INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 28: 153-160, 2011 Alteration of the proteome profile of the pancreas in diabetic rats induced by streptozotocin YONG-LIANG JIANG, YOU NING, XIAO-LEI MA, YAN-YAN LIU, YU WANG, ZENG ZHANG, CHUN-XIAO SHAN, YU-DONG XU, LEI-MIAO YIN and YONG-QING YANG Yue Yang Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200030, P.R. China Received February 18, 2011; Accepted April 12, 2011 DOI: 10.3892/ijmm.2011.696 Abstract. Type 1 diabetes mellitus (T1DM) is receiving Introduction increased attention. To obtain a better understanding of the mechanism underlying T1DM, we performed a proteomic Type 1 diabetes mellitus (T1DM) is a multifactorial disease, study on a rat model induced by streptozotocin. Pancreatic which is characterized by the destruction of the insulin- proteins were separated by two-dimensional gel electro- secreting β cells of the islets of Langerhans in the pancreas. phoresis. Eighteen protein spots were differentially expressed The destruction of β cells leads to severe insulin depletion, (P<0.05) with 2-fold or more increased or decreased intensity which results in hyperglycemia, because of hepatic over- in the diabetic rats as compared with controls, of which 11 production of glucose by glycogenolysis and gluconeogenesis protein spots were up-regulated and 7 protein spots were down- and decreased cellular uptake of glucose from the circulation. regulated. These protein spots were successfully identified by The onset of T1DM is involved in different genetic and/or liquid chromatography-electrospray ionization tandem mass environmental susceptibility determinants, with different spectrometry. The 60 kDa heat shock protein, the carbonyl mechanisms leading to β cell destruction (1). Increased oxidative reductase 1 (Cbr1), the hydroxyacyl-CoA dehydrogenase, stress has been reported to be a participant in the onset of Δ3,5,Δ2,4-dienoyl-CoA isomerase, the elongation factor 1-δ, diabetes (2,3), which can cause heavy damage to β cells because the 26S protease regulatory subunit 7 and the transitional of very low amounts of intrinsic antioxidant enzymes in β endoplasmic reticulum ATPase were up-regulated, while cells (4). It has also been reported that the autoimmune attack the 78 kDa glucose-regulated protein, peroxiredoxin 4 and is involved in β cell destruction of T1DM (5). However, the plakoglobin were down-regulated. The expression change of exact mechanisms of this disease still remain largely unknown. Cbr1 which is closely related to diabetic complications was Animal models are widely used to study the underlying further validated by Western blotting. Our results and those of mechanisms and consequences of diabetes (6). Animal models the bioinformatics analysis suggest that oxidative stress, the for T1DM include genetic models and chemical-induced Wnt pathway, fatty acid degradation and glucose transport models. The genetic models include the non-obese diabetic may be closely related to T1DM. (NOD) mouse and the Bio-Breeding Wistar rat. The models of diabetes with the genetic defects may not be ideal for the study of the disease in humans, because these gene mutations are rare in the general population (7). The commonly used Correspondence to: Professor Yong-Qing Yang, Yue Yang Hospital, chemical-induced models in the mouse and the rat for assessing Shanghai University of Traditional Chinese Medicine, 650 South the mechanism of T1DM, screening potential therapies for Wanping Road, Shanghai 200030, P.R. China this condition, and evaluating therapeutic options are the E-mail: [email protected] streptozotocin (STZ)-induced models (8). The diabetogenic property of STZ is characterized by the selective destruction Abbreviations: T1MD, type 1 diabetes mellitus; NOD, non-obese of β cells of pancreatic islets, causing insulin deficiency, hyper- diabetic; STZ, streptozotocin; SD, Sprague-Dawley; IEF, isoelectric glycemia, polydipsia and polyuria, all of which mimic human focusing; 2-DE, two-dimensional gel electrophoresis; LC-ESI-MS/ T1DM (9), thus making this model better fitted for the study MS, liquid chromatography-electrospray ionization tandem mass of T1DM. spectrometry; PPI, protein-protein interaction; Cbr1, carbonyl reductase 1; HSP60, 60 kDa heat shock protein; GRP78, 78 kDa Proteomics is a powerful tool to describe changes in protein glucose-regulated protein; PRX4, peroxiredoxin 4; HCDH, hydroxy- expression and modification, and holds the promise of providing acyl-CoA dehydrogenase; TER, transitional endoplasmic reticulum major contributions to diabetes research (6). Sanchez et al built the first reference database of the mouse islet proteome Key words: pancreas, proteomics, type 1 diabetes mellitus, strepto- (10). Xie et al performed proteomic analysis of mouse islets zotocin, Sprague-Dawley rats after multiple low-dose STZ injections, and found 4 proteins that were up-regulated and 3 that were down-regulated in diabetic mice (11). In a series of experiments, researchers examined the effect of IL-1β on the proteome profile of rat 154 JIANG et al: PROTEOME PROFILE OF ThE PANCREAS OF DIABETIC RATS islets of Langerhans from Wistar Furth rats (12,13) and later For 2-DE analysis, the protein extracts of each animal in the in Bio Breeding diabetes-prone rats (14,15). They found that same group were equally pooled according to the protein differentially expressed proteins from both rat strains were concentration and stored at -80˚C until use. involved in many pathways, such as energy transduction, redox potentials, glycolysis and the Krebs cycle (15). Yang et al 2-DE. Three independent experiments of 2-DE were performed carried out proteomic analysis of remnants of the pancreas to ensure reproducibility for each group. 2-DE was carried after 90% pancreatectomy, and determined that differentially out using a Bio-Rad 2-DE system following the Bio-Rad expressed proteins in regenerating rat pancreas were involved handbook. Briefly, a 100 µg protein sample was applied for in cell growth, lipid and energy metabolism, protein and amino isoelectric focusing (IEF) using the ReadyStrip IPG strips acid metabolism and signal transduction (16). Due to its (17 cm, nonlinear pH 3-10; Bio-Rad). The strips were placed potential to dissect complex human disorders, proteomics into a Protean IEF cell (Bio-Rad) and were rehydrated at 50 V analysis may be used to add insight into diabetes research (6). for 12 h, and then the proteins were separated based on their In the present study, we carried out proteomic analysis of pI according to the following protocol: 250 V with a linear the pancreas of diabetic Sprague-Dawley (SD) rats induced by climb for 30 min, 1,000 V with a rapid climb for 60 min, STZ. The proteome profile of the pancreas was assessed by 10,000 V with a linear climb for 5 h, 10,000 V with a rapid two-dimensional gel electrophoresis (2-DE) followed by mass climb until 60,000 Vh were reached, and 500 V with a rapid spectrometry analysis (MS) to identify differentially expressed climb for 30 min. After IEF, the IPG strips were equilibrated proteins and bioinformatics analysis with the purpose of for 15 min in a buffer containing 50 mM Tris-HCl, pH 8.8, providing insight into the mechanism of T1DM. 30% glycerol, 7 M urea, 2% SDS and 2% DTT followed by a further equilibration in a similar buffer (but containing 2.5% Materials and methods iodoacetamide instead of DTT) for 15 min and then placed on 13% homogeneous SDS-PAGE gels for electrophoresis using Animals and diabetes induction. Experiments were conducted a Protean II xi Cell system (Bio-Rad) according to the following in 6-week-old male SD rats (210-230 g; SLAC Laboratory protocol: 10 mA/gel for 30 min and 24 mA/gel for 6 h. After Animal Centre, Shanghai, China). Rats were housed in a that, the gels were silver-stained. temperature controlled room (22±1˚C) under a 12:12 h light- dark cycle, with standard rodent chow and water ad libitum. Image analysis and liquid chromatography-electrospray The experiments were conducted in accordance with the ionization tandem mass spectrometry (LC-ESI-MS/MS). The regulations of the State Science and Technology Commission silver-stained gels were scanned by a GS-800 densitometer for the care and use of laboratory animals (State Science and (Bio-Rad) and then analyzed using the PDQuest software Technology Commission Order no. 2, 1988). Rats were randomly (Bio-Rad). The individual protein spot quantity was normalized divided into two groups (n=8), the control group and the as follows: the raw quantity of each spot in a member gel was diabetes group. Diabetes was induced following overnight divided by the total quantity of the valid spots in the gel, and fasting by a single intraperitoneal injection of STZ (60 mg/kg; normalized spot intensities were expressed in ppm. Comparisons Sigma-Aldrich, St. Louis, MO, USA) dissolved in citric acid- were made using the Student's t-test between gel images of the trisodium citrate buffer (0.1 M, pH 4.5; Shanghai Chemical diabetes and control groups. The differentially expressed Reagent Co., Shanghai, China). The induction of diabetes was protein spots (P<0.05) with 2-fold or more increased or considered successful when blood glucose exceeded 16.7 mmol/l decreased intensity between the two groups were selected and 48 h after the STZ injection. The control rats received buffer subjected to further identification by LC-ESI-MS/MS analysis. alone. Blood glucose was measured using a compact glucometer Briefly proteins selected were excised from the gels, washed, (Accu-Check Advantage; Roche Diagnostics, Shanghai, China) dehydrated and digested with 12.5 ng/ml trypsin in 0.1 M on Days 0, 1, 2, 3, 5, 7, 9, 11, 13 and 15. NH4HCO3. LC-ESI-MS/MS analysis was performed using a Finnigan LTQ mass spectrometer (ThermoQuest, San Jose, Histology.