Comment[ArrayExpressAccession] E-GEOD-45554 MAGE-TAB Version 1.1 Public Release Date 2013-10-18 Investigation Title Resistant starch induces catabolic but suppresses immune and cell division pathways and changes the microbiome in proximal colon of male pigs Comment[Submitted Name] Resistant starch induces catabolic but suppresses immune and cell division pathways and changes the microbiome in proximal colon of male pigs Experiment Description Consumption of resistant starch (RS) has been associated with various intestinal health benefits, but knowledge on its effects on global gene expression in the colon is limited. The main objective of the current study was to identify genes affected by RS in the proximal colon to infer which biologic pathways were modulated. Ten 17-wk-old male pigs, fitted with a cannula in the proximal colon for repeated collection of tissue biopsy samples and luminal content, were fed a digestible starch (DS) diet or a diet high in RS (34%) for 2 consecutive periods of 14 d in a crossover design. Analysis of the colonic transcriptome profiles revealed that, upon RS feeding, oxidative metabolic pathways, such as the tricarboxylic acid cycle and β-oxidation, were induced, whereas many immune response pathways, including adaptive and innate immune system, as well as cell division were suppressed. The nuclear receptor peroxisome proliferator-activated receptor γ (PPARG) was identified as a potential key upstream regulator. RS significantly (P < 0.05) increased the relative abundance of several butyrate-producing microbial groups, including the butyrate producers Faecalibacterium prausnitzii and Megasphaera elsdenii, and reduced the abundance of potentially pathogenic members of the genus Leptospira and the phylum Proteobacteria. Concentrations in carotid plasma of the 3 main short-chain fatty acids acetate, propionate, and butyrate were significantly higher with RS consumption compared with DS consumption. Overall, this study provides novel insights on effects of RS in proximal colon and contributes to our understanding of a healthy diet. Ten pigs were fitted with a cannula in the proximal colon for repeated collection of tissue biopsies, and were fed a digestible starch or a resistant starch diet for two consecutive periods of 14 days in a crossover design. After each intervention period a colonic biopsy was taken and subjected to gene expression profiling. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Hooiveld Haenen Müller Hooiveld Person First Name Guido Danielle Michael Guido Person Mid Initials J Person Email guido.hooiveld@wur.nl Person Affiliation Wageningen University Person Address Div. Human Nutrition, Wageningen University, Bomenweg 2, Wageningen, Netherlands Person Roles submitter Protocol Name P-GSE45554-1 P-GSE45554-5 P-GSE45554-6 P-GSE45554-2 P-GSE45554-3 P-GSE45554-4 P-GSE45554-7 Protocol Description Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'Oligo' (v1.22.0). ID_REF = VALUE = RMA signal (as log2) One hundred nanogram of RNA was used for whole transcript cDNA synthesis with the Ambion WT expression kit [catalog number 4411974] (Applied Biosystems/Life Technologies, Nieuwekerk a/d IJssel, The Netherlands). Hybridization of 5.5μg labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridization Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Washing and staining of the arrays were done on an Affymetrix 450 fluidics station using the protocol FS450_0002, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). At 19 weeks of age pigs underwent surgery for the placement of a cannula in the proximal colon. After 4 to 6 d of postsurgical recovery, pigs were gradually switched to one of two dietary treatments (digestible [control] and resistant starch) that lasted for 14 days. On day 14 after start of the first diet intervention (period 1) biopsies were taken from the proximal colon, after which diets were switched (cross-over). After the second intervention period of 14 days (period 2) biopsies were again taken. Male Landrace pigs (barrows) were obtained from the Pig Innovation Centre (Sterksel) of Wageningen University. Pigs were individually housed in metabolism pens of 2 m2, equipped with a feeder. Artificial lights were on from 0500 until 1900 and dimmed during the dark period. Pigs had free access to standard pig feed until surgery, and had free access to tap water during the whole study. Total RNA was prepared from biopsies using TRIzol reagent, whereafter purified total RNA was isolated using Qiagen RNEasy columns. RNA integrity was checked on chip analysis (Agilent 2100 bioanalyzer, Agilent Technologies, Amsterdam, the Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0). Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the Genechip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5). Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name STRAIN OR LINE DIET AGE Experimental Factor Type strain or line diet age Publication Title Resistant Starch Induces Catabolic but Suppresses Immune and Cell Division Pathways and Changes the Microbiome in Proximal Colon of Male Pigs. Publication Author List Haenen D, Souza da Silva C, Zhang J, Koopmans SJ, Bosch G, Vervoort J, Gerrits WJ, Kemp B, Smidt H, M�ller M, Hooiveld GJ PubMed ID 24132577 Publication DOI 10.3945/jn.113.182154 Comment[SecondaryAccession] GSE45554 Comment[GEOReleaseDate] 2013-10-18 Comment[ArrayExpressSubmissionDate] 2013-03-27 Comment[GEOLastUpdateDate] 2013-10-20 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-45554.sdrf.txt