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Compensatory Islet Response to Insulin Resistance Revealed by Quantitative Proteomics

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Compensatory Islet Response to Insulin Resistance Revealed by Quantitative Proteomics Article pubs.acs.org/jpr Compensatory Islet Response to Insulin Resistance Revealed by Quantitative Proteomics † ∥ ‡ ∥ § ∥ † ⊥ † ⊥ Abdelfattah El Ouaamari, , Jian-Ying Zhou, , Chong Wee Liew, , Jun Shirakawa, , Ercument Dirice, , † † † † ‡ Nicholas Gedeon, Sevim Kahraman, Dario F. De Jesus, Shweta Bhatt, Jong-Seo Kim, ‡ ‡ ‡ ‡ Therese R. W. Clauss, David G. Camp, II, Richard D. Smith, Wei-Jun Qian,*, † and Rohit N. Kulkarni*, † Islet Cell & Regenerative Biology, Joslin Diabetes Center, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02215, United States ‡ Biological Sciences Division and Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352, United States § Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612, United States *S Supporting Information ABSTRACT: Compensatory islet response is a distinct feature of the prediabetic insulin-resistant state in humans and rodents. To identify alterations in the islet proteome that characterize the adaptive response, we analyzed islets from 5 month old male control, high-fat diet fed (HFD), or obese ob/ob mice by LC−MS/MS and quantified ∼1100 islet proteins (at least two peptides) with a false discovery rate < 1%. Significant alterations in abundance were observed for ∼350 proteins among groups. The majority of alterations were common to both models, and the changes of a subset of ∼40 proteins and 12 proteins were verified by targeted quantification using selected reaction monitoring and western blots, respectively. The insulin-resistant islets in both groups exhibited reduced expression of proteins controlling energy metabolism, oxidative phosphorylation, hormone processing, and secretory pathways. Conversely, an increased expression of molecules involved in protein synthesis and folding suggested effects in endoplasmic reticulum stress response, cell survival, and proliferation in both insulin-resistant models. In summary, we report a unique comparison of the islet proteome that is focused on the compensatory response in two insulin-resistant rodent models that are not overtly diabetic. These data provide a valuable resource of candidate proteins to the scientific community to undertake further studies aimed at enhancing β-cell mass in patients with diabetes. The data are available via the MassIVE repository, under accession no. MSV000079093. KEYWORDS: Insulin resistance, pancreatic islets, proteome, proliferation, metabolism, function ■ INTRODUCTION While it is still debatable whether therapies should target enhancing insulin secretion from residual β-cells or increasing Type 2 diabetes has reached epidemic proportions worldwide 5 and impacts multiple organ systems. Following the develop- the number of functional insulin-producing cells, insights to design efficient therapeutics might emerge from an under- ment of insulin resistance, the onset of the disease is triggered β when the residual functional β-cells fail to compensate for the standing of the processes by which -cells compensate for a 1 chronic increased demand for insulin. Indeed, obese non- increased metabolic needs of the individual. Despite available β insulin-based and oral hypoglycemic medications, the disease diabetic individuals develop a compensatory islet -cell continues to spread worldwide and is predicted to affect over response to adjust the levels of insulin to counteract insulin 360 million individuals globally by 2030.2 Genome-wide resistance and therefore maintain normoglycemia. Generally association studies revealed that type 2 diabetes-linked genes speaking, humans with insulin resistance (e.g., impaired fasting β 3 glucose or pregnancy) exhibit increased insulin secretion as are involved in regulating -cell mass as well as function, 6 suggesting the relevance of targeting β-cells as a therapeutic compared to that in controls. However, whether this strategy for type 2 diabetes. Although islet transplantation has compensatory response is attributed to structural or functional achieved success in reversing the disease and limiting its complications,4 theshortageofisletsfromdonorshas Received: January 21, 2015 prompted a reconsideration of designing alternative therapies. Published: July 7, 2015 © 2015 American Chemical Society 3111 DOI: 10.1021/acs.jproteome.5b00587 J. Proteome Res. 2015, 14, 3111−3122 Journal of Proteome Research Article adaptation of islet β-cells is incompletely understood. Although Following removal of the buffer, pellets were frozen at −80 °C increased β-cell proliferation in metabolically challenged prior to proteomic analyses. rodents is known as a major structural adaptive response Protein Digestion within islets,7 the proportional contribution of functional changes in islet cells is unclear. Most studies that have Islet samples were homogenized and digested using a 2,2,2- trifluoroethanol (TFE)-based protocol.13 Briefly, islets were investigated islet cell function in the context of insulin μ resistance were performed in vivo,1a where the islet cell mass dissolved in 30 L of 50% TFE/50% 25 mM NH4HCO3 by 3 is a considerable confounder, and fewer in vitro metabolic min sonication in a 5510 Branson ultrasonic water bath studies have been undertaken in rat islets.8 Several proteomics (Branson Ultrasonics, Danbury, CT) with ice-cold water. Protein concentration was determined by BCA assay. About studies were performed on islets derived from insulin-resistant μ diabetic mice.9 However, these studies did not address adaptive 40 g of islet proteins from each mouse was denatured in 50% TFE for 105 min at 60 °C, reduced by 2 mM DTT for 60 min functional molecular changes in islet cells in response to insulin ° β at 37 C, diluted 5-fold with 50 mM NH4HCO3, and digested resistance but rather dysfunction of islet -cells in diabetes. In μ one study, the diabetic MKR (a transgenic mouse with a by 0.8 g of trypsin (1:50 w/w trypsin-to-protein ratio) for 3 h at 37 °C. The digestion was stopped by 0.1% TFA. All peptide dominant-negative IGF-1R in skeletal muscle) mouse was used samplesweredriedinaSpeedVacremoveTFE,and to investigate deleterious effects of insulin resistance on β-cell resuspended in 25 mM NH HCO for LC−MS/MS analysis. function.9c The same group reported a combined proteomic 4 3 and microarray screen to assess defects occurring in insulin- LC−MS/MS Analysis β 9b resistance-induced -cell failure. Interestingly, a proteomics LC−MS/MS analyses were performed on a custom-built screen was used to address the transition from obesity to automated LC system coupled online to an LTQ-Orbitrap diabetes in the Zucker fatty (ZF) and Zucker diabetic fatty mass spectrometer (Thermo Scientific, San Jose, CA) via a 9a (ZDF) rat models. Finally, a two-dimensional gel electro- nanoelectrospray ionization interface as previously described.14 phoresis approach was applied to identify proteomic changes in Briefly, 0.75 μg of peptides was loaded onto a homemade 65 the entire pancreas derived from db/db or C57BL/6J mice cm long reversed-phase capillary column with 75 μm i.d. challenged with high-fat diet (HFD); however, a major packed using 3 μm Jupiter C18 particles (Phenomenex, limitation in these studies was a lack of distinction between Torrance, CA). The mobile phase was held at 100% A (0.1% acinar and islet cells.9d,10 formic acid) for 20 min, followed by a linear gradient from 0 to Herein, we used a comparative proteomics approach to 60% buffer B (0.1% formic acid in 90% acetonitrile) over 85 characterize changes in the islet proteome in two commonly min. The instrument was operated in data-dependent mode used insulin-resistant prediabetic models, ob/ob (small or large with an m/z range of 400−2000, in which a full MS scan with a islets) and HFD mice. Ingenuity Pathway Analysis of the resolution of 100 K was followed by six MS/MS scans. The six significantly altered proteins revealed an intriguing down- most intensive precursor ions were dynamically selected in the regulation of major proteins involved in pathways critical for order of highest to lowest intensity and subjected to collision- hormone secretion, including glucose and amino acid induced dissociation using a normalized collision energy setting metabolism, Krebs cycle, mitochondrial oxidative phosphor- of 35% and a dynamic exclusion duration of 1 min. The heated ylation, hormone biosynthesis, and the final steps of exocytosis, capillary was maintained at 200 °C, and the ESI voltage was suggesting functional maladaptation of islet cells in insulin- kept at 2.2 kV. resistance states. Moreover, increased protein synthesis and MS/MS Data Analysis vesicular transport were observed, indicating endoplasmic − fi reticulum (ER) stress in insulin-resistant islets. Interestingly, LC MS/MS raw data were converted into .dta les using Extract_MSn (version 3.0) in Bioworks Cluster 3.2 (Thermo several proteins known to control cell proliferation and survival fi were upregulated in both HFD and ob/ob islets as compared to Fisher Scienti c, Cambridge, MA), and the SEQUEST algorithm (version 27, revision 12) was used to search all controls. Finally, it is notable that most proteomic changes were fi observed in both models of insulin resistance as well as in both MS/MS spectra against a mouse protein FASTA le that small and large islets. These data provide a comprehensive view contains 16 244 entries (UniProt, released on April 20, 2010). of proteomic changes occurring during obesity-induced islet The search parameters
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