Rapid changes of mRNA-binding protein levels following glucose and 3-isobutyl-1-methylxanthine stimulation of insulinoma INS-1 cells.
Glucose and cAMP-inducing agents such as 3-isobutyl-1-methylxanthine (IBMX) rapidly change the expression profile of insulin-producing pancreatic beta-cells mostly through post-transcriptional mechanisms. A thorough analysis of these changes, however, has not yet been performed. By combining two-dimensional differential gel electrophoresis and mass spectrometry, we identified 165 spots, corresponding to 78 proteins, whose levels significantly change after stimulation of the beta-cell model INS-1 cells with 25 mM glucose + 1 mM IBMX for 2 h. Changes in the expression of selected proteins were verified by one- and two-dimensional immunoblotting. Most of the identified proteins are novel targets of rapid regulation in beta-cells. The transcription inhibitor actinomycin D failed to block changes in two-thirds of the spots, supporting their post-transcriptional regulation. More spots changed in response to IBMX than to glucose alone conceivably because of phosphorylation. Fourteen mRNA- binding proteins responded to stimulation, thus representing the most prominent class of rapidly regulated proteins. Bioinformatics analysis indicated that the mRNA 5'- and 3'-untranslated regions of 22 regulated proteins contain potential binding sites for polypyrimidine tract-binding protein 1, which promotes mRNA stability and translation in stimulated beta-cells. Overall our findings support the idea that mRNA-binding proteins play a major role in rapid adaptive changes in insulin-producing cells following their stimulation with glucose and cAMP-elevating agents.
Experimental Diabetology, Dresden University of Technology, Dresden, Germany.
Christin
C
Süss
Cornelia
C
Czupalla
Christof
C
Winter
Theresia
T
Pursche
Klaus-Peter
KP
Knoch
Michael
M
Schroeder
Bernard
B
Hoflack
Michele
M
Solimena
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology*; D020816:Amino Acid Motifs; D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D019295:Computational Biology; D017124:Conserved Sequence; D015180:Electrophoresis, Gel, Two-Dimensional; D005947:Glucose / Q000494:pharmacology*; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007340:Insulinoma / Q000378:metabolism* / Q000473:pathology*; D013058:Mass Spectrometry; D051379:Mice; D009124:Muscle Proteins / Q000378:metabolism; D009363:Neoplasm Proteins / Q000378:metabolism; D038941:Polypyrimidine Tract-Binding Protein; D040901:Proteomics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism; D020506:Untranslated Regions / Q000235:genetics
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009124:Muscle Proteins; D009363:Neoplasm Proteins; C475523:Ptbp1 protein, rat; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D020506:Untranslated Regions; D038941:Polypyrimidine Tract-Binding Protein; D005947:Glucose; D015056:1-Methyl-3-isobutylxanthine