THYROID ECONOMY

Potential Contribution of Translational Factorsto Triiodo-L-Thyronine-Induced Insulin Synthesis

by Pancreatic Beta Cells

Francemilson Goulart-Silva,1 Silvania da Silva Teixeira,1 Augusto Ducati Luchessi,2

Laila Romagueira Bichara dos Santos,1 Eduardo Rebelato,1

Angelo Rafael Carpinelli,1 and Maria Tereza Nunes1

Background: Thyroid hormones (THs) are known to regulate protein synthesis by acting at the transcriptionallevel and inducing the expression of many genes. However, little is known about their role in protein expressionat the post-transcriptional level, even though studies have shown enhancement of protein synthesis associatedwith mTOR/p70S6K activation after triiodo-l-thyronine (T3) administration. On the other hand, the effects ofTH on translation initiation and polypeptidic chain elongation factors, being essential for activating proteinsynthesis, have been poorly explored. Therefore, considering that preliminary studies from our laboratory havedemonstrated an increase in insulin content in INS-1E cells in response to T3 treatment, the aim of the presentstudy 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 cellswere 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 phosphorylationand the expression of the translational factors eIF2, eIF5A, and eukaryotic elongation factor 1 alpha (eEF1A). Incontrast, total and phosphorylated 4E-BP, as well as total p70S6K and p-eIF2 content, remained unchanged afterT3 treatment.Conclusions: Considering that (i) p70S6K induces S6 phosphorylation of the 40S ribosomal subunit, an essentialcondition 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 transcriptsdecoding, the data presented here lead us to suppose that a part of T3-induced insulin expression in INS-1E cellsdepends on the protein synthesis activation at the post-transcriptional level, as these proteins of the translationalmachinery were shown to be regulated by T3.

Introduction

Thyroid hormones (THs) regulate protein synthesis inmany tissues and cells (1–4); however, the molecular

mechanisms that underlie this effect are not completely un-derstood. Evidence, from the literature, points to an increasein leucine incorporation into total protein after in vitro triiodo-l-thyronine (T3) treatment of cardiomyocytes. This actionrequired the activation of Akt, mTOR, and p70S6K proteins(5), members of a signaling pathway that are committed to the

translational machinery. Indeed, it is known that p70S6K in-duces the S6 phosphorylation of the 40S ribosomal subunit, anessential condition for messenger RNA (mRNA) translationand protein synthesis (6).

It is important to note that, besides p70S6K, there are manyother proteins engaged in the initiation and ending processesof mRNA decoding. These are known as translational factors.The initiation factors promote coupling of the transcripts withthe ribosomal subunits, whereas the elongation factors insertamino acids into the nascent polypeptide chain (7,8).

1Department of Physiology and Biophysics, Institute of Biomedical Sciences, University of Sao Paulo, Sao Paulo, Brazil.2School of Applied Sciences, University of Campinas, Limeira, Brazil.

THYROIDVolume 22, Number 6, 2012ª Mary Ann Liebert, Inc.DOI: 10.1089/thy.2011.0252

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With regard to the initiation factors, the eukaryotic initia-tion factor 2 (eIF2) is one of the most extensively studied.This protein forms a ternary complex with guanosine-5¢-triphosphate and the initiator methionine-transfer RNA (tRNA),which binds to the 40S ribosomal subunit to form the 43Scomplex. This last complex is able to attach to mRNAs (9,10).

mRNAs are coiled molecules that need to be unwoundbefore their coupling with the 43S complex, a process thatrequires specific translational proteins. One of these proteinsis the eIF4E, which is attached to specific cytoplasmic pro-teins, known as eIF4E binding proteins (4E-BPs). This at-tachment inhibits eIF4E and impairs protein synthesisinitiation. Therefore, 4E-BP phosphorylation has been recog-nized as an essential step in mRNA decoding, as its phos-phorylation leads to dissociation between eIF4E and 4E-BP, aprocess that releases the translation factor 4E which initiatesprotein synthesis (11).

After ribosome formation, the next step is the elongationof the polypeptide chain, which depends on the binding ofthe eukaryotic elongation factor 1 alpha (eEF1A) to theaminoacyl tRNAs. This complex is transported to the ribo-somes, and the anticodon sequence of the aminoacyl tRNA iscoupled to the adequate codon of the mRNA molecule, al-lowing the insertion of amino acids to the nascent poly-peptide chain (12,13).

It is known that THs rapidly increase the amount of eEF1Aanchored to cytoskeleton proteins, which may improvemRNA stability and translation (14). Besides eEF1A, otherproteins also participate in the elongation process, such as theeIF5A. Despite being acknowledged as an initiation factor,eIF5A is also involved in the elongation phase of proteinsynthesis. In fact, the elongation factor 2 (eEF2) inhibitor,sordarin, blunts the effect of eIF5A on yeast growth, indicat-ing that eIF5A might function together with eEF2 to promoteribosomal translocation (15,16).

Although studies from our laboratory have shown thatINS-1E cells subjected to T3 treatment did not have alteredproinsulin mRNA content (17), our preliminary data is thatinsulin in this cell type increases after T3 treatment. However,the mechanism of this is unknown.

There have been few studies of the effects of THs on betacells. Knowledge on their actions is limited to the enhance-ment of glucose induced insulin secretion and control ofbeta-cell viability through the regulation of pro-mitotic andpro-apoptotic factors (18,19). Therefore, in this study, weattempted to evaluate the total and phosphorylated proteincontent of translational machinery in pancreatic beta cells asthis might unveil some of the mechanisms by which THsregulate protein, and particularly insulin, synthesis.

Materials and Methods

Materials

T3, Triton X-100, and l-glutamine were purchased fromSigma Chemical Co. (St. Louis, MO, USA). The antibodiesanti-insulin, anti-a-actinin, anti-eIF2, anti-p-eIF2, anti-p70S6K, anti-p-p70S6K, anti-4E-BP, and anti-p-4E-BP werepurchased from Santa Cruz Biotechnology, Inc. (SantaCruz, CA); whereas anti-eEF1A was purchased from Up-state Biotechnology, Inc. (Rochester, NY). The antibodyanti-eIF5A was generously provided by Dr. Augusto Du-cati Luchessi. The anti-mouse, anti-goat, and anti-rabbit

horseradish peroxidase-conjugated antibodies were pur-chased from Jackson Laboratories (Sacramento, CA). So-dium dodecyl sulfate, bovine serum albumin, HEPES, andb-mercaptoethanol were purchased from Invitrogen LifeTechnologies, Inc. (Grand Island, NY). 125I-labeled insulinwas purchased from PerkinElmer (Turku, Finland). En-hanced chemiluminescence (ECL) kit and polyvinylidenedifluoride filters (PVDF Hybond-P) were purchased fromAmersham Biosciences (Picataway, NJ). Protein molecularweight markers and the Bio-Rad protein assay kit werepurchased from Bio-Rad Laboratorios Brasil Ltda (SaoPaulo, Brazil). Heat inactivation was performed on fetalbovine serum (FBS), phosphate-buffered saline, trypsin/ethylenediaminetetraacetic acid (EDTA) solution, RPMI1640 culture medium, and sodium pyruvate. These werepurchased from Vitrocell Comercio de Produtos para La-boratorios Ltda (Campinas, Brazil). Tissue culture materi-als were purchased from TPP Techno Plastic Products AG(Trasadingen, Switzerland). All other reagents were pur-chased from Labsynth (Sao Paulo, Brazil).

Cell culture

The rat INS-1E insulinoma cell line was generously pro-vided by Prof. Dr. Meire Sogayar (University of Sao Paulo,Sao Paulo, Brazil) and cultured in RPMI 1640 with 11 mMglucose, supplemented with 10% FBS, 10 mM HEPES, 1 mMsodium pyruvate, 2 mM l-glutamine, 50 units/mL penicillin,50 lg/mL streptomycin, and 50 lm 2-mercaptoethanol. Cellswere cultured between passages 63 and 72, grown at 37�C in95% humidified air with a 5% CO2 atmosphere, and used forexperiments at 80%–90% confluence. In the experiments cellswere maintained in the presence or absence of T3 (10 - 6 and10 - 8 M) for 12 hours, and afterward, the following para-meters were evaluated: radioimmunoassay for insulin deter-mination in the culture medium and insulin proteinexpression, as well as eIF2, p-eIF2, eIF5A, eEF1A, p70S6K,p-p70S6K, 4E-BP, and p-4E-BP expression by Western blot-ting analysis.

Total protein determination and electrophoresis

Cells were lysed in buffer containing 100 mM Tris (pH 7.5),10 mM EDTA (pH 7.0), 35 mM sodium dodecyl sulfate (SDS),100 mM fluoride, 17 mM pyrophosphate, and 10 mM ortho-vanadate. The homogenate was centrifuged at 12,000 rpm for40 minutes at 4�C, and the supernatant was used for totalprotein determination (20).

One hundred micrograms of protein from each samplewere treated with Laemmli buffer (21), submitted to elec-trophoresis in SDS-–polyacrylamide gel electrophoresis, andtransferred to a nitrocellulose membrane by electroblotting,for 60 minutes at 15 V. Nonspecific protein binding to themembrane was reduced by preincubation with blockingbuffer (5% nonfat dry milk, 2.7 mM KCl, 137 mM NaCl,8 mM NaHPO4, 1.4 mM KPO4, and 0.1% Tween 20), over-night, at 4�C. The membrane was incubated with the specificprimary antibodies in blocking buffer, for at least 3 hours atroom temperature (RT), followed by incubation with theappropriate secondary peroxidase-conjugated antibody(1:5000), in 2.7 mM KCl, 137 mM NaCl, 8 mM NaHPO4,1.4 mM KPO4, and 0.1% Tween 20 buffer, for 90 minutes atRT. The antibodies and acrylamide gel concentration are

638 GOULART-SILVA ET AL.

described in Table 1. After washing the membrane, banddetection was carried out by using the ECL kit.

Blots were analyzed by scanning densitometry and quan-tified using the Image Master-1D-Pharmacia Biotech SWsoftware (Pharmacia Biotech, Uppsala, Sweden). Membraneswere also incubated with monoclonal anti-a-actin antibody,which was used as an internal control. The results were ex-pressed as arbitrary units.

Radioimmunoassay for insulin determination

INS-1E cells culture medium was collected from each flask,and insulin concentration was measured by radioimmuno-assay, as previously described (22).

Statistical analysis

The data were expressed as the mean – standard error ofthe mean and subjected to analysis of variance, followed bythe Student-Newman-Keuls post-test. Differences were con-sidered significant at p < 0.05.

Results

T3 increases insulin protein expression

The insulin content was determined by Western blottingand radioimmunoassay (RIA) analysis, as described. Withregard to the insulin stored in beta cells, an increase ininsulin content after T3 treatment was observed as com-pared with the control cells (Fig. 1A,B). T3 treatment also

increased insulin levels in the cell-culture medium, asshown by the RIA analysis (Fig. 1C).

T3 induces phosphorylation of p70S6K

Total and phosphorylated p70S6K were evaluated. Totalp70S6K remained unchanged in the T3-treated cells, whencompared with the control cells; however, the phosphorylatedp70S6K amount increased in the T3-treated group. These dataare shown in Figure 2.

T3 increases eIF2 protein expression and decreasesthe ratio between phosphorylated and total eIF2

The amount of both total and phosphorylated eIF2 wasevaluated. T3 treatment led to an increase of total eIF2content in beta cells compared with the control group (Fig.3A,C). On the other hand, the phosphorylated eIF2 contentremained unaltered in T3-treated cells versus the controlcells (Fig. 3B,D); however, the ratio between phosphory-lated and total eIF2 in the T3-treated group was shown tobe decreased versus the control (Fig. 3E).

T3 does not affect the total and phosphorylated 4E-BPprotein content

Cells that had been incubated with T3 did not show alter-ations in either total or phosphorylated 4E-BP amount whencompared with the control cells. These data are shown inFigure 4.

Table 1. Description of Antibodies and Acrylamide Gel Concentration

Primary antibody Antibody dilution Secondary antibody Acrylamide gel concentration

Rabbit-anti-insulin 1:1000 Goat-anti-rabbit HRP SDS-PAGE/15%Rabbit-anti-eIF2 1:1000 Goat-anti-rabbit HRP SDS-PAGE/10%Rabbit-anti-p-eIF2 1:1000 Goat-anti-rabbit HRP SDS-PAGE/10%Rabbit-anti-eIF5A 1:250 Goat-anti-rabbit HRP SDS-PAGE/15%Mouse-anti-EF1A 1:1000 Goat-anti-mouse HRP SDS-PAGE/10%Goat-anti-4E-BP 1:500 Donkey-anti-goat HRP SDS-PAGE/15%Goat-anti-p-4E-BP 1:500 Donkey-anti-goat HRP SDS-PAGE/15%Rabbit-anti-p70S6K 1:1000 Goat-anti-rabbit HRP SDS-PAGE/10%Rabbit-anti-p-p70S6K 1:1000 Goat-anti-rabbit HRP SDS-PAGE/10%

HRP, horseradish peroxidase; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; eIF, eukaryotic initiation factor;p-, phosphorylated; 4E-BP, eIF4e binding protein.

FIG. 1. Triiodo-l-thyronine (T3) increasesinsulin content. (A) Representative blots of theinsulin and a-actinin (internal control) wereobtained using the Western blottingtechnique. Total insulin content in (B) INS-1Ecells and in (C) their culture medium wereevaluated by radioimmunoassay (RIA). Thecells were sorted into control and T3-treated(10 - 6 and 10 - 8 M, for 12 hours) groups. Datarepresent the meanGstandard error of themean (SEM) of two independent experiments(n=6 per experiment), and are expressed inarbitrary units (AU). *p < 0.05 vs. control.

T3 MODULATES THE INSULIN SYNTHESIS 639

FIG. 2. T3 induces phosphorylationof p70S6K. Representative blots of total(A, t-) and phosphorylated (B, p-)p70S6K as well as of the a-actinin(internal control) were obtained usingthe Western blotting technique. Thetotal (C) and phosphorylated (D)p70S6K content in beta cells wereevaluated by RIA. The cells weresorted into control and T3-treated(10 - 6 and 10 - 8 M, for 12 hours)groups. Data represent themeanGSEM of three independentexperiments (n = 5 per experiment),and are expressed in AU. *p < 0.05vs. control.

FIG. 3. T3 increases eukaryotic initiation factor 2 (eIF2) protein expression and decreases the ratio between phosphorylatedand total eIF2. Representative blots of total (A) and phosphorylated (B) eIF2 as well as of the a-actinin (internal control) wereobtained using the Western blotting technique. The symbol # indicates a nonspecific band (23). Total (C) and phosphorylated(D) eIF2 content in beta cells were evaluated by RIA, and the ratio between the phosphorylated and total eIF2 content (E)was calculated. The cells were sorted into control and T3-treated (10 - 6 and 10 - 8 M, for 12 hours) groups. Data represent themeanGSEM of two independent experiments (n = 4 per experiment), and are expressed in AU. *p < 0.05 vs. control.

FIG. 4. T3 does not regulate eIF4Ebinding protein (4E-BP) expression andphosphorylation. Representative blotsof total (A) and phosphorylated (B) 4E-BP as well as of the a-actinin (internalcontrol) were obtained using theWestern blotting technique. Total (C)and phosphorylated (D) 4E-BP contentin the beta cells were evaluated by RIA.The cells were sorted into control andT3-treated (10 - 6 and 10 - 8 M, for12 hours) groups. Data represent themeanGSEM of two independentexperiments (n = 5 per experiment),and are expressed in AU.

640 GOULART-SILVA ET AL.

T3 increases eEF1A and eIF5A protein expression

Compared with the control, the T3-treated cells showed anincrease in both eEF1A and eIF5A translational factors, whichare committed to the elongation phase of protein synthesis.These data are shown in Figure 5.

Discussion

This study focuses on the modulation of the expression andactivities of translational factors by TH (T3); this may relate tothe stimulatory T3 action on insulin production. Insulin is themajor product synthesized by pancreatic beta cells and one ofthe most important hormones involved in the regulation ofcarbohydrate metabolism (24). Insulin synthesis and secretionare regulated by several factors, including metabolic, neural,and hormonal ones. With regard to the latter, we have shownthat T3 is one of the hormonal factors that is capable of reg-ulating insulin production and secretion, as insulin contentinside beta cells, as well as in the culture medium, increasesafter T3 administration.

These data indicate a clear stimulatory effect of THs oninsulin synthesis. This has not yet been properly characterizedin other tissues and cells (25,26). It is known that T3 inducesthe activation of p70S6K, thus stimulating protein synthesis inmany tissues and cells (5,27). This led us to postulate thatproteins from translational machinery may be targets for theregulatory action of THs on protein synthesis.

Indeed, we found that T3 modulates p70S6K activity inbeta cells; evidence for this was the increase in the amount ofphosphorylated p70S6K, an effect that apparently does notrequire new protein synthesis, as the total p70S6K contentremained unchanged. It is worth noting that p70S6K activatesthe ribosomal S6 protein by phosphorylation, enhancing thedecoding of several mRNA species into proteins (6); this mayexplain, in part, the increase in insulin expression observed inthese cells.

The translation of transcripts is a complex process involv-ing several proteins. One of them is eIF2, which is essential forthe initiation of mRNA translation. In this study, we foundthat eIF2 expression is under the control of THs, as T3 ad-ministration led to an increase in its content. This may accel-erate insulin synthesis, as more ternary complexes are formed,

allowing more transcripts to be directed to ribosomes andenabling translation into protein. Moreover, T3 did not affecteIF2 phosphorylation. This is known to reduce the activity ofeIF2 and, as a consequence, protein synthesis (28). Therefore,it seems that the decreased ratio of phosphorylated eIF2 tototal eIF2 as observed in T3-treated cells, compared withcontrol cells, reinforces the stimulatory T3 effect on insulinsynthesis.

Usually, the stimulation of p70S6K activities is accompa-nied by phosphorylation of 4E-BPs. This process leads to theinhibition of 4E-BPs and increases protein synthesis, due tothe consequent release of eIF4E, which allows its interactionwith specific regions of the transcripts, an important step inmRNA translation. Despite the increase in p70S6K phos-phorylation, T3 treatment was not associated with a change ineither 4E-BP phosphorylation or its total protein levels, indi-cating that, in this beta-cell line, 4E-BPs do not mediate theeffect of THs on insulin expression.

Furthermore, T3 also modulates other classes of moleculesthat are involved in polypeptide chain elongation. One of theseproteins is the eEF1A, which binds to the aminoacyl-tRNA andtransports it to ribosomes, where the tRNA anticodon’s tripletsequence can base-pair to one or more codons, starting theelongation stage of polypeptide chain formation (12,13). Thisphase is crucial to protein synthesis, and seems to be regulatedby THs, as eEF1A content significantly increased after T3treatment. Moreover, T3 increased the expression of the elon-gation factor eIF5A, which is thought to stimulate the ribosometo move on the mRNA molecule, as previously shown for eEF2(15,16). Thus, the increase in eEF1A and eIF5A expression re-inforces the concept that THs act on insulin production at theelongation phase, which is supported by the increase in theamount of amino acids addressed to ribosomes and the accel-eration of the ribosome movement on the mRNA molecule.

In summary, the data presented here lead us to concludethat the stimulatory effect of T3 on insulin expression de-pends, at least in part, on activation of protein synthesis, as theexpression of translational factors, as well as p70S6K phos-phorylation, was modulated by T3. In addition, this studyhelps us understand some of the molecular mechanismswhereby T3 regulates protein synthesis. It should be notedthat, even though we have focused our studies on insulin

FIG. 5. T3 increases eukaryoticelongation factor 1 alpha (eEF1A) andeIF5A expression. Representative blots oftotal eEF1A (A), total eIF5A (B), and a-actinin (internal control) were obtainedusing the Western blotting technique.Total eEF1A (C) and total eIF5A (D)content in the beta cells were evaluated byRIA. The cells were sorted into controland T3-treated (10 - 6 and 10 - 8 M, for12 hours) groups. Data represent themeanGSEM of two independentexperiments (n = 4 per experiment), andare expressed in arbitrary units. *p < 0.05vs. control.

T3 MODULATES THE INSULIN SYNTHESIS 641

synthesis, we cannot exclude the possibility that other pro-teins in these cells we studied might also be similarly alteredby T3 treatment.

Acknowledgments

This work was supported by a grant from the Fundacao deAmparo a Pesquisa do Estado de Sao Paulo (FAPESP: 08/56446-9). The authors thank Leonice Lourenco Poyares for theexcellent technical assistance rendered.

Disclosure Statement

The authors declare that no competing financial interestsexist.

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Address correspondence to:Maria Tereza Nunes, Ph.D.

Department of Physiology and BiophysicsInstitute of Biomedical Sciences

University of Sao PauloAv. Prof. Lineu Prestes 1524

Sao Paulo, SP 05508-900Brazil

E-mail: mtnunes@icb.usp.br

642 GOULART-SILVA ET AL.