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You are free to: Share— copy and redistribute the material in any medium or format Under the following terms: Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use. NonCommercial — You may not use the material for commercial purposes. NoDerivatives — If you remix, transform, or build upon the material, you may not distribute the modified material. http://creativecommons.org.nz/licences/licences-explained/ Proceedings of the New Zealand Society of Animal Production 2009. Vol 70: 33-38 33 Effect of growth hormone on the liver transcriptome profile during established lactation in the dairy cow D. PACHECO, K. NONES, Q. SCIASCIA and S.A. MCCOARD* AgResearch Grasslands, Private Bag 11-008, Palmerston North 4442, New Zealand *Corresponding author: [email protected] ABSTRACT Milk production involves a coordinated metabolic response from multiple tissues. Administration of growth hormone (GH) to lactating cows provides an endocrine signal that redirects nutrients towards milk production. Hepatic responses to GH define the nutrient supply to peripheral organs both directly and indirectly, through processes such as gluconeogenesis and endocrine modulation. However, the mechanisms controlling these processes in the liver itself have not been completely elucidated. Our objective was to evaluate the effect of GH on gene expression in the liver of lactating dairy cows. Using a 22,690 expressed sequence tags (EST) bovine cDNA microarrays, we examined differences in liver mRNA transcript profiles between four cows treated with a slow-release formulation of GH and four control cows treated with saline solution. We identified 38 unique transcripts in liver that met the criteria of having expression changes greater than +/- 1.2 fold and false discovery rates <0.05. Of these genes affected by GH, 29 were down-regulated and nine were up-regulated. The pathways most affected by GH were carbohydrate and lipid metabolism, molecule transport and small molecule biochemistry. This preliminary study provides further insights into the molecular changes involved in the effects of GH on liver gene expression in the lactating dairy cow. Keywords: bovine; liver; gene expression; milk; growth hormone. INTRODUCTION The objective of this study was to gain a better understanding of the effects of GH on nutrient Lactation is an orchestrated process in which partitioning in support of lactation, by evaluating the the metabolic activity of multiple tissues is mRNA transcript profile (transcriptome), in the liver modulated to direct nutrients towards the mammary using cDNA microarrays. gland. Growth hormone (GH) is a critical part of the endocrine controls modulating the utilisation of MATERIALS AND METHODS nutrients among different organs and tissues. In the lactating dairy cow, this modulation increases milk All procedures involving animals were carried production by partitioning nutrients towards out in compliance with the guidelines of the lactation (Bauman, 1992). The liver is an important AgResearch Grasslands Animal Ethics Committee. organ in this process, as it is the point of confluence Liver tissue was collected from lactating dairy of endocrine signals, nutrient-rich blood flow from cows treated with either commercially available GH the gastrointestinal tract and blood flow from (Treatment) or saline (Control) as previously peripheral tissues, including the mammary gland. described (Hayashi et al., 2009). Briefly, eight non- The contribution of liver metabolism to lactation is pregnant, second parity spring-calved Jersey cows exemplified by the fact that hepatic gluconeogenesis (300 ± 9 kg; 189 ± 11 d postpartum) were housed provides most of the glucose requirement for lactose indoors, milked twice daily, and offered ad libitum a synthesis in a lactating dairy cow (Etherton & total mixed ration diet consisting of approximately Bauman, 1998). The liver also acts as a mediator of 6.3 kg DM of pasture baleage (11.5 MJ ME/kg the responses of muscle and adipose tissue through DM), plus approximately 2.7 kg of milking cow its role in lipid and amino acid metabolism (Burton concentrate (11.5 MJ of ME/kg DM). The diet, et al., 1994; Rhoads et al., 2007). However, milk which was formulated to exceed by 10% the production is not just the result of hepatic nutrient National Research Council’s recommendations for “spill over” (Doepel et al., 20090, but rather the metabolisable energy, protein and essential amino mammary gland, liver and other organs influence acid requirements (National Research Council, each other in a complex nutrient and endocrine 2001), was offered thrice daily. Following a two- coordination. week adaptation period, four cows were randomly Although the metabolic responses to GH in assigned to each treatment groups and received lactating ruminants have been extensively studied, either a slow-release formula of commercially the molecular pathways coordinating inter-organ available recombinant GH (Lactotropin®, 500 mg nutrient metabolism and endocrine signals are not GH per cow; Elanco Animal Health, South Africa) fully understood. or sterile saline solution (0.9% sodium chloride; 34 Pacheco et al. - GH effect on bovine liver transcriptome Baxter Healthcare, Australia) via a single FIGURE 1: Diagramatic representation of the loop subcutaneous injection on Day 0. Liver tissue was design used for the microarray experiment. Three harvested on Day 6 (Treatment) or Day 7 (Control) treatments were included (A, B, C), with four cows within five minutes of euthanasia by barbiturate each (eg. A1, A2). Each arrow symbolises a overdose (Pentobarb, Provet, New Zealand). Tissue microarray slide, and letters connected by the arrows was snap frozen in liquid nitrogen and stored at indicate the pair of samples hybridised in each slide. -85°C for later analysis. Round end and point end of the arrow indicate which Microarrays sample in the pair was hybridised to Cy3 and Cy5 dyes, respectively. Only contrasts between RNA extraction treatments A (Control) and B (GH Treatment) are Total RNA was extracted using Trizol® presented in this manuscript. Reagent (Invitrogen Life Technologies, Carlsbad, California, USA) according to the manufacturer’s protocol. Extracted total RNA was quantified and quality checked using a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, California, USA). Only samples with OD260 to OD280 ratios greater than 2.0 were used for microarray hibridisations. cDNA generation, slide hybridisation and scanning Bovine cDNA microarrays were prepared as described by Diez-Tascón et al. (2005). The microarrays contained 22,690 unique amplified cDNA expressed sequence tags (EST), representing 52 tissue libraries, including the liver, and 17,307 unique genes. The liver libraries represented 465 automatic flagging of spurious spots. For this ESTs. Fluorescently-labelled cDNA (SuperScript experiment, 18 microarray slides were used in Indirect cDNA Labeling System, Invitrogen Life modification of the loop design described by Technologies, Carlsbad, California, USA; Cy5 or Oleksiak et al. (2002) with a balanced, partial dye Cy3 dyes, Amersham Biosciences, Piscataway, New swap (Figure 1). Only results from the Control and Jersey, USA) was generated from 10 µg total RNA GH treatments are presented herein. per tissue sample and purified to remove Statistical analysis of microarray data unincorporated dyes using the QIA-Quick PCR All data quality checks, normalisation and tests purification kit (Qiagen, Hilden, Germany). of treatment effects were conducted in R 2.7.0 using Microarray slides were pre-hybridised in a the IPLOTS (http://www..iPlots.org) and LIMMA preheated (42°C) 0.22 µm filtered solution of 5x (Smyth, 2005) packages. Data diagnostics included SSC (3.0 M sodium chloride; 0.30 sodium citrate), boxplots of foreground and background readings per 0.1% SDS and 0.25% bovine serum albumin (Sigma dye, within and across slides. Data flagged, internal A-788) for 20 minutes. Slides were rinsed twice in negative controls (bacterial genes) and blank spots deionised water, once in isopropanol and air dried were weighed zero prior normalisation of the data prior to hybridisation. Samples were heat-denatured using the print-tip-loess method with no background (95°C x 10 minutes) and 60 µL pre-warmed (68°C) correction. A modified t-test was performed for each SlideHyb No1 (Ambion, Foster City, California, EST and