Diabetes 1 Comparison of Human and Murine Enteroendocrine Cells by Transcriptomic and Peptidomic Profiling Geoffrey P. Roberts,1,2 Pierre Larraufie,1 Paul Richards,1,3 Richard G. Kay,1 Sam G. Galvin,1 Emily L. Miedzybrodzka,1 Andrew Leiter,4 H. Joyce Li,4 Leslie L. Glass,1 Marcella K.L. Ma,1 Brian Lam,1 Giles S.H. Yeo,1 Raphaël Scharfmann,3 Davide Chiarugi,1 Richard H. Hardwick,2 Frank Reimann,1 and Fiona M. Gribble1 https://doi.org/10.2337/db18-0883 GENETICS/GENOMES/PROTEOMICS/METABOLOMICS Enteroendocrine cells (EECs) produce hormones such the quality and quantity of ingested foods. They produce at as glucagon-like peptide 1 and peptide YY that regulate least 20 different hormones, mostly peptides, that act in food absorption, insulin secretion, and appetite. Based concert to coordinate digestion, peripheral nutrient dis- on the success of glucagon-like peptide 1–based ther- posal, and appetite through actions at local and distant apies for type 2 diabetes and obesity, EECs are them- target tissues. In the field of human metabolism, glucagon- selves the focus of drug discovery programs to enhance like peptide 1 (GLP-1) and peptide YY (PYY) have raised gut hormone secretion. The aim of this study was to particular interest because of their central and pancreatic identify the transcriptome and peptidome of human EECs actions controlling food intake and insulin secretion. GLP- and to provide a cross-species comparison between 1–based drugs are widely used for the treatment of type humans and mice. By RNA sequencing of human EECs – purified by flow cytometry after cell fixation and staining, 2 diabetes and obesity, and new gut hormone based we present a first transcriptomic analysis of human EEC therapeutics are under development, aiming to mimic the populations and demonstrate a strong correlation with unrivalled effectiveness of gastric bypass surgery on weight murine counterparts. RNA sequencing was deep enough loss and type 2 diabetes resolution (1). to enable identification of low-abundance transcripts such Recent years have witnessed substantial progress in our as G-protein–coupled receptors and ion channels, reveal- understanding of murine EEC physiology, facilitated by the ing expression in human EECs of G-protein–coupled recep- generation of transgenic mice with fluorescently labeled EECs tors previously found to play roles in postprandial nutrient that enable cell identification and functional characterization detection. With liquid chromatography–tandem mass spec- through a range of approaches including fluorescence-acti- trometry, we profiled the gradients of peptide hormones vated cell sorting (FACS), transcriptomics, and live-cell imaging along the human and mouse gut, including their sequences (2–7). Our knowledge of human EECs, however, is limited by fi and posttranslational modi cations. The transcriptomic a lack of methods to identify and characterize this scattered fi and peptidomic pro les of human and mouse EECs and cell population that only comprises ;1% of the intestinal cross-species comparison will be valuable tools for drug epithelium (2). A number of G-protein–coupled receptors discovery programs and for understanding human metab- (GPCRs) have been identified and characterized in murine olism and the endocrine impacts of bariatric surgery. EECs that represent promising candidates for therapeutic approaches to enhance endogenous gut hormone secretion, Enteroendocrine cells (EECs) are specialized hormone- but tools to predict the translatability of these findings from secreting cells in the intestinal epithelium that monitor mouse to humans would be a major advance in this field (8). 1Wellcome Trust-MRC Institute of Metabolic Science, University of Cambridge, This article contains Supplementary Data online at http://diabetes Cambridge, U.K. .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0883/-/DC1. 2 ’ Cambridge Oesophago-Gastric Centre, Addenbrooke s Hospital, Cambridge, U.K. G.P.R., P.L., and P.R. are joint first authors. F.R. and F.M.G. are joint senior authors. 3INSERM U1016, Institut Cochin, Université Paris-Descartes, Paris, France © 2018 by the American Diabetes Association. Readers may use this article as 4Division of Gastroenterology, Department of Medicine, University of Massachu- long as the work is properly cited, the use is educational and not for profit, and the setts Medical School, Worcester, MA work is not altered. More information is available at http://www.diabetesjournals Corresponding authors: Fiona M. Gribble, [email protected], and Frank Reimann, .org/content/license. [email protected] Received 14 August 2018 and accepted 23 October 2018 Diabetes Publish Ahead of Print, published online February 7, 2019 2 Enteroendocrine Transcriptomics and Peptidomics Diabetes The objectives of this study were to generate tran- cells were washed twice in nuclease free 1% w/v BSA in scriptomic profiles of human EECs and to compare mouse PBS, and if a FACS facility was not immediately available, and human EECs at the transcriptomic and peptidomic they were suspended in 1% w/v BSA and 4% v/v RNAsin levels. We sequenced EECs from humans and mice at plus RNAse inhibitor (Promega, Fitchburg, WI) in PBS at a depth sufficient for the identification of low-abundance 4°C overnight. transcripts, including GPCRs and ion channels. With liquid Cells were permeabilized with either a single 30-min chromatography–tandem mass spectrometry (LC-MS/MS), incubation with 0.1% v/v Triton 3100 (Sigma-Aldrich) in we mapped the exact sequences of different gut peptides 1% w/v BSA in PBS prior to antibody staining or by the produced along the gastrointestinal (GI) tract in humans addition of 0.1% w/v Saponin (Sigma-Aldrich) to solutions and mice. in all steps from this point until after the first wash postsecondary antibody staining, with identical results. RESEARCH DESIGN AND METHODS Primary antibody staining was for 1 h in 4% v/v Ethics RNAsin, 1% w/v BSA, 1% v/v goat anti-GLP-1 (sc7782; Santa Cruz, Dallas, TX), 2% v/v rabbit anti-chromogranin This study was conducted in accordance with the princi- A (CHGA) (Ab15160; Abcam, Cambridge, U.K.), and 0.25% ples of the Declaration of Helsinki and good clinical v/v rabbit anti-secretogranin 2 (SCG2) (Ab12241; Abcam) practice. Human ethics approvals were given by Cambridge Central and South Research Ethics Committees (ref: in PBS at 4°C. Cells were then washed twice in 1% w/v BSA and 1% v/v RNAsin, and secondary antibody staining was 09/H0308/24, 16/EE/0338, 15/EE/0152) and the INSERM for 30 min in 4% v/v RNAsin, 1% w/v BSA, 0.2% v/v ethics committee and Agence de la Biomédecine (ref: donkey anti-goat Alexa 555, and 0.2% v/v donkey anti- PFS16–004). Animal work was regulated under the Animals rabbit Alex 647 in PBS at 4°C. Cells were washed twice then (Scientific Procedures) Act 1986 Amendment Regulations suspended in 4% v/v RNAsin and 1% w/v BSA in PBS on ice 2012 and conducted following ethics review by the Univer- for FACS. sity of Cambridge Animal Welfare and Ethical Review Body. FACS Human Tissue Transcriptome Cell populations were sorted on a BD FACS ARIA III in the Sample Collection National Institute for Health Research (NIHR) Cambridge Jejunal tissue was obtained from 11 human participants BRC Cell Phenotyping Hub or at Institut Cochin (Paris, (Supplementary Table 1). Samples of human jejunum France). Single cells positive for Alexa 647 but not Alexa 2 2 discarded during surgery were collected during total gas- 555 (i.e., CHGA/SCG2+ /GLP-1 e) were classified as GCG trectomy for treatment or prophylaxis of gastric cancer or enteroendocrine cells. Single cells positive for both Alexa Roux-en-Y gastric bypass for obesity. All were from the 647 and Alexa 555 were classified as GCG+ enteroendo- point of enteroenterostomy 50 cm distal to the ligament of crine cells. At least 5,000 cells were collected for each Treitz. Two matched samples of jejunum and terminal positive population. Twenty thousand double-negative ileum were collected during organ procurement from cells were collected as the negative (i.e., nonenteroendo- transplant donors. Data were collected on age, sex, and crine) cell population. Cells were sorted into 2% v/v BMI, and participants stratified as lean versus obese RNAsin in PBS at 4°C. (BMI .30 kg/m2). Tissue samples from different regions of the human GI tract for LC-MS/MS were obtained from RNA Extraction Addenbrooke’s Human Research Tissue Bank and the RNA was extracted using the Ambion RecoverAll Total Cambridge Biorepository for Translational Medicine. Nucleic Acid Isolation Kit for FFPE (Ambion, Foster City, fi Samples were immediately placed in cold Leibovitz’s CA) with modi cations to the protocol as below. The FACS- L-15 media (Thermo Scientific, Waltham, MA) and pro- sorted cell suspension was centrifuged at 3,000g for 5 min m cessed to the point of fixation or homogenized and stored at 4°C, and the pellet resuspended in 200- L digestion m at 270°C within 6 h. buffer with 4- L protease and incubated at 50°C for 3 h. The solution was then stored at 270°C for at least 12 h Tissue Preparation for FACS prior to further extraction. After thawing, RNA was FACS and RNA extraction from fixed human cells followed extracted using the manufacturer’s protocol (including a modified version of the MARIS protocol (9). Intestine a DNase step), with the exception of performing 23 was rinsed in cold PBS and the muscular coat was removed. 60-mL elutions from the filter column in the final step. Diced mucosa was digested twice in 0.1% w/v collage- The RNA solution was concentrated using a RNEasy nase XI (Sigma-Aldrich, St. Louis, MO) in Hanks’ Buffered MinElute cleanup kit (Qiagen, Hilden, Germany).