Beta-cell death and survival in models for type 1 diabetes: proteomic analysis

T1D is an autoimmune disease characterized by a selective destruction of the insulin-producing ß-cells in the pancreas. We used 2D-DIG E as proteomic platform to elucidate the effects at the protein level of ß-cell death inducing agents. One of the biases of every proteomic research is the inability to study the complete proteome at the same time, due to its inherent bewilderingl y complex nature. By creating a reference map (chapter 4) of the INS-1E cells, we sought to gain insight in the proportion of the proteome readi ly accessible to us by 2D-GE. We identified 592 spots on the gel, and cl assified the proteins according to function and subcellular localization . This reference map will serve as a starting point and guide for future proteomic analyses in diabetes-related research. Future quantitative ap proaches under different experimental conditions will hopefully provide new insights in the mechanisms involved in altered ß-cell func tion and viability associated with diabetes development. ß-cell destruction is partially mediated through cytokines secreted by immune cells causing activation of ER stress and pro-apoptotic pathw ays. We sought for the mechanisms involved in ß-cell destructi on and the changes evoked at the protein level. We analyzed the effect o f the cytokines IL-1ß and IFN-γ on ß-cell apoptosis and proteome. We firstly (chapter 5) studied the effect on the insulin-p roducing INS-1E cells, a rat model cell line for ß-cells. The individual cytokines did not generate profound effects on apoptosis indu ction in this cell line. We mainly identified proteins with a protective or anti-apoptotic role in INS-1E cells treated with IFN-γ alone. W hen we considered the effects of IL-1ß alone, the proteome changes in the INS-1E cells were indicative of imbalanced cell functionality. Th e combination of IL-1ß and IFN-γ elicited profound cellular ch anges, translated in a high rate of apoptosis induction and numerous pro teins affected. Different cellular mechanisms were affected by the cytok ine treatment. Metabolism, as observed through differential expressed en zymes involved in the Krebs cycle and glycolysis, was impaired, as well as cytoskeleton formation. Chaperones, necessary for proper protein fold ing, were inactivated or downregulated. The proteins responsible for sta bilization of insulin mRNA and proper processing of the insulin protein displayed a decreased expression, as was the case for proteins with prot ective roles in ROS quenching. Bioinformatical network analysis demonstr ated a close interconnectivity of these diverse effects. In a follow-up study (chapter 6), we used mouse islets deficient in two important members of the IFN-γ signaling pathway. Disruption of the signaling pathway at the level of STAT-1 completely prevented cytokine- induced apoptosis, while disruption more downstream, at the level of IRF -1, was responsible for a partial protection. Wild-type mouse islets sho wed a similar response at the protein level as compared to INS-1E cells. Mouse islets deficient in STAT-1 or IRF-1 displayed an alteration in th eir proteome in concordance with their reduced sensitivity for cytokine- induced apoptosis. Proteins with a protective effect which were downregu lated in wild-type mouse islets did not display such a regulation in the disrupted mouse islets. Moreover, the PTM and inactivation of GRP78, an important chaperone in ß-cells, as observed in INS-1E cells a nd wild-type mouse islets, did not occur in the disrupted mouse islets. We identified a protein, namely GRP75, whose function in the ß -cell and expression profile in the different mouse strains could explai n the differences observed in sensitivity to cytokine-induced cell death in STAT-1-/- and IRF-1-/- mouse islets. Taken together, we identified t he proteins which were STAT-1 or IRF-1 dependent and had a role in the p rotection of the islets against cytokine-induced apoptosis. With these s tudies, we were able to validate and complement previous research on cyt okine-induced apoptosis in ß-cells, one of the mechanisms invo lved in T1D. We identified the proteomic profile of a ß-cell g oing into apoptosis by cytokines, and proposed some proteins with possib le key roles in the road to ß-cell apoptosis or survival. We c hose not only to further focus on the important JAK/STAT pathway for&nbs p;ß-cell death upon cytokine challenge, but also a more mechanistic level was investigated. IL-1ß and IFN-γ cause ß-cel l apoptosis partially through ER stress. We used the reversible ER stres sor CPA in order to elucidate the changes at the protein level this ER s tressor generates. Low levels of ER stress were insufficient to trigger apoptosis, but we detected at the proteome level the picture of an imbal anced cell, with the inherent profile of a cell destined to go into apop tosis. At later time points however, this proteomic profile reversed and apoptosis was averted. Key proteins for this reversal were identified ( 14-3-3 proteins and the modification status of GRP78). At elevated level s of ER stress, still not toxic to other cell types, INS-1E cells demons trated a high rate of apoptosis. We identified the changes imposed on th e proteome using a high dose of CPA. Proteins responsible for cell metab olism, cytoskeleton, protein synthesis and RNA synthesis are all downreg ulated with no recovery at a later time point.Abbreviation list VII 1. Introduction 1 1.1 Type 1 diabetes mellitus 1 1.1.1 General 1 1.1.2 The INS-1E cell line as a model for β-cells 4 1.2 β-cell apoptosis and type 1 diabetes 6 1.2.1 General 6 1.2.2 Cytokines 9 1.2.2.1 Interleukin 1 beta 9 1.2.2.2 Interferon gamma 10 1.2.2.3 Tumor necrosis factor alpha 11 1.2.3 NF-κB pathway 12 1.2.4 Signal transducer and activator of transcription 1 pathway 14 1.2.5 Interferon regulatory factor 1 pathway 17 1.3 Endoplasmic reticulum stress and type 1 diabetes 19 1.3.1 General 19 1.3.2 The Unfolded Protein Response 20 1.4 Proteomics 25 1.4.1 General 25 1.4.2 Gel-based proteomics 27 1.4.2.1 Introduction 27 1.4.2.2 Gel-based proteomic techniques: visualization 31 1.4.2.3 Gel-based proteomic techniques: 2D-DIGE 32 1.4.3 Gel-free proteomics 35 1.4.4 Proteomics in diabetes 36 1.4.5 Studies in animal models 37 1.4.6 Studies on human material 38 2. Aims of the study 41 2.1 General objective 41 2.2 Specific aims 42 3. Materials and methods 45 3.1 Cell culture conditions 45 3.2 Animal models 45 3.3 Treatment conditions 46 3.3.1 Cytokine treatment in INS-1E cells 46 3.3.2 Cytokine treatment in mouse islets 46 3.3.3 CPA treatment in INS-1E and PC12 cells 46 3.4 Cell death analysis 47 3.5 Nitrite determination 47 3.6 2D-DIGE analysis 48 3.6.1 Sample preparation 48 3.6.2 First and second dimension 48 3.6.3 Scanning and analysis of the gels 49 3.6.4 Spot digestion and protein identification by MALDI-TOF/TOF analysis 49 3.7 Real-time qRT-PCR 52 3.8 Western blotting 53 3.9 ELISA 54 3.10 Interactome network analysis 54 3.11 Statistical analysis 54 4. Two dimensional gel proteome reference map of INS-1E cells 57 4.1 Introduction 57 4.2 Experimental design 58 4.3 Results and discussion 58 4.4 Concluding remarks 66 5. Cytokine-induced dysfunction and apoptosis in INS-1E cells: alterations in protein profile 67 5.1 Introduction 67 5.2 Experimental design 69 5.3 Results 69 5.3.1 Effects of cytokines on cell viability and nitric oxide production 69 5.3.2 Differential proteomic profile of cytokine-exposed INS-1E cells 70 5.3.3 Identification and classification of differentially expressed proteins 71 5.3.4 Network analysis 76 5.4 Discussion 86 6. Involvement of STAT-1 and IRF-1 in cytokine- mediated dysfunction and apoptosis in mouse islets 93 6.1 Introduction 93 6.2 Experimental design 95 6.3 Results 95 6.3.1 Susceptibility to cell death upon cytokine treatment 95 6.3.2 2D-DIGE analysis 96 6.3.2.1 C57Bl/6J islets and INS-1E cells 96 6.3.2.2 Effects of the JAK/STAT pathway on the islet proteome 97 6.3.2.3 The different impact of Stat1 and Irf1 disruption on the islet proteome 98 6.4 Discussion 112 7. Endoplasmic reticulum stress in INS-1E cells: alterations in protein profile 117 7.1 Introduction 117 7.2 Experimental design 118 7.3 Results 118 7.3.1 Effect of CPA on apoptosis susceptibility of INS-1E cells, compared to PC12 cells 118 7.3.2 Effects of high concentration of CPA (25 µM) on INS-1E cells protein expression 121 7.3.2.1 Alterations in proteomic profile: 2D-DIGE analysis 121 7.3.2.2 Interactome-network 123 7.3.2.3 Confirmation of the observed alterations in chaperone levels: western blotting and quantitative RT-PCR 124 7.3.2.4 Alterations in proteins involved in insulin processing: functional implications 125 7.3.3 Effects of low concentration of CPA (6.25µM) on INS-1E cells protein expression 127 7.3.3.1 Alterations in proteomic profile: 2D-DIGE analysis 127 7.3.3.2 Interactome-network analysis of altered proteins by CPA 130 7.3.3.3 Alterations in chaperone levels: confirmation by western blotting and quantitative RT-PCR 132 7.3.3.4 Effects on insulin processing: glucose stimulated insulin secretion 133 7.4 Discussion 133 8. General conclusions and perspectives 139 9. Summary 145 10. Samenvatting 149 11. References 153 Supplemental data 183 List of Publications 217nrpages: 218status: publishe

Type 1 diabetes: entering the proteomic era

During the last decade, a major breakthrough in the field of proteomics has been achieved. This review describes available techniques for proteomic analyses, both gel and non-gel based, particularly concentrating on relative quantification techniques. The principle of the different techniques is discussed, highlighting the advantages and drawbacks of recently available visualization methods in gel-based assays. In addition, recent developments for quantitative analysis in non-gel-based approaches are summarized. This review focuses on applications in Type 1 diabetes. These mainly include proteomic studies on pancreatic islets in animal models and in the human situation. Also discussed are mass spectrometry-based studies on T-cells, and studies on the development of diagnostic markers for diabetic nephropathology by capillary electrophoresis coupled to mass spectrometry.status: publishe

Proteomics as a tool to discover biomarkers for the prediction of diabetic complications

BACKGROUND: The incidence of diabetes is increasing rapidly, owing to unhealthy lifestyles. The human suffering and healthcare burden caused by diabetes are mainly a consequence of its microvascular and macrovascular complications. Late diagnosis and lack of strict metabolic control lead to increased morbidity and mortality. A problem is the insidious course of the complications, diagnosed in advanced stages, when end organ damage is already present. OBJECTIVE: Discovery of early markers of microangiopathy and macroangiopathy may help to identify patients at risk of organ damage. METHODS: This review focuses on recent developments in proteomics, performed on body fluids and different tissues implicated in microvascular and macrovascular complications. RESULTS: The results provide important information for the early detection of diabetic complications as well as for better understanding of their pathophysiology.status: publishe

A proteome reference map of INS-1E cells

Diabetes is an emerging global epidemic disease that can be traced back to rapid increases in overweight, obesity and physical inactivity. Total deaths are projected to rise by more than 50% in the next 10 years. Two major types can be discriminated. Type 1 diabetes is characterized by a total insulin deficiency, caused by immune-mediated beta-cell destruction and type 2 diabetes results from beta-cell dysfunction and peripheral insulin resistance, leading to a relative insulin deficiency. The aim of this study was to provide a useful tool in facilitating proteomic research in diabetes. We used the rat insulin-producing cell line, INS-1E (provided by Claes Wolheim), a widely used model to study different aspects of diabetes. Total cell lysate was separated in the first dimension on 24 cm pH 4-7 strips and on a 12.5% SDS-polyacrylamide gel in the second dimension. Spots were picked using a an Ettan™ Spot Picker, digested with Trypsin Gold and identified with an AB 4800 MALDI TOF/TOF. Up till now, 424 protein spots out of an approximate 2200 spots have been identified with a MOWSE score higher than the cut-off score indicating identity or extensive homology (p<0.05). This resulted in 271 unique proteins. Proteins were grouped according to their Gene Ontology classification. Major groups identified include: metabolic processes of all kind, protein folding, response to stress and ubiquitin-dependent protein catabolic processes. The full reference map will help us and others to speed up differential analyses in the quest for understanding diabetes.status: accepte

In silico identification of new secretory peptide genes in Drosophila melanogaster

Bioactive peptides play critical roles in regulating most biological processes in animals. The elucidation of the amino acid sequence of these regulatory peptides is crucial for our understanding of animal physiology. Most of the (neuro)peptides currently known were identified by purification and subsequent amino acid sequencing. With the entire genome sequence of some animals now available, it has become possible to predict novel putative peptides. In this way, BLAST (Basic Local Alignment Searching Tool) analysis of the Drosophila melanogaster genome has allowed annotation of 36 secretory peptide genes so far. Peptide precursor genes are, however, poorly predicted by this algorithm, thus prompting an alternative approach described here. With the described searching program we scanned the Drosophila genome for predicted proteins with the structural hallmarks of neuropeptide precursors. As a result, 76 additional putative secretory peptide genes were predicted in addition to the 43 annotated ones. These putative (neuro)peptide genes contain conserved motifs reminiscent of known neuropeptides from other animal species. Peptides that display sequence similarities to the mammalian vasopressin, atrial natriuretic peptide, and prolactin precursors and the invertebrate peptides orcokinin, prothoracicotropic hormones, trypsin modulating oostatic factor, and Drosophila immune induced peptides (DIMs) among others were discovered. Our data hence provide further evidence that many neuropeptide genes were already present in the ancestor of Protostomia and Deuterostomia prior to their divergence. This bioinformatic study opens perspectives for the genome-wide analysis of peptide genes in other eukaryotic model organisms.status: publishe

Two-dimensional gel proteome reference map of INS-1E cells

The insulin-producing INS-1E rat cell line is widely used as a model for studying β-cells. It is a well-characterized cell line, mainly used in diabetes research. We established a 2-DE reference map for INS-1E cells. Using MALDI-TOF/TOF-MS/MS, we identified 546 spots. These included various proteins with an important role in β-cell physiology and with known roles as crucial proteins for diabetes development. We believe that the availability of this reference map will enhance our knowledge of β-cell physiology.status: publishe

Identification of new immune induced molecules in the haemolymph of Drosophila melanogaster by 2D-nanoLC MS/MS

Antimicrobial peptides (AMPs) play an important role in the innate immunity of insects. In Drosophila 17 additional immune induced molecules (DIMs) were found in the haemolymph of adult flies upon septic injury. Previous studies using MALDI mass spectrometry combined with Edman degradation, detected AMPs and DIMs of a predominantly large size. By means of 2D-nanoLC ESI MS/MS, 43 DIMs were identified in this study from the haemolymph of Drosophila third instar larvae 12h after challenge with a mixture of Micrococcus luteus and Escherichia coli. Most peptides were derived from known AMP or DIM precursors, but only four peptides were purified and identified before. The majority of the peptides that we detected were smaller in size. Interestingly, two previously unknown peptide precursors were found and hereby related to immune defense. These include CG7738 and CG32185. Many of the identified peptides are post-translationally modified by an N-terminal pyroglutamic acid and/or a C-terminal amide. Haemolymph of control larvae was treated in the same way and revealed only one peptide.status: publishe

Neuropeptides of the islets of Langerhans: a peptidomics study

Neuropeptides from the endocrine pancreas (the islets of Langerhans) play an important role in the regulation of blood glucose levels. Therefore, our aim is to identify the "peptidome" (the in vivo peptide profile at a certain time) of the pancreatic islets, which is beneficial for medical progress related to the treatment of diabetes. So far, there are few neuropeptides isolated and sequenced from the endocrine pancreas and mainly in situ hybridisation and immunocytochemical techniques have been used to demonstrate the occurrence of peptides in the pancreas. These techniques do not allow for unequivocal identification of peptides. In contrary, mass spectrometry identifies peptides unambiguously. We have analysed the peptidome of the islets using peptidomics, i.e. a combination of liquid chromatography, mass spectrometry and bioinformatics. We are able to identify the peptidome of islets extracts. We not only confirm the presence of peptides with a well-known effect on blood glucose levels, but also identify new peptides, which are unknown to affect blood glucose levels.status: publishe

High glucose induces dysfunction in insulin secretory cells by different pathways: a proteomic approach.

Chronic hyperglycemia is a hallmark of type 2 diabetes and can contribute to progressive beta cell dysfunction and death. The aim of the present study was to identify pathways mediating high glucose-induced beta cell demise by a proteomic approach. INS-1E cells were exposed to 25 mM glucose for a sustained period of 24 h. Protein profiling of INS-1E cells was done by two-dimensional difference gel electrophoresis, covering the pH ranges 4-7 and 6-9 (n = 4). Differentially expressed proteins (P < 0.05) were identified by MALDI-TOF/TOF and proteomic results were confirmed by functional assays. High glucose levels impaired glucose-stimulated insulin secretion and decreased insulin content. 2D-DIGE analysis revealed 100 differentially expressed proteins that were involved in different pathways. Chaperone proteins were down-regulated, protein biosynthesis and ubiquitin-related proteasomal degradation were attenuated and perturbations in intracellular trafficking and vesicle transport and secretion could be observed. Moreover, several pathways were confirmed by functional assays and a direct role for eEF2 in insulin biosynthesis was demonstrated. The present findings provide new insights in glucotoxicity and identify key target proteins for the prevention and treatment of beta cell dysfunction in type 2 diabetes.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe