Potential contribution of translational factors to triiodo-L-thyronine-induced insulin synthesis by pancreatic beta cells.
BACKGROUND
Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptional level and inducing the expression of many genes. However, little is known about their role in protein expression at the post-transcriptional level, even though studies have shown enhancement of protein synthesis associated with mTOR/p70S6K activation after triiodo-L-thyronine (T3) administration. On the other hand, the effects of TH on translation initiation and polypeptidic chain elongation factors, being essential for activating protein synthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory have demonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the present study was to investigate if proteins of translational nature might be involved in this effect.


METHODS
INS-1E cells were maintained in the presence or absence of T3 (10(-6) or 10(-8) M) for 12 hours. Thereafter, insulin concentration in the culture medium was determined by radioimmunoassay, and the cells were processed for Western blot detection of insulin, eukaryotic initiation factor 2 (eIF2), p-eIF2, eIF5A, EF1A, eIF4E binding protein (4E-BP), p-4E-BP, p70S6K, and p-p70S6K.


RESULTS
It was found that, in parallel with increased insulin generation, T3 induced p70S6K phosphorylation and the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). In contrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged after T3 treatment.


CONCLUSIONS
Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essential condition for protein synthesis; (ii) eIF2 is essential for the initiation of messenger RNA translation process; and (iii) eIF5A and eEF1A play a central role in the elongation of the polypeptidic chain during the transcripts decoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cells depends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translational machinery were shown to be regulated by T3.
Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of São Paulo, SP, Brazil.
Francemilson
F
Goulart-Silva
Silvania da Silva
Sda S
Teixeira
Augusto Ducati
AD
Luchessi
Laila Romagueira Bichara
LR
Dos Santos
Eduardo
E
Rebelato
Angelo Rafael
AR
Carpinelli
Maria Tereza
MT
Nunes
D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D004305:Dose-Response Relationship, Drug; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D050417:Insulin-Secreting Cells / Q000187:drug effects* / Q000378:metabolism* / Q000473:pathology; D007340:Insulinoma / Q000378:metabolism / Q000473:pathology; D047908:Intracellular Signaling Peptides and Proteins; D023421:Models, Animal; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology; D010448:Peptide Initiation Factors / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D014176:Protein Biosynthesis; D016601:RNA-Binding Proteins / Q000378:metabolism*; D051381:Rats; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D014284:Triiodothyronine / Q000494:pharmacology*; D000097574:Eukaryotic Translation Initiation Factor 5A
D002352:Carrier Proteins; D015852:Eukaryotic Initiation Factor-2; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; D010448:Peptide Initiation Factors; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D014284:Triiodothyronine; D000097574:Eukaryotic Translation Initiation Factor 5A; C499526:Eif4ebp1 protein, rat