Impact of embryo number and maternal undernutrition around the time of conception on insulin signaling and gluconeogenic factors and microRNAs in the liver of fetal sheep

Abstract

-This study aimed to determine whether exposure of the oocyte and/or embryo to maternal undernutrition results in the later programming of insulin action in the liver and factors regulating gluconeogenesis. To do this, we collect livers from singleton and twin fetal sheep that were exposed to periconceptional (PCUN; Ϫ60 to 7 days) or preimplantation (PIUN; 0 -7 days) undernutrition at 136 -138 days of gestation (term ϭ 150 days). The mRNA and protein abundance of insulin signaling and gluconeogenic factors were then quantified using qRT-PCR and Western blotting, respectively, and global microRNA expression was quantified using deep sequencing methodology. We found that hepatic PEPCK-C mRNA (P Ͻ 0.01) and protein abundance and the protein abundance of IRS-1 (P Ͻ 0.01), p110␤ (P Ͻ 0.05), PTEN (P Ͻ 0.05), CREB (P Ͻ 0.01), and pCREB (Ser 133 ; P Ͻ 0.05) were decreased in the PCUN and PIUN singletons. In contrast, hepatic protein abundance of IRS-1 (P Ͻ 0.01), p85 (P Ͻ 0.01), p110␤ (P Ͻ 0.001), PTEN (P Ͻ 0.01), Akt2 (P Ͻ 0.01), p-Akt (Ser 473 ; P Ͻ 0.01), and p-FOXO-1 (Thr24) (P Ͻ 0.01) was increased in twins. There was a decrease in PEPCK-C mRNA (P Ͻ 0.01) but, paradoxically, an increase in PEPCK-C protein (P Ͻ 0.001) in twins. Both PCUN and PIUN altered the hepatic expression of 23 specific microRNAs. We propose that the differential impact of maternal undernutrition in the presence of one or two embryos on mRNAs and proteins involved in the insulin signaling and gluconeogenesis is explained by changes in the expression of a suite of specific candidate microRNAs.pregnancy; nutrition; fetus; epigenetic IT HAS BEEN DEMONSTRATED in a range of epidemiological and experimental studies that exposure of the oocyte, embryo, or fetus to a range of environmental stressors, including poor maternal nutrition, results in poor metabolic and cardiovascular outcomes in postnatal life (8,9,13,22,25,41,42,53,56). In sheep, maternal undernutrition from 60 days before to 30 days after conception resulted in an impairment of the insulin and glucose responses to a glucose tolerance test at 10 mo after birth (49). The effects of exposure to maternal undernutrition during early gestation were also more pronounced in singleton than in twin offspring. However, it is not known whether exposure to maternal undernutrition limited to around the time of conception alone is sufficient to program changes in the insulin-signaling pathway in tissues of metabolic importance such as the liver or whether there is a differential impact of maternal undernutrition in the periconceptional period in singletons and twins.Insulin acts through the insulin receptor (IR), which is stabilized by caveolin-1 (Cav-1), resulting in a series of activations by phosphorylation of the insulin receptor substrate-1 (IRS-1) or -2 (IRS-2), phosphatidylinositol 3-kinase (PI3K), and conversion of phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3). Conversion of PIP2 to PIP3 is negatively regulated by phosphatase and tensin homolog (...

Influence of moderate maternal nutrition restriction on the fetal baboon metabolome at 0.5 and 0.9 gestation

“…This increase was shown to be due to an enhanced activity of PEPCK which results from decreased methylation of the gene's promoter [13]. Formerly, it was assumed that substantial gluconeogenesis only starts to occur` at birth [22], but more recent publications, using mammalian animal models of intrauterine growth restriction and inadequate maternal nutrition, point towards intra-uterine initiation of gluconeogenesis [13,23,24]. In fetal baboons it was shown that the PEPCK protein, a key enzyme in gluconeogenesis, was upregulated in MNR fetuses [13].…”

Impact of maternal undernutrition around the time of conception on factors regulating hepatic lipid metabolism and microRNAs in singleton and twin fetuses

“…We have also reported recently that in singleton fetal sheep exposed to maternal undernutrition during either the periconceptional or preimplantation period, there was lower abundance of key insulin-signaling molecules in the liver compared with control fetuses. In contrast, protein abundance of the key insulin-signaling molecules was higher in the liver of twin fetal sheep exposed to periconceptional or preimplantation undernutrition compared with controls (26).…”

“…All procedures were approved by the University of Adelaide Animal Ethics Committee and by the Primary Industries and Resources South Australia Animal Ethics Committee (26,49).…”

“…South Australian Merino ewes were fed a diet that consisted of lucerne chaff and pellets containing cereal hay, lucerne hay, barley, oats, almond shells, lupins, oat bran, lime, and molasses (Johnsons & Sons, Kapunda, South Australia, Australia), as described previously (26,49). All ewes received 100% of the nutritional requirements to provide sufficient energy for the maintenance of a nonpregnant ewe as defined by the Agricultural and Food Research Council (Energy and Protein Requirements of Ruminants) in 1993.…”

“…We have also determined the impact of PCUN or PIUN on the expression of candidate miRs that may play a role in the regulation of protein abundance of the key factors within the lipid metabolism pathway. We hypothesized, based on our findings on the abundance of insulin-signaling and gluconeogenic molecules in the fetal liver of the PCUN and PIUN groups (26), that exposure to PCUN and PIUN would result in decreased abundance of fatty acid ␤-oxidation molecules in singletons but increased abundance of fatty acid ␤-oxidation molecules in twins.…”