In vitro profiling of endocrine cell death using UCHL1 and GAD65 as soluble biomarkers

2016 ◽  
Author(s):  
Benedicte Brackeva ◽  
Sarah Roels ◽  
Geert Stangé ◽  
Gamze Ates ◽  
Olivier R. Costa ◽  
...  
Keyword(s):  
Cell Death ◽  
Beta Cell ◽  
Beta Cells ◽  
Islet Transplantation ◽  
High Sensitivity ◽  
Low Grade ◽  
Variable Degree ◽  
Beta Cell Death

AbstractBACKGROUNDPancreatic islet grafts are cultured in vitro prior to transplantation and this is associated to a variable degree of beta cell loss. Optimization of culture conditions is currently hampered by the lack of a specific and sensitive in vitro indicator of beta cell death.METHODSWe developed a high-sensitivity duplex bead-based immunoassay for two protein-type biomarkers of beta cell destruction, GAD65 and UCHL1, and investigated its proficiency for in vitro toxicity profiling on rodent and human beta cells, as compared to a semi-automatic and manual image-based assessment of beta cell death, and in vivo after intraportal islet transplantation.RESULTSBoth GAD65 and UCHL1 were discharged by necrotic and apoptotic beta cells proportionate to the number of dead beta cells as counted by microscopic methods. In vitro, UCHL1 was superior to GAD65, in terms of biomarker stability providing more sensitive detection of low grade beta cell death. In vivo, however, GAD65 was consistently detected after islet transplantation while UCHL1 remained undetectable.CONCLUSIONThe use of soluble biomarkers represents a fast, selective and sensitive method for beta cell toxicity profiling in vitro. UCHL1 is superior to GAD65 in vitro but not in vivo.

scholarly journals The microRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts

2021 ◽  
Author(s):  
Geert Antoine Martens ◽  
Geert Stange ◽  
Lorenzo Piemonti ◽  
Jasper Anckaert ◽  
Zhidong Ling ◽  
...  
Keyword(s):  
Cell Death ◽  
Beta Cell ◽  
Beta Cells ◽  
Islet Transplantation ◽  
Beta Cell Death ◽  
Beta Cell Destruction ◽  
Cell Destruction ◽  
Graft Outcome ◽  
Plasma Microrna

Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. MicroRNA-375 (miR-375) ranks among top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 hour before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to identification of 8 microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients, by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.

scholarly journals Deletion of protein kinase Cδ in mice modulates stability of inflammatory genes and protects against cytokine-stimulated beta cell death in vitro and in vivo

Diabetologia ◽  
2010 ◽  
Vol 54 (2) ◽  
pp. 380-389 ◽  
Author(s):  
J. Cantley ◽  
E. Boslem ◽  
D. R. Laybutt ◽  
D. V. Cordery ◽  
G. Pearson ◽  
...  
Keyword(s):  
Cell Death ◽  
Protein Kinase ◽  
Beta Cell ◽  
Beta Cell Death ◽  
Inflammatory Genes ◽  
Protein Kinase Cδ

scholarly journals The MicroRNA Landscape of Acute Beta Cell Destruction in Type 1 Diabetic Recipients of Intraportal Islet Grafts

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1693
Author(s):  
Geert A. Martens ◽  
Geert Stangé ◽  
Lorenzo Piemonti ◽  
Jasper Anckaert ◽  
Zhidong Ling ◽  
...  
Keyword(s):  
Cell Death ◽  
Beta Cell ◽  
Beta Cells ◽  
Islet Transplantation ◽  
Beta Cell Death ◽  
Beta Cell Destruction ◽  
Cell Destruction ◽  
Graft Outcome ◽  
Plasma Microrna

Ongoing beta cell death in type 1 diabetes (T1D) can be detected using biomarkers selectively discharged by dying beta cells into plasma. microRNA-375 (miR-375) ranks among the top biomarkers based on studies in animal models and human islet transplantation. Our objective was to identify additional microRNAs that are co-released with miR-375 proportionate to the amount of beta cell destruction. RT-PCR profiling of 733 microRNAs in a discovery cohort of T1D patients 1 h before/after islet transplantation indicated increased plasma levels of 22 microRNAs. Sub-selection for beta cell selectivity resulted in 15 microRNAs that were subjected to double-blinded multicenter analysis. This led to the identification of eight microRNAs that were consistently increased during early graft destruction: besides miR-375, these included miR-132/204/410/200a/429/125b, microRNAs with known function and enrichment in beta cells. Their potential clinical translation was investigated in a third independent cohort of 46 transplant patients by correlating post-transplant microRNA levels to C-peptide levels 2 months later. Only miR-375 and miR-132 had prognostic potential for graft outcome, and none of the newly identified microRNAs outperformed miR-375 in multiple regression. In conclusion, this study reveals multiple beta cell-enriched microRNAs that are co-released with miR-375 and can be used as complementary biomarkers of beta cell death.

scholarly journals A Model for Human Islet Transplantation to Immunodeficient Streptozotocin-Induced Diabetic Mice

2018 ◽  
Vol 27 (11) ◽  
pp. 1684-1691 ◽  
Author(s):  
Elisabet Estil·les ◽  
Noèlia Téllez ◽  
Montserrat Nacher ◽  
Eduard Montanya
Keyword(s):  
Cell Death ◽  
Beta Cell ◽  
Beta Cells ◽  
Islet Transplantation ◽  
Cell Mass ◽  
Human Islet ◽  
Diabetic Mice ◽  
Beta Cell Death ◽  
Human Beta Cells ◽  
Human Islet Transplantation

Streptozotocin (STZ) is a cytotoxic glucose analogue that causes beta cell death and is widely used to induce experimental diabetes in rodents. The sensitivity of beta cells to STZ is species-specific and human beta cells are resistant to STZ. In experimental islet transplantation to rodents, STZ-diabetes must be induced before transplantation to avoid destruction of grafted islets by STZ. In human islet transplantation, injection of STZ before transplantation is inconvenient and costly, since human islet availability depends on organ donation and frail STZ-diabetic mice must be kept for unpredictable lapses of time until a human islet preparation is available. Based on the high resistance of human beta cells to STZ, we have tested a new model for STZ-diabetes induction in which STZ is injected after human islet transplantation. Human and mouse islets were transplanted under the kidney capsule of athymic nude mice, and 10–14 days after transplantation mice were intraperitoneally injected with five consecutive daily doses of STZ or vehicle. Beta-cell death increased and beta-cell mass was reduced in mouse islet grafts after STZ injection. In contrast, in human islet grafts beta cell death and mass did not change after STZ injection. Mice transplanted with rodent islets developed hyperglycemia after STZ-injection. Mice transplanted with human islets remained normoglycemic and developed hyperglycemia when the graft was harvested. STZ had no detectable toxic effects on beta cell death, mass and function of human transplanted islets. We provide a new, more convenient and cost-saving model for human islet transplantation to STZ-diabetic recipients in which STZ is injected after islet transplantation.

scholarly journals Pharmacological Inhibition of NOX4 Improves Mitochondrial Function and Survival in Human Beta-Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (12) ◽  
pp. 1865
Author(s):  
Andris Elksnis ◽  
Jing Cen ◽  
Per Wikström ◽  
Per-Ola Carlsson ◽  
Nils Welsh
Keyword(s):  
Cell Death ◽  
Beta Cell ◽  
Insulin Release ◽  
Mitochondrial Function ◽  
High Glucose ◽  
Human Islet ◽  
Sodium Palmitate ◽  
Nadph Oxidase 4

Previous studies have reported beneficial effects of NADPH oxidase 4 (NOX4) inhibition on beta-cell survival in vitro and in vivo. The mechanisms by which NOX4 inhibition protects insulin producing cells are, however, not known. The aim of the present study was to investigate the effects of a pharmacological NOX4 inhibitor (GLX7013114) on human islet and EndoC-βH1 cell mitochondrial function, and to correlate such effects with survival in islets of different size, activity, and glucose-stimulated insulin release responsiveness. We found that maximal oxygen consumption rates, but not the rates of acidification and proton leak, were increased in islets after acute NOX4 inhibition. In EndoC-βH1 cells, NOX4 inhibition increased the mitochondrial membrane potential, as estimated by JC-1 fluorescence; mitochondrial reactive oxygen species (ROS) production, as estimated by MitoSOX fluorescence; and the ATP/ADP ratio, as assessed by a bioluminescent assay. Moreover, the insulin release from EndoC-βH1 cells at a high glucose concentration increased with NOX4 inhibition. These findings were paralleled by NOX4 inhibition-induced protection against human islet cell death when challenged with high glucose and sodium palmitate. The NOX4 inhibitor protected equally well islets of different size, activity, and glucose responsiveness. We conclude that pharmacological alleviation of NOX4-induced inhibition of beta-cell mitochondria leads to increased, and not decreased, mitochondrial ROS, and this was associated with protection against cell death occurring in different types of heterogeneous islets. Thus, NOX4 inhibition or modulation may be a therapeutic strategy in type 2 diabetes that targets all types of islets.

scholarly journals Imatinib protects against human beta-cell death via inhibition of mitochondrial respiration and activation of AMPK

2021 ◽  
Author(s):  
Andris Elksnis ◽  
Tomas A Schiffer ◽  
Fredrik Palm ◽  
Yun Wang ◽  
Jing Cen ◽  
...  
Keyword(s):  
Cell Death ◽  
Tyrosine Kinase ◽  
Beta Cell ◽  
Mitochondrial Respiration ◽  
Kinase Inhibitor ◽  
Direct Interaction ◽  
Ampk Activation ◽  
Beta Cell Death ◽  
Compound C

The protein tyrosine kinase inhibitor imatinib is used in the treatment of various malignancies, but may also promote beneficial effects in the treatment of diabetes. The aim of the present investigation was to characterize the mechanisms by which imatinib protects insulin producing cells. Treatment of NOD mice with imatinib resulted in increased beta-cell AMPK phosphorylation. Imatinib activated AMPK also in vitro, resulting in decreased ribosomal protein S6 phosphorylation and protection against IAPP-aggregation, TXNIP upregulation and beta-cell death. AICAR mimicked and compound C counteracted the effect of imatinib on beta-cell survival. Imatinib-induced AMPK activation was preceded by reduced glucose/pyruvate-dependent respiration, increased glycolysis rates, and a lowered ATP/AMP ratio. Imatinib augmented the fractional oxidation of fatty acids/malate, possibly via a direct interaction with the beta-oxidation enzyme ECHS1. In non-beta cells, imatinib reduced respiratory chain complex I and II-mediated respiration and ACC phosphorylation, suggesting that mitochondrial effects of imatinib are not beta-cell specific. In conclusion, tyrosine kinase inhibitors modestly inhibit mitochondrial respiration, leading to AMPK activation and TXNIP downregulation, which in turn protects against beta-cell death.

Comparative toxicity of alloxan, N-alkylalloxans and ninhydrin to isolated pancreatic islets in vitro

1997 ◽  
Vol 155 (2) ◽  
pp. 283-293 ◽  
Author(s):  
A Jorns ◽  
R Munday ◽  
M Tiedge ◽  
S Lenzen
Keyword(s):  
Beta Cell ◽  
Pancreatic Islets ◽  
Beta Cells ◽  
Pancreatic Beta Cells ◽  
Ultrastructural Changes ◽  
Cell Necrosis ◽  
High Concentrations ◽  
Beta Cell Necrosis

The in vitro toxicity of the diabetogenic agent alloxan as documented by the induction of beta cell necrosis was studied in isolated ob/ob mouse pancreatic islets. The effect of alloxan has been compared with that of a number of N-alkyl alloxan derivatives and with that of the structurally related compound, ninhydrin. Alloxan and its derivatives were selectively toxic to pancreatic beta cells, with other endocrine cells and exocrine parenchymal cells being well preserved, even at high concentration. In contrast, ninhydrin was selectively toxic to pancreatic beta cells only at comparatively low concentration, destroying all islet cell types at high concentrations. The ultrastructural changes induced by all the test compounds in pancreatic beta cells in vitro were very similar to those observed during the development of alloxan diabetes in vivo. The relative toxicity of the various compounds to pancreatic beta cells in vitro was not, however, related to their ability to cause diabetes in vivo. Indeed, the non-diabetogenic substances ninhydrin, N-butylalloxan and N-isobutylalloxan were very much more toxic to isolated islets than the diabetogenic compounds alloxan and N-methylalloxan. These results suggest that the differences in diabetogenicity among alloxan derivatives are not due to intrinsic differences in the susceptibility of the pancreatic beta cells to their toxicity, but may reflect differences in distribution or metabolism. High concentrations of glucose protected islets against the harmful effects of alloxan and its derivatives, but not those of ninhydrin. Low levels of glucose, and non-carbohydrate nutrients, afforded little protection, indicating that the effect of glucose is not due to the production of reducing equivalents within the cell, 3-O-Methylglucose, which protects against alloan diabetes in vivo, did not protect against alloxan toxicity in vitro. Since 3-O-methylglucose is known to prevent uptake of alloxan by pancreatic beta cells, it appears that uptake of alloxan by the cell is not a prerequisite for the induction of beta cell necrosis.

scholarly journals The adaptor protein NumbL is involved in the control of glucolipotoxicity-induced pancreatic beta cell apoptosis

2020 ◽  
Author(s):  
Halesha D. Basavarajappa ◽  
Jose M. Irimia ◽  
Patrick T. Fueger
Keyword(s):  
Cell Death ◽  
Beta Cell ◽  
Beta Cells ◽  
Cell Apoptosis ◽  
Molecular Mechanisms ◽  
Pancreatic Beta Cell ◽  
Adaptor Protein ◽  
Beta Cell Apoptosis ◽  
Beta Cell Death ◽  
Egf Signaling

AbstractAvoiding loss of functional beta cell mass is critical for preventing or treating diabetes. Currently, the molecular mechanisms underlying beta cell death are partially understood, and there is a need to identify new targets for developing novel therapeutics to treat diabetes. Previously, our group established that Mig6, an inhibitor of EGF signaling, mediates beta cell death under diabetogenic conditions. The objective of this study was to clarify the mechanisms linking diabetogenic stimuli to beta cell death by investigating Mig6-interacting proteins. Using co-immunoprecipitation and mass spectrometry, we evaluated the binding partners of Mig6 under both normal glucose (NG) and glucolipotoxic (GLT) conditions in beta cells. We identified that Mig6 interacts dynamically with NumbL; whereas Mig6 associates with NumbL under NG, this interaction is disrupted under GLT conditions. Further, we demonstrate that siRNA-mediated suppression of NumbL expression in beta cells prevented apoptosis under GLT conditions by blocking activation of NF-κB signaling. Using co-immunoprecipitation experiments we observed that NumbL’s interactions with TRAF6, a key component of NFκB signaling, are increased under GLT conditions. The interactions among Mig6, NumbL, and TRAF6 are dynamic and context-dependent. We propose a model wherein these interactions activate pro-apoptotic NF-κB signaling while blocking pro-survival EGF signaling under diabetogenic conditions, leading to beta cell apoptosis. These findings indicate that NumbL should be further investigated as a candidate anti-diabetic therapeutic target.

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