Butyrate-Induced Transcriptional Changes in Human Colonic Mucosa Steven A. L. W. Vanhoutvin1,2*, Freddy J. Troost1,2, Henrike M. Hamer1,2, Patrick J. Lindsey3, Ger H. Koek2, Daisy M. A. E. Jonkers1,2, Andrea Kodde1,2, Koen Venema1,4, Robert J. M. Brummer1,2,5 1 TI Food and Nutrition, Wageningen, The Netherlands, 2 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Maastricht University, Maastricht, The Netherlands, 3 Department of Population Genetics, Genomics and Bioinformatics, Maastricht University, Maastricht, The Netherlands, 4 Department of Bio Sciences, TNO Quality of life, Zeist, The Netherlands, 5 School of Health and Medical Sciences, O¨ rebro University, O¨ rebro, Sweden Abstract Background: Fermentation of dietary fiber in the colon results in the production of short chain fatty acids (mainly propionate, butyrate and acetate). Butyrate modulates a wide range of processes, but its mechanism of action is mostly unknown. This study aimed to determine the effects of butyrate on the transcriptional regulation of human colonic mucosa in vivo. Methodology/Principal Findings: Five hundred genes were found to be differentially expressed after a two week daily butyrate administration with enemas. Pathway analysis showed that the butyrate intervention mainly resulted in an increased transcriptional regulation of the pathways representing fatty acid oxidation, electron transport chain and oxidative stress. In addition, several genes associated with epithelial integrity and apoptosis, were found to be differentially expressed after the butyrate intervention. Conclusions/Significance: Colonic administration of butyrate in concentrations that can be achieved by consumption of a high-fiber diet enhances the maintenance of colonic homeostasis in healthy subjects, by regulating fatty acid metabolism, electron transport and oxidative stress pathways on the transcriptional level and provide for the first time, detailed molecular insight in the transcriptional response of gut mucosa to butyrate. Citation: Vanhoutvin SALW, Troost FJ, Hamer HM, Lindsey PJ, Koek GH, et al. (2009) Butyrate-Induced Transcriptional Changes in Human Colonic Mucosa. PLoS ONE 4(8): e6759. doi:10.1371/journal.pone.0006759 Editor: Stefan Bereswill, Charite´-Universita¨tsmedizin Berlin, Germany Received January 21, 2009; Accepted July 9, 2009; Published August 25, 2009 Copyright: ß 2009 Vanhoutvin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was funded by Top Institute of Food and Nutrition, Wageningen, The Netherlands. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction modulate numerous processes. It induces cell differentiation and strongly inhibits cell proliferation in tumor cell lines [7–13]. Short-chain fatty acids (SCFAs) are derived from the microbial Colonocytes use butyrate as their primary energy source and in fermentation of undigested dietary fibers in the colon. As micro- the absence of butyrate they undergo apoptosis, but opposite organisms preferably ferment carbohydrates, most saccharolytic effects were seen in transformed cells, suggesting a possible anti- fermentation occurs in the proximal colon. Depletion of carcinogenic effect of butyrate [13–15]. Furthermore, butyrate carbohydrate sources in the distal colon leads to a switch from mayhaveaneffectoninflammation[13],oxidativestress[13], saccharolytic to proteolytic fermentation, which is less favorable intestinal barrier function [13,16,17], visceral perception and due to the formation of potentially toxic products. Both these rectal compliance [18] and may play a role in satiety [19,20]. toxic products and the lower availability of SCFAs in the distal Transcriptional responses of butyrate were studied mostly in cell colon are hypothesized to be involved in the pathogenesis of lines [14,15,21–32] and some studies were performed in animals gastro-intestinal disorders such as ulcerative colitis (UC) and and human patients [3,30,33–35]. In vitro and animal studies cancer [1–3]. The amount of SCFAs (mainly acetate, propionate showed that butyrate downregulates the expression of genes and butyrate) produced in the colon depends on the site of associated with proliferation and oxidative stress and upregulates fermentation, the diet and the composition of the microbiota, and the expression of Mucin associated genes (Muc 1–4), tight junction can account for up to 5–15% of the total energy requirements of proteins (zonulin and occludin) and the butyrate transporter humans [4]. Fecal concentrations of acetate, propionate and monocarboxylate transporter-1 (MCT1). In UC patients, butyrate butyrate are found in a molar ratio of approximately 60:20:20 was shown to increase the expression of the butyrate transporter [5,6], but limited data about luminal concentrations in specific MCT-1 and to decrease inflammation by inhibition of the parts of the colon are only available from sudden death patients. activation of NF-kB. Effects of butyrate on global, genome-wide Due to rapid absorption and metabolism, actual concentrations transcriptional responses of human intestinal mucosa were not may differ. Among the different SCFAs, butyrate is known to described previously. PLoS ONE | www.plosone.org 1 August 2009 | Volume 4 | Issue 8 | e6759 Effects of Butyrate in Humans The aim of this study was to determine the in vivo genome-wide seconds at maximum speed using a minibeadbeater. 200 ml transcriptional response to local administration of butyrate in the Chloroform was added and after 3 minutes of incubation, the distal colon in healthy volunteers in order to identify the biological samples were centrifuged for 15 minutes, 21000 g at 4uC. 500 ml processes mediated by butyrate, providing new leads for clinical was taken from the upper colorless phase and mixed with 500 ml and mechanistic studies. 70% ethanol. RNA was further purified with an RNeasy mini kit (Qiagen, Venlo, The Netherlands) combined with a DNase Materials and Methods treatment using the RNase-Free DNase set (Qiagen, Venlo, The Netherlands) according to manufacturers protocol. Objectives Quantity and purity of the RNA samples was determined using To determine the in vivo transcriptional response of a local the Nanodrop ND-1000 spectrophotometer (Nanodrop Technol- administration of butyrate in the distal colon in healthy volunteers. ogies, Wilmington, USA) and RNA integrity was determined using the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, USA). Participants Sixteen healthy volunteers (12 females and 4 males, 18 to 62yrs) Microarray hybridization participated in this study. Exclusion criteria were signs of bowel Total RNA (150 ng) was amplified using the two-cycle cDNA dysfunction, gastrointestinal surgery, age over 65 years, or use of any synthesis kit (Affymetrix, Santa Clara, USA) in combination with medication, probiotics or prebiotics three months prior to inclusion, the MEGAscript T7 in vitro transcription system (Ambion). were excluded from participation. All participants signed an Double-stranded cDNA was biotin labeled with the GeneChip in informed consent prior to participation to the study, which was vitro transcription IVT labeling kit (Affymetrix, Santa Clara, USA). approved by the Ethical Committee of the University Hospital Following fragmentation, 11 mg of biotin-labeled cRNA were Maastricht, the Netherlands, and conducted in full accordance with hybridized for 16 hour at 45uC on Affymetrix Human Genome the principles of the ‘Declaration of Helsinki’ (52nd WMA General U133 Plus 2.0 Arrays. assembly, Edinburgh, Scotland, Oct 2000). The study has been GeneChips were washed and stained in the Affymetrix Inc. registered in the US National Library of Medicine (http://www. Fluidics Station 450 (Affymetrix, Santa Clara, USA) and clinicaltrials.gov) with reference code NCT00693355. hybridized. Cyclic RNA was detected using streptavidin coupled to phycoerythrin. GeneChips were scanned using GeneChip Design Scanner 3000/7G and GeneChip Operating System (GCOS, Affymetrix, Santa Clara, USA) using Affymetrix default settings. The study was performed according to a double-blind randomized placebo-controlled cross-over design. The protocol comprised of two experimental periods of two weeks each with a Microarray analysis wash-out period of two weeks in between (Figure 1). During each Images of the Human Genome U133 Plus 2.0 arrays were experimental period, the subjects self-administered an enema quantified with GCOS software (Affymetrix). The chip description containing 100 mM of butyrate or placebo (60 ml, pH 7.0), file (CDF) used for the analysis was an update created and freely respectively, once daily prior to sleeping. The local hospital distributed by the microarray lab of the university of Michigan pharmacy department prepared all enemas. The volunteers were (http://brainarray.mbni.med.umich.edu) [36] based on UniGenes asked to defecate, if possible, prior to instillation of the enema. At (version 8). A more detailed description of this analysis is shown in the the end of each experimental period, a sigmoidoscopy was supplementary data (Statistics S1). Briefly, the genes were analyzed