Presented at the Neonatal Society 2009 Spring Meeting .
Hyde MJ1, Clarke L2, Kemp PR3
1 Neonatal Medicine, Imperial College London, Chelsea and Westminster Hospital, 369 Fulham Road, London, SW10 9NH, UK
2 School of Agriculture, Policy & Development, University of Reading, RG6 6AR, UK
3 Section of Molecular Medicine, Sir Alexander Fleming Building, Imperial College London, South Kensington, SW7 2AZ, UK
Background: We have previously reported that caesarean section (CS) delivered piglets accumulate more hepatic lipid after 7 days on total parenteral nutrition (TPN), than vaginally delivered (VD) animals (1). The fact that alterations in hepatic metabolism persist 7 days post-partum between CS and VD piglets on the same diet suggests underlying changes in gene expression. To identify such changes, gene array analysis of hepatic RNA was performed.
Methods: Experiments were conducted under licence with relevant ethical approval. Piglets born by CS (≈3 days preterm) or VD (at term), had bilateral jugular catheters inserted 3 hours postpartum. Piglets (CS n = 5, VD n = 4) received TPN (including Intralipid 20%) for ≈7 days, after which they were killed and tissue sampled (2). Hepatic lipid was determined gravimetrically. Hepatic RNA was analysed using Affymetrix Porcine GeneChip® Arrays by the MRC CSC-IC Microarray Centre (Hammersmith Hospital, London). Genes showing a 2 fold change between the groups at p < 0.01 (two-way ANOVA followed by Benjamini-Hochberg (FDR) multiple test correction) were identified as differentially expressed. Plasma Cholesterol was determined using a commercial kit.
Results: After 7 days post partum, liver lipid content was higher (p < 0.05) in CS vs. VD piglets (CS 7.6±0.8; VD 4.2±0.2 % (w/w)). Plasma cholesterol was elevated in the VD vs. CS piglets (CS 1.04±0.25; VD 1.73±0.32 mmol∙L-1, p < 0.05). The gene array showed that 118 genes were up-regulated in excess of 2 fold (including 32 genes >4 fold higher) and 243 genes were down-regulated at least 2 fold (including 46 genes >4 fold lower) in the livers of VD vs. CS piglets. Significant changes were noted in three areas, viz. those associated with: interferon response, immature hepatocytes and cholesterol metabolism. 20 genes were elevated in VD piglets and 8 genes in CS piglets associated with inflammation. Of these, 14 up-regulated in the VD group were interferon inducible genes, (including 3 of the 4 most highly up-regulated genes). Furthermore, interferon-γ expression was increased in the VD animals. Expression of two orexigenic peptides, galanin-like peptide and angiopoietin-like 4, were significantly up-regulated with VD, interestingly, the promoters of these genes contain consensus interferon responsive elements. The CS piglets also had increased expression of genes previously described as markers of hepatocyte stem cells (3) (including PEG3, PEG10, and alpha fetoprotein). A number of genes associated with cholesterol metabolism and transport also showed significant differences between CS and VD piglets, including Apo-A1, StAR-related lipid transfer protein (START), cytosolic HMG-CoA synthase and acyltransferase ACAT2.
Conclusion: These data suggest that VD has a role in priming of the hepatic innate immune system, in the modification of cholesterol metabolism and in the normal maturation of the liver at birth either directly or as a result of the altered lipid storage. This apparent failure of CS piglets to activate the hepatic innate immune system (4) and the immaturity of the hepatocytes may have long term metabolic and immunological implications of CS.
Acknowledgements: The authors wish to thank John Laws, Anne Corson, Kate Perkins and Jennie Litten, for their assistance with animal husbandry. MJH is funded by a BBSRC Studentship.
References
1. Hyde MJ, Griffin JL, Clarke L, Kemp PR.. Neonatal Society Spring Meeting 2008; Institute of Child Health, Guildford Street, London p. 3.
2. Hyde MJ, Amusquivar E, Laws J, Corson AM, Geering RR, Lean IJ, et al. Neonatology2008; 93(2):77-86.
3. Nierhoff D, Levoci L, Schulte S, Goeser T, Rogler LE, Shafritz DA. Hepatology2007; 46(2):535-47.
4. Li Z, Diehl AM. Curr Opin Gastroenterol 2003; 19(6):565-71.