 0.001) of DNA methylation in livers of fetuses of MNR mothers. DNA methylation varied among the nine CpG residues; 6 out of the 9 CpG dinucleotides, CpG −82, −30, −5, +31, +99 and +105, showed significant loss of DNA methylation in fetal livers of MNR mothers compared with CTR livers (Fig. 4).

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

Figure 4

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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.

Discussion

The moderate degree of maternal nutrient reduction imposed on the pregnant baboons and their fetuses in this study had only minor effects on overall fetal growth or growth of specific fetal organs (Nijland et al. 2007). Despite these limited effects on external phenotype, we have reported marked effects on detailed structure and gene and protein abundance in the placenta, fetal liver and kidney (Cox et al. 2006; Li et al. 2007; Nijland et al. 2007; Schlabritz-Loutsevitch et al. 2007). Similar studies of maternal nutrient reduction in pregnant and lactating rats have demonstrated major effects on short and long term functional outcomes while pup weight at birth was not always reduced (Armitage et al. 2004; Zambrano et al. 2005a,b;). Thus our findings further sustain the view that weight, either of the whole body or individual organs, is an inadequate measure of compromised fetal development and more refined measures of body composition are needed to assess consequences of maternal nutrient reduction on fetal and neonatal phenotype. Our findings further emphasize the view that detailed studies of cellular structure and function at the molecular level are required to determine the nature and mechanisms responsible for fetal adaptations resulting from maternal challenges such as reduced nutrient availability.

We previously reported that fetal renal mammalian target of rapamycin (mTOR), considered a sensor of the level of cellular nutrient stress, is decreased in the kidney following a 30% reduction in nutrient availability in baboon pregnancy. This finding provides evidence of the primate fetus’ ability to sense the decrease of nutrient delivery imposed (Nijland et al. 2007). There has been extensive study of adaptive metabolic changes induced in the developing rodent (Langley & Jackson, 1994; Armitage et al. 2004; Zambrano et al. 2005a,b; Guzman et al. 2006) and sheep during a variety of challenges to fetal growth and nutrition (Gentili et al. 2006; Yakubu et al. 2007; Symonds et al. 2009). In contrast, data from non-human primate pregnancy, indispensible for determining similarities and differences in outcomes and mechanisms relevant to human development, are scarce. We have reported an increase in liver glycogen in fetuses of mothers challenged, as in this study, by a 30% global reduction in food intake from 0.16G to 0.5G (Li et al. 2007). A similar rise in fetal sheep glycogen occurs during the challenges of chronic maternal hypoglycaemia and maternal hyperthermia (Regnault et al. 2006; Rozance et al. 2007). An increased concentration of fetal liver glycogen in the presence of lowered energy availability suggests fetal initiation of mechanisms that conserve energy by reduction of glycolysis or that increase glucose availability by gluconeogenesis.

Feeding pregnant rats a low protein, high carbohydrate, isocaloric diet results in increased overall enzyme activity of fetal liver PEPCK at day 20 of gestation especially if the challenge is exerted in late pregnancy (Franko et al. 2009). In another study in rats, similar offspring responses to a low protein diet were observed when the low protein diet was fed either for the whole of pregnancy or restricted to the first 4.25 days of gestation, the preimplantation period, indicating that there are potentially multiple mechanisms of activation of the increase in fetal hepatic PEPCK other than direct stimulation of more mature fetal systems (Kwong et al. 2007). The duration and extent of the challenge may be important since fetal hepatic PCK1 mRNA did not increase in response to the challenge of a slightly greater degree of nutrient reduction (50%) in ovine pregnancy from 110 days gestation to term (Hyatt et al. 2008). Alternatively there may well be species differences.

Although we know of no data that support a direct association between circulating cortisol and DNA methylation, cortisol has been shown to be a key regulator of PEPCK mediated via a glucocorticoid response element (GRE) in the PCK-1 gene promoter (Cassuto et al. 2005). In one early study, infusion of dexamethasone was shown to increase fetal hepatic glycogen in rhesus monkeys (Epstein et al. 2009). Infusing cortisol intravenously to fetal sheep for 5 days increases glycogen deposition and the activities of G6Pase and PEPCK in the fetal liver (Fowden et al. 1993). In contrast, in one study, maternally administered dexamethasone had no effect on hepatic PEPCK enzyme activity in the fetal liver but did increase glucose-6-phosphatase. The authors concluded that dexamethazone's effects on the fetal sheep liver are primarily on glycogenolysis rather than gluconeogenesis. However, fetal liver samples were obtained only 34 h after the initial exposure to dexamethasone. As a result it is possible that an insufficient time may have elapsed to allow for the transcriptional and translational processes necessary to synthesize enough protein for PEPCK activity to increase. However, there was sufficient time for the stimulation of both transcription and translation of glucose-6-phosphatase to enhance activity (Franko et al. 2007). Deaxamethasone given to pregnant rats daily for the last 7 days of gestation results in increased PEPCK enzyme activity in adult offspring (Nyirenda et al. 2001). Fetal cortisol increases spontaneously at term and plays a role in maturation of fetal organs such as the lung (Liggins, 1969). However, factors other than cortisol probably play a role in the late gestation increase in gluconeogenic capacity of the developing liver since levels of other key glucoenogenic enzymes such as alanine aminotransferase are not elevated by cortisol infusion but do increase with gestational age (Fowden et al. 1993).

Epigenetic gene modification has been shown to play a central role in developmental programming. When rats are fed a 50% reduced protein, isocaloric diet during gestation with a normal diet during lactation, methylation of the peroxisome proliferator-activated receptor alpha (PPARα) promoter in the offspring liver at day 34 of postnatal life was 26% lower than controls due to specific reduction at four CpG dinucleotides by from 33–48%. These changes persisted until studied at 80 days of postnatal life. The involvement of one carbon cycle methylation was indicated by the ability to reverse the methylation changes by the addition of high doses of folate to the maternal low protein diet (Lillycrop et al. 2008).

To address the potential impact of DNA methylation on PCK1 gene activation in livers of MNR baboon fetuses, we performed bisulfite sequencing analysis of genomic DNA. The multiple well-defined cis-regulatory elements that are essential for transcriptional activation of PCK1 have been identified in the promoter region (Chakravarty et al. 2005; Hall et al. 2007) and methylation of CpGs in the promoter is a primary mechanism by which transcription factor binding is modulated (Watt & Malloy, 1988; Suzuki & Bird, 2008) Our methylation study focused on the CpG-rich region spanning the transcriptional start site of PCK1. We found 4 out of the 9 CpG sites (CpG −50, +31, +74 and +99) are conserved between baboons and humans. An overall comparison of genomic DNA from CTR and MNR fetal livers showed a significant reduction (28%, P < 0.001) of DNA methylation in MNR livers. The degree of DNA methylation varied among the 9 CpG residues; examining DNA methylation profiles of the individual CpG dinucleotides, we found that 6 out of the 9 CpG dinucleotides (CpG −82, −30, −5, +31, +99 and +105) exhibited a significant loss of DNA methylation. The PCK1 promoter is stimulated by cyclic adenosine monophosphate (cAMP) and contains a CREB-binding site (CRE) which is recruited when the gene is activated by glucagon and glucocorticoids (Wilson et al. 2002).

The differentially methylated CpG motifs are located close to the binding sites for the transcriptional activators CREB and E2F, both of whose activities are potentially modulated by DNA methylation (Iguchi-Ariga & Schaffner, 1989; Campanero et al. 2000), and suggests one mechanism whereby methylation status of the PCK1 promoter may influence PCK1 transcriptional activation in 0.9G baboon livers. Altered methylation is now considered a significant mechanism in modifying gene expression as a result of nutritional challenges during development. Interestingly, neonatal overfeeding achieved by reducing rat litters to three pups with the contol litters consisting of 12 pups resulted in hypermethylation of the promoter of the main anorexigenic neurohormone, proopiomelanocortin (POMC), at CpG dinucleotides within the Sp1 and NFκB binding sequences in overfed offspring (Plagemann et al. 2009).

Poor delivery of nutrition during development has been shown to have major effects on the fetus and newborn in a wide variety of species. Many of these studies have been conducted postnatally in rodents and involve challenges to systems that develop prenatally in precocial species. It is important to keep in mind that the environment acting on the gene during fetal life differs greatly from the postnatal environment. Of special significance are the lower fetal Inline graphic (about half the tension in the blood of the extra-uterine neonate), lower plasma glucose concentrations in the fetal compartment compared with postnatal mammals and importantly differences in the current level of activity of glucocorticoids (Koenen et al. 2002). To our knowledge this is the first demonstration of an epigenetic alteration of a gene that plays a key role in intermediary metabolism and preparation for an independent extra-uterine life in response to decreased nutrient availability in a non-human primate. The increased PCK1 mRNA expression is likely to reflect the decreased methylation of the PCK1 promoter. It remains to be seen whether these changes persist into postnatal life as shown for similar gene methylation changes in altricial species.(Ozanne et al. 1996).

Acknowledgments

This work was supported by NIH HD 21350. We gratefully acknowledge the technical assistance provided by Jeremy P. Glenn, Kenneth Lange and Myrna Miller. This investigation was conducted in facilities constructed with support from Research Facilities Improvement Program Grant Numbers C06 RR015456 and C06 RR013556 from the National Center for Research Resources, National Institutes of Health. We would like to thank Karen Moore for her help with the preparation of this manuscript.

Glossary

Abbreviations

CTR

control

IHC

immunohistochemistry

MNR

maternal nutrient reduction

PEPCK

phospho-enolpyruvate carboxykinase

Author contributions

M.J.N., K.M., P.W.N. and L.A.C. contributed to the conception, design and interpretation of the studies; K.M., C.L., T.J.M. and L.A.C. contributed to analysis and interpretation of the data; all authors contributed to the drafting and revision of the manuscript and approved the version to be published.

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