130 research outputs found
Glucagon-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
Pancreatic Alpha-Cells Contribute Together With Beta-Cells to CXCL10 Expression in Type 1 Diabetes
No full textC-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine specifically recognized by the ligand receptor CXCR3 which is mostly expressed in T-lymphocytes. Although CXCL10 expression and secretion have been widely associated to pancreatic islets both in non-obese diabetic (NOD) mice and in human type 1 diabetic (T1D) donors, the specific expression pattern among pancreatic endocrine cell subtypes has not been clarified yet. Therefore, the purpose of this study was to shed light on the pancreatic islet expression of CXCL10 in NOD, in C57Bl/6J and in NOD-SCID mice as well as in human T1D pancreata from new-onset T1D patients (DiViD study) compared to non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD). CXCL10 was expressed in pancreatic islets of normoglycaemic and new-onset diabetic NOD mice but not in C57Bl/6J and NOD-SCID mice. CXCL10 expression was increased in pancreatic islets of new-onset diabetic NOD mice compared to normoglycaemic NOD mice. In NOD mice, CXCL10 colocalized both with insulin and glucagon. Interestingly, CXCL10-glucagon colocalization rate was significantly increased in diabetic vs. normoglycaemic NOD mouse islets, indicating an increased expression of CXCL10 also in alpha-cells. CXCL10 was expressed in pancreatic islets of T1D patients but not in non-diabetic donors. The analysis of the expression pattern of CXCL10 in human T1D pancreata from DiViD study, revealed an increased colocalization rate with glucagon compared to insulin. Of note, CXCL10 was also expressed in alpha-cells residing in insulin-deficient islets (IDI), suggesting that CXCL10 expression in alpha cells is not driven by residual beta-cells and therefore may represent an independent phenomenon. In conclusion, we show that in T1D CXCL10 is expressed by alpha-cells both in NOD mice and in T1D patients, thus pointing to an additional novel role for alpha-cells in T1D pathogenesis and progression.sponsorship: This project has received funding from the Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under Grant Agreement No. 115797-INNODIA and No. 945268 INNODIA HARVEST. 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. FD was supported by the Italian Ministry of University and Research (No. 2017KAM2R5_003). GS was supported by the Italian Ministry of University and Research (No. 201793XZ5A_006). (Innovative Medicines Initiative 2 (IMI2) Joint Undertaking|115797, Innovative Medicines Initiative 2 (IMI2) Joint Undertaking|945268, Union's Horizon 2020 research and innovation programme, EFPIA, JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Italian Ministry of University and Research|2017KAM2R5_003, Italian Ministry of University and Research|201793XZ5A_006)status: Publishe
IL-17A increases the expression of proinflammatory chemokines in human pancreatic islets
No full textAIMS/HYPOTHESIS:
Cytotoxic T cells and macrophages contribute to beta cell destruction in type 1 diabetes at least in part through the production of cytokines such as IL-1β, IFN-γ and TNF-α. We have recently shown the IL-17 pathway to be activated in circulating T cells and pancreatic islets of type 1 diabetes patients. Here, we studied whether IL-17A upregulates the production of chemokines by human pancreatic islets, thus contributing to the build-up of insulitis.
METHODS:
Human islets (from 18 donors), INS-1E cells and islets from wild-type and Stat1 knockout mice were studied. Dispersed islet cells were left untreated, or were treated with IL-17A alone or together with IL-1β+IFN-γ or TNF-α+IFN-γ. RNA interference was used to knock down signal transducer and activator of transcription 1 (STAT1). Chemokine expression was assessed by quantitative RT-PCR, ELISA and histology. Cell viability was evaluated with nuclear dyes.
RESULTS:
IL-17A augmented IL-1β+IFN-γ- and TNF-α+IFN-γ-induced chemokine mRNA and protein expression, and apoptosis in human islets. Beta cells were at least in part the source of chemokine production. Knockdown of STAT1 in human islets prevented cytokine- or IL-17A+cytokine-induced apoptosis and the expression of particular chemokines, e.g. chemokine (C-X-C motif) ligands 9 and 10. Similar observations were made in islets isolated from Stat1 knockout mice.
CONCLUSIONS/INTERPRETATION:
Our findings indicate that IL-17A exacerbates proinflammatory chemokine expression and secretion by human islets exposed to cytokines. This suggests that IL-17A contributes to the pathogenesis of type 1 diabetes by two mechanisms, namely the exacerbation of beta cell apoptosis and increased local production of chemokines, thus potentially aggravating insulitis
SARS-CoV-2 Receptor Angiotensin I-Converting Enzyme Type 2 (ACE2) Is Expressed in Human Pancreatic <i>β</i>-Cells and in the Human Pancreas Microvasculature
No full textIncreasing evidence demonstrated that the expression of Angiotensin I-Converting Enzyme type 2 (ACE2) is a necessary step for SARS-CoV-2 infection permissiveness. In light of the recent data highlighting an association between COVID-19 and diabetes, a detailed analysis aimed at evaluating ACE2 expression pattern distribution in human pancreas is still lacking. Here, we took advantage of INNODIA network EUnPOD biobank collection to thoroughly analyze ACE2, both at mRNA and protein level, in multiple human pancreatic tissues and using several methodologies. Using multiple reagents and antibodies, we showed that ACE2 is expressed in human pancreatic islets, where it is preferentially expressed in subsets of insulin producing β-cells. ACE2 is also highly expressed in pancreas microvasculature pericytes and moderately expressed in rare scattered ductal cells. By using different ACE2 antibodies we showed that a recently described short-ACE2 isoform is also prevalently expressed in human β-cells. Finally, using RT-qPCR, RNA-seq and High-Content imaging screening analysis, we demonstrated that pro-inflammatory cytokines, but not palmitate, increase ACE2 expression in the β-cell line EndoC-βH1 and in primary human pancreatic islets. Taken together, our data indicate a potential link between SARS-CoV-2 and diabetes through putative infection of pancreatic microvasculature and/or ductal cells and/or through direct β-cell virus tropism.sponsorship: The work is supported by the Innovative Medicines Initiative 2 (IMI2) Joint Undertaking under grant agreement No. 115797-INNODIA and No. 945268 INNODIA HARVEST. 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. FD was supported by the Italian Ministry of University and Research (2017KAM2R5_003). GS was supported by the Italian Ministry of University and Research (201793XZ5A_006). (Innovative Medicines Initiative 2 (IMI2) Joint Undertaking|115797-INNODIA, Innovative Medicines Initiative 2 (IMI2) Joint Undertaking|945268 INNODIA HARVEST, Union's Horizon 2020 research and innovation programme, EFPIA, JDRF, Leona M. and Harry B. Helmsley Charitable Trust, Italian Ministry of University and Research|2017KAM2R5_003, Italian Ministry of University and Research|201793XZ5A_006)status: Publishe
Discovery of molecular pathways mediating 1,25-dihydroxyvitamin D3 protection against cytokine-induced inflammation and damage of human and male mouse islets of Langerhans.
No full textProtection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported. The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion and β-cell survival were analyzed by glucose-stimulated insulin release and electron microscopy or Hoechst/propidium iodide staining, respectively. Gene expression profiles were assessed by Affymetrix microarrays. Nuclear factor-κB activity was tested, whereas effects on secreted chemokines/cytokines were confirmed by ELISA and migration studies. Cytokine exposure caused a significant increase in β-cell apoptosis, which was almost completely prevented by 1,25(OH)2D3. In addition, 1,25(OH)2D3 restored insulin secretion from cytokine-exposed islets. Microarray analysis of murine islets revealed that the expression of approximately 4000 genes was affected by cytokines after 6 and 24 hours (n = 4; >1.3-fold; P < .02), of which nearly 250 genes were modified by 1,25(OH)2D3. These genes belong to functional groups involved in immune response, chemotaxis, cell death, and pancreatic β-cell function/phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3 against the induction of autoimmune diabetes.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jSCOPUS: ar.jinfo:eu-repo/semantics/publishe
IL-17A increases the expression of proinflammatory chemokines in human pancreatic islets.
No full textCytotoxic T cells and macrophages contribute to beta cell destruction in type 1 diabetes at least in part through the production of cytokines such as IL-1β, IFN-γ and TNF-α. We have recently shown the IL-17 pathway to be activated in circulating T cells and pancreatic islets of type 1 diabetes patients. Here, we studied whether IL-17A upregulates the production of chemokines by human pancreatic islets, thus contributing to the build-up of insulitis.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe
Cluster analysis of rat pancreatic islet gene mRNA levels after culture in low-, intermediate- and high-glucose concentrations
No full textAIMS/HYPOTHESIS: Survival and function of insulin-secreting pancreatic beta cells are markedly altered by changes in nutrient availability. In vitro, culture in 10 rather than 2 mmol/l glucose improves rodent beta cell survival and function, whereas glucose concentrations above 10 mmol/l are deleterious. METHODS: To identify the mechanisms of such beta cell plasticity, we tested the effects of 18 h culture at 2, 5, 10 and 30 mmol/l glucose on the transcriptome of rat islets pre-cultured for 1 week at 10 mmol/l glucose using Affymetrix Rat 230 2.0 arrays. RESULTS: Culture in either 2-5 or 30 mmol/l instead of 10 mmol/l glucose markedly impaired beta cell function, while little affecting cell survival. Of about 16,000 probe-sets reliably detected in islets, some 5,000 were significantly up- or downregulated at least 1.4-fold by glucose. Analysis of these probe-sets with GeneCluster software identified ten mRNA profiles with unidirectional up- or downregulation between 2 and 10, 2 and 30, 5 and 10, 5 and 30 or 10 and 30 mmol/l glucose. It also identified eight complex V-shaped or inverse V-shaped profiles with a nadir or peak level of expression in 5 or 10 mmol/l glucose. Analysis of genes belonging to these various clusters using Onto-express and GenMAPP software revealed several signalling and metabolic pathways that may contribute to induction of beta cell dysfunction and apoptosis after culture in low- or high- vs intermediate-glucose concentration. CONCLUSIONS/INTERPRETATION: We have identified 18 distinct mRNA profiles of glucose-induced changes in islet gene mRNA levels that should help understand the mechanisms by which glucose affects beta cell survival and function under states of chronic hypo- or hyperglycaemia
Digital as the new popular in African cinema? Case studies from the continent
No full textThis paper considers different forms of contemporary filmmaking on the African continent in order to show how Africa has embraced digital technology as a crucial factor toward more, and increasingly varied, forms of popular cinema. The case studies are chosen to show a variety of production, exhibition, and distribution methods utilizing digital technology. The focus of the paper is on popular cinema as a form of popular culture, as theorized by Karin Barber. We review Barber's pioneering work on African popular culture and subsequent work by her and other scholars to determine and argue how the new forms of digital cinema discussed in this paper can be considered part of African popular culture
Initiation and execution of lipotoxic ER stress in pancreatic beta-cells.
No full textFree fatty acids (FFA) cause apoptosis of pancreatic beta-cells and might contribute to beta-cell loss in type 2 diabetes via the induction of endoplasmic reticulum (ER) stress. We studied here the molecular mechanisms implicated in FFA-induced ER stress initiation and apoptosis in INS-1E cells, FACS-purified primary beta-cells and human islets exposed to oleate and/or palmitate. Treatment with saturated and/or unsaturated FFA led to differential ER stress signaling. Palmitate induced more apoptosis and markedly activated the IRE1, PERK and ATF6 pathways, owing to a sustained depletion of ER Ca(2+) stores, whereas the unsaturated FFA oleate led to milder PERK and IRE1 activation and comparable ATF6 signaling. Non-metabolizable methyl-FFA analogs induced neither ER stress nor beta-cell apoptosis. The FFA-induced ER stress response was not modified by high glucose concentrations, suggesting that ER stress in primary beta-cells is primarily lipotoxic, and not glucolipotoxic. Palmitate, but not oleate, activated JNK. JNK inhibitors reduced palmitate-mediated AP-1 activation and apoptosis. Blocking the transcription factor CHOP delayed palmitate-induced beta-cell apoptosis. In conclusion, saturated FFA induce ER stress via ER Ca(2+) depletion. The IRE1 and resulting JNK activation contribute to beta-cell apoptosis. PERK activation by palmitate also contributes to beta-cell apoptosis via CHOP.Journal ArticleResearch Support, N.I.H. ExtramuralResearch Support, Non-U.S. Gov'tinfo:eu-repo/semantics/publishe
Beta-cel dood en overleving in modellen voor type-1 diabetes: proteomische analyse
No full textT1D 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.status: Publishe
- …
