Protein expression changes in a cell system of beta-cell maturation reflect an acquired sensitivity to IL-1?

Abstract

Aim/hypothesis. Type 1 diabetes mellitus (T1DM) is caused by specific destruction of the pancreatic beta cells in the islets of Langerhans. Increased sensitivity to cytokines, in particular to interleukin-1β (IL-1β) seems to be an acquired trait during beta-cell maturation. In response to cytokines both protective and deleterious mechanisms are induced in beta cells, and when the deleterious prevail, T1DM develops. The aims of this study were to identify perturbation in protein patterns (PiPP) associated with beta-cell maturation, and compare these changes to previous analyses of IL-1β exposed rat islets. For this purpose, proteome analyses were carried out using a cell-line, which matures from a glucagon-producing pre-beta-cell phenotype (NHI-glu) to an insulin-producing beta-cell phenotype (NHI-ins). We have previously shown that this maturation is accompanied by acquired sensitivity to the toxic effects of IL-1β.Methods. 2D-gel electrophoresis was used to separate the proteins and MALDI-MS and database searches were performed to identify the proteins. Results. During beta-cell maturation 135 protein spots out of 2239 detectable changed expression levels. Of these, 74 were down-regulated, 44 up-regulated, 16 were suppressed and 1 was expressed de novo. Using MALDI-MS, positive identification was obtained for 93 out of the 135 protein-spots revealing 97 different proteins. Of these, 22 proteins were in common with changes identified in previous proteome analysis of perturbation in protein pattern in IL-1β exposed rat islets. Several of the proteins were present in more than one spot suggesting post-translational modification. Conclusion/interpretation. Several proteins and protein modifications were identified that could be critically involved in beta-cell maturation, insulin-gene expression and the acquired IL-1β sensitivity. [Diabetologia (2004) A. E. Karlsen ( ✉ ), Steno Diabetes Center, Niels Steensensvej 2, 2820 Gentofte, Denmark E-mail: aek@novonordisk.com Abbreviations: T1DM, Type 1 diabetes mellitus; PiPP, perturbation in protein pattern; NO, nitric oxide; iNOS, inducible nitric oxide synthase; 2D-GE, 2 dimensional gel electrophoresis; IEF, isoelectric focusing; NEPHGE, non-equilibrium pH-gradient electrophoresis; WF, Wistar Furth; BB, Bio Breeding; PDX-1, pancreatic duodenum homeobox 1; ASS, argininosuccinate synthetase; HSP, heat shock protein; Picot, PKC-interacting cousin of thioredoxin; JNK, Jun N-terminal kinase; VDAC, voltage-dependent anion channel; GST, glutathione-Stransferase; Mw, molecular weight; pI, isoelectric point. Diabetologia (2004) 47:62-74 DOI 10.1007/s00125-003-1277 Protein expression changes in a cell system of beta-cell maturation reflect an acquired sensitivity to IL-1β Autoimmune Type 1 diabetes mellitus (T1DM) is caused by specific destruction of the insulin-producing beta cells in the islets of Langerhans in the pancreas [1]. During this process the islets are infiltrated with macrophages and lymphocytes, and these cells release a mixture of cytokines, such as inte...

Gene expression profiles during beta cell maturation and after IL-1? exposure reveal important roles of Pdx-1 and Nkx6.1 for IL-1? sensitivity

“…Comparison of the present array data with our recent proteome analyses of beta cell maturation using the same cellular cell system surprisingly revealed only two mRNA and proteins expression changes in common (creatine kinase and argininosuccinate synthetase) [19]. Several explanations may account for this, including the different methods of assay/sample preparation, different detection sensitivity, translational regulation, alternative splicing of certain mRNAs, selective degradation or excretion of proteins, time discrepancy between gene and protein expression, expression level and post-translational modification.…”

“…Down-regulation of hexokinase (FC −11; Panel A, group 3), one of three enzymes that controls glycolysis and up-regulation of fructose-1,6-bisphosphase (FC 14.9), which stimulates gluconeogenesis, suggests a reduced level of pyruvate in the beta cells, which may lead to decreased energy generation. Decreased energy generation was also evident from our previous proteome analysis using this cell system of beta cell maturation [19]. In contrast, pyruvate kinase (FC 2.8) and aldolase B (FC 9.7) involved in glycolysis were both up-regulated during beta cell maturation.…”

“…This was substantiated by proteome analyses, demonstrating changes in protein expression levels affecting pathways previously shown to be of importance in IL-1β-induced beta cell toxicity [19]. We hypothesise that changes in the mRNA expression profile, leading to susceptibility to IL-1β damage, are reflected in the transcriptome changes during beta cell maturation.…”

“…In previous proteome analyses [19,21,22] the level of significance was chosen at p<0.01, whereas in previously published array analyses [23,24,25] the level of significance has been based on fold changes (≥2.5 or ≥3), probably due to the very low number of independent experiments reported, not allowing statistical interpretation. The present array analyses are based on four independent experiments and classical statistical analysis.…”

“…Several explanations may account for this, including the different methods of assay/sample preparation, different detection sensitivity, translational regulation, alternative splicing of certain mRNAs, selective degradation or excretion of proteins, time discrepancy between gene and protein expression, expression level and post-translational modification. Furthermore in the proteome analysis, at least 37% of the protein-changes during beta cell maturation represented post-translationally modified forms not reflected at the transcript level [19]. In addition, some transcript proteins may not be present on the GeneChip and therefore not detected as significantly changed transcripts.…”