140 research outputs found
Interactie tussen de beta-cel en het immuunsysteem in type 1 diabetes: rol voor post-translationele modificaties
No full textDiabetes mellitus is one of the most common endocrine diseases in the world and the incidence is still increasing rapidly. Clinical characteristic of diabetes include hyperglycemia and glucosuria, as a result of relative or absolute shortage in insulin. This is the hormone secreted by the beta-cell in the islets of Langerhans upon elevated blood glucose concentrations after a meal. Where the most prevalent type, T2D, is rather considered as a metabolic disease involving insulin resistance and beta-cell dysfunction, T1D, accounting for 5-10% of all diabetic patients, is an autoimmune disease where the body’s own immune system attacks the insulin-secreting pancreatic beta-cells.
The mechanisms responsible for loss of tolerance against the beta-cells are not yet completely understood. The immune system is supposed to be of major importance, but more and more evidence point to the beta-cells themselves as active partners in their own destruction. Aspecific inflammation in the islets, indicated as the first trigger for T1D, results in upregulation of HLA-I molecules on the surface and release of chemokines and cytokines. In addition, ER and oxidative stress are induced, resulting in beta-cell dysfunction, apoptosis and the generation of molecular changes like alternative splicing and PTMs. This PhD project, with the general objective to study how the interaction between the beta-cell and immune system participates in the development of T1D, put specifically the importance of PTMs of beta-cell antigens in the spotlight.
In the first project (Chapter 3) we investigated if at early age before onset of insulitis, there are differences in islets of Langerhans from NOD mice compared to two healthy mouse strains namely the NOR mice, showing 88% sequence similarity with NOD mice, and the more distant C57Bl/6 mice, that could potentially contribute to an intrinsic higher risk for auto-immune attack or dysfunction. Analysis of the transcriptome and proteome, combined with integrated pathway and network analyses, indicated that islets of NOD mice are subjected to higher levels of stress due to an inefficient supply of energy and by a higher probability that misfolded proteins will accumulate, mainly due to lower expression of PDIA3, PDIA4 and PDIA6, enzymes implicated in S-S bond formation. However, the most remarkable finding in NOD islets at this early pre-insulitic age was the high expression of Padi2 mRNA. Padi2 is one of the five Ca2+ dependent enzymes responsible for the modification of arginine to citrulline, known as citrullination. This modification that highly impacts protein conformation and HLA binding, is known to be implicated in the generation of autoantigens is several autoimmune diseases, most importantly RA, but also in T1D citrullination of GAD65 results in autoantigen generation.
In order to examine both aspects of the interaction between the beta-cell and immune system, the aim of the second project (Chapter 4) was to investigate how inflammation affects the beta-cell proteome resulting in an active contribution to T1D development. We build further on a previous finding from our lab that demonstrated PTM of GRP78 in a rat beta-cell line (INS-1E) upon exposure to pro-inflammatory cytokines, a model that is used to mimic T1D ex-vivo. Further, we characterized this PTM as a citrullination on arginine 510 of GRP78. In addition, it was also shown that cytokines induce the translocation of GRP78 from the ER to the plasma-membrane of INS-1E cells and mouse islets, a process mediated by ER stress. While intracellularly we identified 3 isoforms of GRP78, much more modified isoforms of GRP78 were expressed on the cell surface. Finally, citrullination of GRP78 results in loss of immune tolerance in diabetic NOD mice by the presence of autoantibodies and auto-reactive T-cells.
The aim of the third project (Chapter 5) was to translate these findings from INS-1E cells and NOD mice to the human situation. This pointed to a similar PTM of GRP78 in human islets exposed to pro-inflammatory cytokines in three out of five healthy donors. Furthermore, proteome analysis by LC-MS/MS indicated that inflammation in general induced citrullination and deamidation, another PTM known to be involved in autoantigen generation. The identification of two citrullinated and deamidated peptides of GRP78 showed evidence for the implication of these PTMs in human islets as well. To investigate the relevance in terms of autoantigen recognition, modified GRP78 epitopes were designed that were predicted to bind to the T1D susceptibility HLA-DR4 molecule and these peptides were further on used in immunological assays. The first indication for the implication of citrullinated GRP78 as autoantigen in human T1D patients came from the presence of reactive CD4+ T-cells in infiltrated T1D islets. Next, higher CD4+ T-cell frequencies recognizing a citrullinated epitope were also present in peripheral blood of T1D patients compared to healthy controls, indicating a possible application as biomarker for T1D.
To conclude, this PhD project clearly demonstrated the importance of the interaction that connects beta-cells and immune system in the pathogenesis of T1D. PTMs of beta-cell antigens, especially citrullination, appeared to have substantial importance for the break of tolerance. Furthermore, the ability to detect autoreactive T-cells against citrullinated GRP78 in blood of NOD mice and T1D patients emphasizes its possible use as a biomarker and opens the road to the development of new therapeutic applications.status: Publishe
Therapieën in Type 1 Diabetes: het onderzoek van de interactie tussen bèta-cel en het immuunsysteem door gebruik te maken van pre-klinische modellen
No full textstatus: Publishe
Quantification of chemokines by real-time reverse transcriptase PCR: applications in type 1 diabetes
No full textGlucagon-Like Peptide-1 Protects Human Islets against Cytokine-Mediated β-Cell Dysfunction and Death: A Proteomic Study of the Pathways Involved
No full textGlucagon-like peptide-1 (GLP-1) has been shown to protect pancreatic β-cells against cytokine-induced dysfunction and destruction. The mechanisms through which GLP-1 exerts its effects are complex and still poorly understood. The aim of this study was to analyze the protein expression profiles of human islets of Langerhans treated with cytokines (IL-1β and IFN-γ) in the presence or absence of GLP-1 by 2D difference gel electrophoresis and subsequent protein interaction network analysis to understand the molecular pathways involved in GLP-1-mediated β-cell protection. Co-incubation of cytokine-treated human islets with GLP-1 resulted in a marked protection of β-cells against cytokine-induced apoptosis and significantly attenuated cytokine-mediated inhibition of glucose-stimulated insulin secretion. The cytoprotective effects of GLP-1 coincided with substantial alterations in the protein expression profile of cytokine-treated human islets, illustrating a counteracting effect on proteins from different functional classes such as actin cytoskeleton, chaperones, metabolic proteins, and islet regenerating proteins. In summary, GLP-1 alters in an integrated manner protein networks in cytokine-exposed human islets while protecting them against cytokine-mediated cell death and dysfunction. These data illustrate the beneficial effects of GLP-1 on human islets under immune attack, leading to a better understanding of the underlying mechanisms involved, a prerequisite for improving therapies for diabetic patients
De rol van dieet in ontwikkeling en remissie van type 2 diabetes
No full textType 2 diabetes is a chronic disease, characterized by insulin resistance and beta-cell dysfunction. It is thought to be a consequence of both genetic predisposition and environmental factors. Although sedentary lifestyle and Western diet are the main drivers of the rising prevalence of type 2 diabetes, environmental pollutants have been reported to play a role as well.
In the first part of this thesis, we evaluated the role of the cereulide toxin in beta cell failure and the onset of type 2 diabetes, as cereulide had been implicated in beta-cell toxicity and it had been found frequently in low concentrations in starchy dishes in Belgian restaurants. We confirmed the increased sensitivity to cereulide of different beta-cell models when compared to other mammalian cell lines. Rat and mouse beta-cell lines (INS-1E and MIN6) and freshly isolated murine pancreatic islets showed very high rates of apoptosis after exposure to 5 ng/ml cereulide for 24 h, whereas human hepatocellular carcinoma (HepG2) and renal fibroblast cells (COS-1) remained viable when exposed to the same concentration. Hoechst/propidium Iodide staining and electron microscopy evaluation revealed that the observed cell death was mainly due to apoptosis, which was further supported by increased caspase 3/7 activation, elevated cytochrome C release into the cytoplasm and upregulation of pro-apoptotic mRNA markers in MIN6 cells. Transmission electron microscopy showed that mitochondria of the MIN6 cells were swollen and disintegrated after exposure to 0.5 ng/ml cereulide. At this dose, reactive oxygen species culminated more than twofold, and basal respiration rate was reduced to half, as compared to unexposed MIN6 cells. Presumably, such high mitochondrial toxicity explains the reduced insulin secretion by MIN6 cells or whole mouse islets after cereulide exposure.Taken together, we show that cereulide causes apoptotic beta-cell death at low concentrations and impairs beta-cell function at even lower concentrations, with mitochondrial dysfunction underlying these defects.
In the second part of the thesis, we investigated the impact of Roux-en-Y Gastric Bypass (RYGB) on pancreatic islet mass and function in a mouse model. Diet-induced obese C57BL/6J mice underwent RYGB or sham surgery at the age of 22 weeks. Postoperatively, the mice consumed the same high fat diet (HFD) as before, and sham mice were either pair-fed (PFS) or weight-matched (WMS) to the RYGB mice and compared to age-matched normal chow fed LEAN control mice. PFS mice regained body weight quickly after sham surgery to become obese and glucose intolerant by the end of the 8 week postsurgical follow-up period. RYGB and WMS on the other hand, had a similar body weight and body fat percentage as LEAN mice. Total energy expenditure was higher in RYGB compared to WMS mice. In vitro glucose stimulated insulin secretion was not significantly altered in isolated islets after RYGB, compatible with the hypothesis that changes in the gastrointestinal tract are necessary for the RYGB-specific observed effects on beta-cell function. Histological examination showed similar islet numbers in LEAN, WMS, RYGB and PFS mice. However, islets of PFS mice were larger than islets of LEAN mice. Interestingly, the islet size in RYGB and WMS also tended to be larger than in LEAN mice. We speculate that the islet size increased during the high fat feeding that rendered the mice obese. Subsequently, islet partial involution occurs when weight is lost by either RYGB or caloric restriction. We did not detect differences in islet composition, in particular in terms of relative insulin content, between experimental groups. Ki67-staining showed very low proliferation of islet cells in all groups, as can be expected in 30 week old mice at steady state. Pathway analysis of the RYGB islet transcriptome suggested no difference in inflammation or endoplasmic reticulum stress in islets post-RYGB compared to WMS mice. However, several proliferation/differentiation markers were upregulated in islets following RYGB and most members of the Reg gene family were expressed in the pancreases of the RYGB but not the WMS mice. Overexpression of Reg2 and Reg3b in MIN6 cells line, did not affect the insulin secretion. As no difference in proliferation was noted in the histology samples, the relevance of the Reg gene upregulation in islets after RYGB remains to be determined and will be subject of further research. In this chapter, we concluded that islet histology, function and gene profile did not differ dramatically between the mice that underwent RYGB or lost similar weight by caloric restriction.
In the third part of the thesis we investigated the metabolic impact after different weight loss strategies. Twenty two weeks old diet-induced obese C57BL/6J mice underwent RYGB-HF or caloric restriction on either normal chow (RESTR-NC) or HFD (RESTR-HF), in order to attain a similar body weight as the LEAN control mice on normal chow at the end of the 8 weeks intervention period. Thirty weeks old HFD fed mice were used as age-matched OBESE controls. Oxygen consumption, heat production and total energy expenditure decreased with weight loss through adaptive thermogenesis and there was no significant difference between RESTR-NC and RESTR-HF mice. The epididymal white adipose tissue (eWAT) pads and individual adipocytes of the RESTR-HF mice were significantly larger than in RESTR-NC and RYGB-HF mice. The adipocytes in RESTR-NC mice were not enlarged anymore, and the increased density compared to NC-CO mice suggests that adipocyte size rather than number is affected by weight loss. In addition, mRNA markers of inflammation were at normal levels in RESTR-NC and RYGB-HF mice, but were increased in the eWAT of OBESE mice and RESTR-HF mice. Both histological examination and triglyceride (TG) content evaluation of hepatic tissues showed massive steatosis in all OBESE mice, but also signs of steatosis in RESTR-HF and RYGB-HF mice, while RESTR-NC and LEAN were free of fatty liver disease. The eWAT size and hepatic TG content was tightly correlated with the glucose and insulin tolerance tests. Actually, the intraperitoneal glucose tolerance tests revealed that the RESTR-HF mice were only slighty less glucose intolerant than the OBESE mice, despite a major weight loss. If the same weight loss was achieved on a normal chow diet on the other hand (RESTR-NC) or by RYGB the glucose tolerance was restored. Thus, we clearly show that metabolic improvement is not solely weight driven. We demonstrate that shifting to lower fat foods has a greater metabolic benefit than losing weight by restricting calories or surgery without modifications of the food content. Food with lower fat content causes less fat disposition and by consequence less glucose intolerance than weight loss achieved through restriction on fat.
The overall conclusion of the present thesis is that external pollutants can contribute to type 2 diabetes by direct detrimental effects on beta-cell survival and function and that the nature of weight loss strategies matters by its impact on fat disposition and insulin resistance, but has no direct effect on beta-cells.status: Publishe
Understanding dendritic cell biology and its role in immunological disorders through proteomic profiling
No full textUnderstanding type 1 diabetes through proteomics
No full textAuto-immunity against pancreatic beta-cells leads to an absolute shortage of the hormone insulin, resulting in hyperglycemia and the onset of type 1 diabetes (T1D). Proteomic approaches have been used to elucidate the mechanisms of beta-cell dysfunction and death. Areas covered: In the present review, we discuss discoveries in the beta-cell proteome that have contributed to better insights in the role of the beta-cell in T1D. Techniques, such as 2D-DIGE and MALDI imaging, together with new approaches for sample preparation, including laser capture microdissection and immunopeptidomics, have resulted in novel mechanistic insights in the pathogenesis of T1D. We describe how proteomic studies in beta-cell lines as well as isolated islets from animal models and humans have discovered intracellular signaling pathways leading to beta-cell destruction, the generation of neo-antigens through post-translational modifications of beta-cell antigens as well as better biomarkers of disease progression. Expert commentary: Proteomics has contributed to the discovery of beta-cell neo-autoantigen generation through post-translational modifications, hybrid insulin peptide formation and the generation of defective ribosomal gene products. These concepts are revolutionizing our insights in the pathogenesis of T1D, acknowledging a central role for the beta-cell in its own destruction.sponsorship: This work was supported by grants from the KU Leuven (GOA 12/24), the IMI2-JU under grant agreement No 115797 (INNODIA). This Joint Undertaking receives support from the Union's Horizon 2020 research and innovation programme and 'EFPIA', 'JDRF' and 'The Leona M. and Harry B. Helmsley Charitable Trust'; and a PhD fellowship from Agentschap voor Innovatie door Wetenschap en Technologie. (KU Leuven|GOA 12/24, IMI2-JU|115797, Union's Horizon 2020 research and innovation programme, EFPIA, JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Agentschap voor Innovatie door Wetenschap en Technologie)status: Publishe
A proteome reference map of INS-1E cells
No full textDiabetes 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
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