Interleukin-1beta induced changes in the protein expression of rat islets: a computerized database.
Insulin-dependent diabetes mellitus is caused by an autoimmune destruction of the beta-cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to beta-cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, previous studies have found an association of beta-cell destruction with alterations in protein synthesis. Thus, two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins may be an important tool facilitating studies of the molecular pathogenesis of insulin-dependent diabetes mellitus. 2-D gel electrophoresis of islet proteins may lead to (i) the determination of qualitative and quantitative changes in specific islet proteins induced by cytokines, (ii) the determination of the effects of agents modulating cytokine action, and (iii) the identification of primary islet protein antigen(s) initiating the immune destruction of the beta-cells. Therefore, the aim of this study was to create databases (DB) of all reproducibly detectable protein spots on 10% and 15% acrylamide 2-D gels of neonatal rat islets (10% and 15% DB), labeled under standardized culture conditions. 1235 and 557 spots were present in 5 of 5 gels in the 15% isoelectric focusing (IEF) and nonequilibrium pH gradient electrophoresis (NEPHGE) DB, respectively, whereas 995 and 378 spots were present in 5 of 5 gels in the 10% IEF and NEPHGE DB, respectively, yielding a reproducibility of spot detection between 75.2% and 91.7%. In both DBs, the average coefficient of variation of the percentage of integrated optical density (CV% of %IOD) for spots present in all gels was between 42.4% and 45.7%. When the same sample was analyzed in consecutive sets of gels on different days (interassay analysis), the average CV% of %IOD was 35.5%-36.1%. When the same sample was analyzed repeatedly in one set of gels (intra-assay analysis), the average CV% of %IOD was 30.2% in the IEF gels, while the average CV% of %IOD was 45.7% in the NEPHGE gels. Addition of interleukin-1beta (IL-1beta) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2-D gel protein databases of neonatal rat islets of Langerhans and demonstrate its usage to identify proteins altered in expression by IL-1beta.
Andersen
H U
HU
Steno Diabetes Center, Gentofte, Denmark.
Fey
S J
SJ
Larsen
P M
PM
Nawrocki
A
A
Hejnaes
K R
KR
Mandrup-Poulsen
T
T
Nerup
J
J
D000818:Animals; D000831:Animals, Newborn; D015925:Cryopreservation; D046508:Culture Techniques; D016208:Databases, Factual; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000378:metabolism*; D007525:Isoelectric Focusing; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D051381:Rats; D011920:Rats, Inbred WF; D011994:Recombinant Proteins / Q000494:pharmacology; D012044:Regression Analysis; D015203:Reproducibility of Results
D007375:Interleukin-1; D011506:Proteins; D011994:Recombinant Proteins