2188 Diabetes Volume 66, August 2017 Temporal Transcriptomic and Proteomic Landscapes of Deteriorating Pancreatic Islets in Type 2 Diabetic Rats Junjie Hou,1 Zonghong Li,1,2 Wen Zhong,1,3 Qiang Hao,1 Lei Lei,1 Linlin Wang,1,4 Dongyu Zhao,1 Pingyong Xu,1 Yifa Zhou,2 You Wang,1 and Tao Xu1,3,4 Diabetes 2017;66:2188–2200 | https://doi.org/10.2337/db16-1305 Progressive reduction in b-cell mass and function com- of which appear to play a primary role in b-cell function prise the core of the pathogenesis mechanism of type 2 rather than to affect insulin resistance, further highlighting diabetes. The process of deteriorating pancreatic islets, the importance of b-cells in the pathogenesis of T2D (5). in which a complex network of molecular events is in- T2D is a complex disease, and b-cell failure is likely caused volved, is not yet fully characterized. We used RNA sequenc- by altered expression of many genes and their products. ing and tandem mass tag–based quantitative proteomics Therefore, the use of system-oriented strategies is critical technology to measure the temporal mRNA and protein to investigate the complex changes that occur in b-cells or expression changes of pancreatic islets in Goto-Kakizaki pancreatic islets, which primarily comprise b-cells. Hence, (GK) rats from 4 to 24 weeks of age. Our omics data set large-scale and unbiased omics technologies, particularly outlines the dynamics of the molecular network during the microarray-based transcriptomics and mass spectrometry deterioration of GK islets as two stages: The early stage (MS)–based proteomics, have been used to analyze islets (4–6 weeks) is characterized by anaerobic glycolysis, in- isolated from various T2D animal models and human ca- flammation priming, and compensation for insulin synthe- b sis, and the late stage (8–24 weeks) is characterized by daver donors to elucidate the mechanisms underlying -cell inflammation amplification and compensation failure. Fur- failure (summarized in Supplementary Table 1). b-Cell fail- ISLET STUDIES ther time course analysis allowed us to reveal 5,551 dif- ure during diabetes progression is a gradual process that ferentially expressed genes, a large portion of which have undergoes various stages (6,7) wherein different molecule not been reported before. Our comprehensive and tem- events occur in chronological order. However, current stud- poral transcriptome and proteome data offer a valuable ies have focused primarily on single time points at relatively resource for the diabetes research community and for late stages of the disease, so mapping the order in which quantitative biology. these events occur and distinguishing causal molecular events (leading to diabetes) from those that occur as a consequence of glucolipotoxicity associated with diabetic Type 2 diabetes (T2D) is a major public health issue conditions are impossible. For this reason, prospective characterized by pancreatic islet b-cell failure in the pres- studies investigating the evolution of molecular events ence of insulin resistance. Accumulating evidence suggests in islet b-cells at various stages of T2D are of interest. that progressive deterioration of pancreatic b-cell function The study of b-cells in humans with T2D often has and gradual loss of b-cell mass could be the core events been hindered by the limited accessibility of human islets during T2D development, regardless of therapy status and by ethical considerations. In this context, appropriate (1–4). Genome-wide association and sequencing studies rodent models are essential for the identification of diabetic have identified multiple risk variants for T2D, the majority mechanisms (8). The Goto-Kakizaki (GK) rat, one of the 1National Laboratory of Biomacromolecules, CAS Center for Excellence in Bio- Received 18 November 2016 and accepted 17 May 2017. macromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, This article contains Supplementary Data online at http://diabetes China .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1305/-/DC1. 2School of Life Sciences, Northeast Normal University, Changchun, China J.H., Z.L., and W.Z. contributed equally to this work. 3College of Life Science and Technology, HuaZhong University of Science and Technology, Wuhan, China © 2017 by the American Diabetes Association. Readers may use this article as 4College of Life Sciences, University of Chinese Academy of Sciences, Beijing, long as the work is properly cited, the use is educational and not for profit, and the China work is not altered. More information is available at http://www.diabetesjournals .org/content/license. Corresponding author: You Wang, [email protected], and Tao Xu, xutao@ ibp.ac.cn. diabetes.diabetesjournals.org Hou and Associates 2189 best-characterized animal models of spontaneous T2D (9), instructions from the manufacture (Thermo Fisher Scien- shares many characteristics with human patients with di- tific). TMT-labeled peptide mixtures were equally pooled, abetes (10). Similar to human T2D, the core cause under- separated by offline high pH reversed-phase chromatogra- lying hyperglycemia in GK rats is b-cell failure (11,12). phy, and repeatedly analyzed using a nano–liquid chro- In the current study, to understand the process of matography-tandem MS technique (Supplementary Fig. deteriorating pancreatic islets at the molecular level, we 1A). The raw MS data were processed with Proteome Dis- used RNA sequencing (RNA-seq) and tandem mass tag coverer 1.4 software. The peptide confidence value was set (TMT)–based quantitative proteomics technology to gener- as 0.01. At the protein level, a precursor intensity fraction ate integrated transcriptomic and proteomic profiles of of 50% was selected as an optimal trade-off value for both pancreatic islets in GK rats after the establishment of hyper- identification and quantification (Supplementary Fig. 1B). A glycemia (from 4 to 24 weeks). Subsequent bioinformatics pseudocount representing relative protein abundance was analysis in a time course fashion revealed the chronological calculated by using the TMT ratio and the normalized spec- order of T2D-related molecular events during the deterio- tral abundance factor (15). ration of pancreatic islets. This large quantitative data set Bioinformatics Analysis represents a valuable resource that provides a comprehen- Both mRNA raw counts and protein pseudocounts were sive picture of the mechanisms responsible for islet dysfunc- normalized by using the remove unwanted variation approach tion and will allow us to identify potential interventions to (Supplementary Fig. 1C) (16). Differentially expressed (DE) prevent b-cell failure and deterioration in human T2D. genes were assessed by ANOVA with a false discovery rate , RESEARCH DESIGN AND METHODS of 0.01. The Database for Annotation, Visualization and Integrated Discovery (DAVID) Web service API Perl Client Brief descriptions of key experimental procedures are (17,18) was used to perform gene ontology (GO) functional provided below. More details are provided in the Supple- enrichment analysis (with a false discovery rate of ,0.05). mentary Data. The k-means clustering algorithm was used to classify dy- Animals namic gene expression patterns. Kyoto Encyclopedia of Founders of GK/Jcl diabetic rats were purchased from RIKEN Genes and Genomes (KEGG) signaling pathway enrichment BioResource Center (Ibaraki, Japan). All GK/Jcl diabetic rats analysis was carried out by using the Generally Applicable and Wistar (WST) rats were maintained under specific Gene Set Enrichment (GAGE) package in R software (with pathogen-free conditions and were used between 4 and an adjusted P , 0.05) (19). 24 weeks of age in accordance with the animal experimental Data Resources guidelines set forth by the Institutional Animal Care and For this work, 92.4 gigabyte sequencing data were gener- Use Committee of the Institute of Biophysics, Chinese ated. All RNA-seq data were deposited in the National Academy of Sciences. Center for Biotechnology Information Gene Expression Preparation of Pancreatic Islets From GK and WST Rats Omnibus under accession number GSE 81811. The MS data Pancreatic islets from male GK and age-matched control were deposited in the ProteomeXchange Consortium through fi WST rats were isolated through collagenase digestion. After the PRIDE (Proteomics Identi cations) (20) partner reposi- fi separation on a Ficoll density gradient, the islets were tory with the data set identi er PXD004709. handpicked in Hanks’ buffer under a dissection microscope. RESULTS N-Acetyl-L-Cysteine Treatment Experiments Transcriptomic and Proteomic Profiles of Rat SixteenmaleGKratswereusedinthefollowingexperi- Pancreatic Islets Over Time ments. Littermates of GK rats were randomly divided into To investigate the global molecular dynamics of T2D islets, N-acetyl-L-cysteine (NAC) and control groups. Four-week- we analyzed the transcriptomes and proteomes of pancre- old rats were orally administered NAC 200 mg/kg of body atic islets isolated from male GK rats and age- and sex- weight (616-91-1; Sigma) or drinking water by gavage once matched control WST rats at five consecutive time points a day for 12 weeks. Random blood glucose assay and glucose (weeks 4, 6, 8, 16, and 24) (Fig. 1A). Transcriptomes and tolerance, insulin tolerance, and glucose-stimulated insulin proteomes of islets were measured by using the MAPS- secretion (GSIS) tests were performed as described in the based RNA-seq technique and TMT labeling–based proteo- Supplementary Data. mic method, respectively. Combined