Glucagon-Like Peptide-1 Protects Human Islets against Cytokine-Mediated β-Cell Dysfunction and Death: A Proteomic Study of the Pathways Involved

Glucagon-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.status: publishe

Non-muscle myosin IIA is involved in focal adhesion and actin remodelling controlling glucose-stimulated insulin secretion

Actin and focal adhesion (FA) remodelling are essential for glucose-stimulated insulin secretion (GSIS). Non-muscle myosin II (NM II) isoforms have been implicated in such remodelling in other cell types, and myosin light chain kinase (MLCK) and Rho-associated coiled-coil-containing kinase (ROCK) are upstream regulators of NM II, which is known to be involved in GSIS. The aim of this work was to elucidate the implication and regulation of NM IIA and IIB in beta cell actin and FA remodelling, granule trafficking and GSIS

The beta-cell in type 1 diabetes: What have we learned from proteomic studies?

Pancreatic beta-cells have a crucial role in the regulation of blood glucose homeostasis by the production and secretion of insulin. In type 1 diabetes (T1D), an autoimmune reaction against the beta-cells together with the presence of inflammatory cytokines and ROS in the islets leads to beta-cell dysfunction and death. This review gives an overview of proteomic studies that lead to better understanding of beta-cell functioning in T1D. Protein profiling of isolated islets and beta-cell lines in health and T1D contributed to the unraveling of pathways involved in cytokine-induced cell death. In addition, by studying the serological proteome of T1D patients, new biomarkers and beta-cell autoantigens were discovered, which may improve screening tests and follow-up of T1D development. Interestingly, an important role for PTMs was demonstrated in the generation of beta-cell autoantigens. To conclude, proteomic techniques are of indispensable value to improve the knowledge on beta-cell function in T1D and the search toward therapeutic targets.status: publishe

Tumour necrosis factor induces phosphorylation primarily of the nitric-oxide-responsive form of glyoxalase I

We have previously shown that TNF (tumour necrosis factor) induces phosphorylation of GLO1 (glyoxalase I), which is required for cell death in L929 cells. In the present paper, we show that the TNF-induced phosphorylation of GLO1 occurs primarily on the NO (nitric oxide)-responsive form of GLO1. In addition, analysis of several cysteine mutants of GLO1 indicated that Cys-138, in combination with either Cys-18 or Cys-19, is a crucial target residue for the NO-mediated modification of GLO1. Furthermore, the NO-donor GSNO (S-nitrosogluthathione) induces NO-mediated modification of GLO1 and enhances the TNF-induced phosphorylation of this NO-responsive form. GSNO also strongly promotes TNF-induced cell death. By the use of pharmacological inhibition of iNOS (inducible NO synthase) and overexpression of mutants of GLO1 that are deficient for the NO-mediated modification, we have shown that the NO-mediated modification of GLO1 is not a requirement for TNF-induced phosphorylation or TNF-induced cell death respectively. In summary, these data suggest that the TNF-induced phosphorylation of GLO1 is the dominant factor for cell death

Phosphorylation on Thr-106 and NO-modification of glyoxalase I suppress the TNF-induced transcriptional activity of NF-kappa B

Glyoxalase I (GLO1), together with glyoxalase II and the co-factor GSH, comprise the glyoxalase system, which is responsible for the detoxification of the cytotoxic glycolytic-derived metabolite methylglyoxal (MG). We, and others, have previously reported that GLO1 is subjected to several post-translational modifications, including a NO-mediated modification and phosphorylation. In this study, we demonstrate that GLO1 is a substrate for calcium, calmodulin-dependent protein kinase II (CaMKII). Site-directed mutagenesis of several serine and threonine residues revealed that CaMKII induced phosphorylation of GLO1 at a single site Thr-106. Mutagenesis of Thr-106 to Ala in GLO1 completely abolished the CaMKII-mediated phosphorylation. A phosphopeptide bracketing phosphothreonine-106 in GLO1 was used as an antigen to generate polyclonal antibodies against phosphothreonine-106. By using this phospho-specific antibody, we demonstrated that TNF induces phosphorylation of GLO1 on Thr-106. Furthermore, we investigated the role of NO-mediated modification and phosphorylation of GLO1 in the TNF-induced transcriptional activity of NF-kappa B. Overexpression of WT GLO1 suppressed TNF-induced NF-kappa B-dependent reporter gene expression. Suppression of the basal and TNF-induced NF-kappa B activity was significantly stronger upon expression of a GLO1 mutant that was either deficient for the NO-mediated modification or phosphorylation on Thr-106. However, upon overexpression of a GLO1 mutant that was deficient for both types of modification, the suppressive effect of GLO1 on TNF-induced NF-kappa B activity was completely abolished. These results suggest that NO-modification and phosphorylation of GLO1 contribute to the suppression of TNF-induced NF-kappa B-dependent reporter gene expression. In line with this, knock-down of GLO1 by siRNA significantly increased TNF-induced NF-kappa B-dependent reporter gene expression. These findings suggest that phosphorylation and NO-modification of glyoxalase I provides another control mechanism for modulating the basal and TNF-induced expression of NF-kappaB-responsive genes.status: publishe

Novel Mechanistic Link between Focal Adhesion Remodeling and Glucose-stimulated Insulin Secretion

Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic beta cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the beta cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary beta cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin beta 1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between beta 1 integrins and the extracellular matrix with an anti-beta 1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by beta 1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion.status: publishe

Novel mechanistic link between focal adhesion remodeling and glucose-stimulated insulin secretion

Actin cytoskeleton remodeling is well known to be positively involved in glucose-stimulated pancreatic β cell insulin secretion. We have observed glucose-stimulated focal adhesion remodeling at the β cell surface and have shown this to be crucial for glucose-stimulated insulin secretion. However, the mechanistic link between such remodeling and the insulin secretory machinery remained unknown and was the major aim of this study. MIN6B1 cells, a previously validated model of primary β cell function, were used for all experiments. Total internal reflection fluorescence microscopy revealed the glucose-responsive co-localization of focal adhesion kinase (FAK) and paxillin with integrin β1 at the basal cell surface after short term stimulation. In addition, blockade of the interaction between β1 integrins and the extracellular matrix with an anti-β1 integrin antibody (Ha2/5) inhibited short term glucose-induced phosphorylation of FAK (Tyr-397), paxillin (Tyr-118), and ERK1/2 (Thr-202/Tyr-204). Pharmacological inhibition of FAK activity blocked glucose-induced actin cytoskeleton remodeling and glucose-induced disruption of the F-actin/SNAP-25 association at the plasma membrane as well as the distribution of insulin granules to regions in close proximity to the plasma membrane. Furthermore, FAK inhibition also completely blocked short term glucose-induced activation of the Akt/AS160 signaling pathway. In conclusion, these results indicate 1) that glucose-induced activation of FAK, paxillin, and ERK1/2 is mediated by β1 integrin intracellular signaling, 2) a mechanism whereby FAK mediates glucose-induced actin cytoskeleton remodeling, hence allowing docking and fusion of insulin granules to the plasma membrane, and 3) a possible functional role for the Akt/AS160 signaling pathway in the FAK-mediated regulation of glucose-stimulated insulin secretion

A proteomic study of the regulatory role for STAT-1 in cytokine-induced beta-cell death

Signal transducer and activator of transcription 1 (STAT-1) plays a crucial role in cytokine-induced beta-cell destruction. However, its precise downstream pathways have not been completely clarified. We performed a proteome analysis of cytokine-exposed C57Bl/6 and STAT-1(-/-) mouse islets and prioritized proteins for their potential in relation to type 1 diabetes (T1D).status: publishe