Acute T3 administration induces reorganization of somatotroph\'s cytoskeleton of hypothyroid rats: potential effect on 6H mRNA stability and translation and 6H secretion.

O T3 aumenta a poliadenilação e estabilidade do GH mRNA. O citoesqueleto (Cy) participa da estabilidade e tradução de mRNAs, pois fatores, como o EF 1a, ligam alguns transcritos a ele, aumentando sua estabilidade e tradução. Cy também participa dos processos secretores celulares. Observamos que no hipotiroidismo (Tx), há um desarranjo do Cy nos somatotrofos que é revertido pela administração aguda de T3. Neste estudo avaliamos a ligação do EF 1a e do GH mRNA ao Cy e, deste aos polissomos, na hipófise, e a expressão do IGF-I mRNA hepático, em ratos controle e Tx tratados com T3 ou salina, e sacrificados após 30 min. Observamos redução da F-actina, da ligação do EF 1a e GH mRNA a ela, do GH mRNA nos polissomos, e da expressão de IGF-I mRNA hepático, nos ratos Tx, o que indicou redução da síntese e secreção do GH. A administração de T3 estimulou esses processos, aumentando a estabilidade, tradução do GH mRNA e a secreção de GH, o que ocorreu em paralelo ao rearranjo do Cy, indicando uma ação não genômica do T3.T3 increases GH mRNA poly-A tail and stability. Cytoskeleton (Cy) plays a part on mRNA stability and translation, since factors, like EF 1a, can bind some transcripts to it, improving stability and translation efficiency. Cy is also involved in cellular secretory process. We showed that somatotropes Cy is disrupted in hypothyroidism (Tx), and rearranged by acute T3 treatment. In this study we investigated the binding of EF 1a and GH mRNA to Cy and of GH mRNA to polysomes in pituitary, as well as the liver IGF-I mRNA content, in control and Tx rats treated with T3 or saline, and killed 30 min thereafter. We observed that Tx reduced F-actin content, EF 1a and GH mRNA binding to it, GH mRNA recruitment to polysomes, in pituitary, and IGF-I mRNA expression in liver, which indicates that GH synthesis and secretion are impaired. Acute T3 treatment stimulated all these process, indicating that stability, translation of GH mRNA and GH secretion were restored. These events occurred in parallel to the Cy rearrangement, which strongly indicates a non genomic effect of T3

T3 rapidly modulates TSH beta mRNA stability and translational rate in the pituitary of hypothyroid rats

Whereas it is well known that T3 inhibits TSH beta gene transcription, its effects on TSH beta mRNA stability and translation have been poorly investigated. This study examined these possibilities, by evaluating the TSH beta transcripts poly(A) tail length, translational rate and binding to cytoskeleton, in pituitaries of thyroidectomized and sham-operated rats treated with T3 or saline, and killed 30 min thereafter. The hypothyroidism induced an increase of TSH beta transcript poly(A) tail, as well as of its content in ribosomes and attachment to cytoskeleton. The hypothyroid rats acutely treated with T3 exhibited a reduction of TSH beta mRNA poly(A) tail length and recruitment to ribosomes, indicating that this treatment decreased the stability and translation rate of TSH beta mRNA. Nevertheless, acute T3 administration to sham-operated rats provoked an increase of TSH beta transcripts binding to ribosomes. These data add new insight to an important role of T3 in rapidly regulating TSH gene expression at posttranscriptional level. (C) 2010 Elsevier Ireland Ltd. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)[06/61713-0]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Pesquisa e Desenvolvimento (CNPq)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

New insights about the posttranscriptional mechanisms triggered by iodide excess on sodium/iodide symporter (NIS) expression in PCCl3 cells

Iodide excess acutely downregulates NIS mRNA expression, as already demonstrated. PCCl3 cells treated or not with Nal, Nal + NaClO4 or Nal + Methimazole, for 30 min to 24 h, were used to further explore how iodide reduces NIS gene expression. NIS mRNA expression was evaluated by Real-Time PCR; its poly(A) tail length, by RACE-PAT; its translation rate, by polysome profile; total NIS content, by Western blotting. NIS mRNA decay rate was evaluated in actinomycin-D-treated cells, incubated with or without Nal for 0-6 h. Iodide treatment caused a reduction in NIS mRNA expression, half-life, poly(A) tail length, recruitment to ribosomes, as well as NIS protein expression. Perchlorate, but not methimazole, prevented these effects. Therefore, reduced poly(A) tail length of NIS mRNA seems to be related to its decreased half-life, in addition to its translation impairment. These data provide new insights about the molecular mechanisms involved in the rapid and posttranscriptional inhibitory effect of iodide on NIS expression. (C) 2011 Elsevier Ireland Ltd. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2009/50175-6, 2010/05503-2, 2008/56446-9, 2009/17834-6]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP

Triiodothyronine Acutely Stimulates Glucose Transport into L6 Muscle Cells Without Increasing Surface GLUT4, GLUT1, or GLUT3

Background: Thyroid hormones (THs) act genomically to stimulate glucose transport by elevating glucose transporter (Slc2a) expression and glucose utilization by cells. However, nongenomic effects of THs are now emerging. Here, we assess how triiodothyronine (T-3) acutely affects glucose transport and the content of GLUT4, GLUT1, and GLUT3 at the surface of muscle cells, and possible interactions between T-3 and insulin action. Methods: Differentiated L6 myotubes transfected with myc-tagged Slc2a4 (L6-GLUT4myc) or Slc2a1 (L6-GLUT1myc) and wild-type L6 myotubes were studied in the following conditions: control, hypothyroid (Tx), Tx plus T3, Tx plus insulin, and Tx plus insulin and T-3. Results: Glucose uptake and GLUT4 content at the cell surface decreased in the Tx group relative to controls. T-3 treatment for 30 minutes increased glucose transport into L6-GLUT4myc cells without altering surface GLUT4 content, which increased only thereafter. The total amount of GLUT4 protein remained unchanged among the groups studied. The surface GLUT1 content of L6-GLUT1myc cells also remained unaltered after T-3 treatment; however, in these cells glucose transport was not stimulated by T-3. In wild-type L6 cells, although T-3 treatment increased the total amount of GLUT3, it did not change the surface GLUT3 content. Moreover, within 30 minutes, T-3 stimulation of glucose uptake was additive to that of insulin in L6-GLUT4myc cells. As expected, insulin elevated surface GLUT4 content and glucose uptake. However, interestingly, surface GLUT4 content remained unchanged or even dropped with T-3 plus insulin. Conclusions: These data reveal that T-3 rapidly increases glucose uptake in L6-GLUT4myc cells, which, at least for 30 minutes, did not depend on an increment in GLUT4 at the cell surface yet potentiates insulin action. We propose that this rapid T-3 effect involves activation of GLUT4 transporters at the cell surface, but cannot discount the involvement of an unknown GLUT.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [FAPESP: 08/56446-9, 2006/52830-3]Fundacao de Amparo a Pesquisa do Estado de Sao PauloConselho Nacional de Pesquisa e Desenvolvimento (CNPq), BrazilCNPq Conselho Nacional de Pesquisa e Desenvolvimento (Brazil)Canadian Institutes of Health [MT 12601]Canadian Institutes of Healt

Potential contribution of translational factors to triiodo-l-hyronine-induced Insulin synthesis by pancreatic beta cells

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. 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. 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. 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 T3226637642FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP08/56446-

Effects of metformin on insulin resistance and metabolic disorders in tumor-bearing rats with advanced cachexia

Metformin (MET) is widely used in the correction of insulin (INS) resistance and metabolic abnormalities in type 2 diabetes. However, its effect on INS resistance and metabolic disorders associated with cancer cachexia is not established. We investigated the MET effects, isolated or associated with INS, on INS resistance and metabolic changes induced by Walker-256 tumor in rats with advanced cachexia. MET (500 mg.kg-1, oral) and MET + INS (NPH, 1.0 IU.kg-1, sc) were administered during 12 days, starting on the day of tumor cell inoculation. Tumor-bearing rats showed adipose and muscle mass wasting, weight loss, anorexia, decreased Akt phosphorylation in retroperitoneal and mesenteric adipose tissue, peripheral INS resistance, hypoinsulinemia, reduced INS content and secretion from pancreatic islets and also inhibition of glycolysis, gluconeogenesis and glycogenolysis in liver. MET and MET + INS treatments did not prevent these changes. It can be concluded that treatments with MET and MET + INS did not prevent the adipose and muscle mass wasting and weight loss of tumor-bearing rats possibly by not improving INS resistance. Therefore, MET, used for the treatment of INS resistance in type 2 diabetes, is not effective in improving INS resistance in the advanced stage of cancer cachexia, evidencing that the drug does not have the same beneficial effect in these two diseases.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

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.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo [FAPESP: 08/56446-9]Fundacao de Amparo a Pesquisa do Estado de Sao Paul

Iodide Transport Defect: Functional Characterization of a Novel Mutation in the Na(+)/I(-) Symporter 5 `-Untranslated Region in a Patient with Congenital Hypothyroidism

Context: Iodide transport defect (ITD) is an autosomal recessive disorder caused by impaired Na(+)/I(-) symporter (NIS)-mediated active iodide accumulation into thyroid follicular cells. Clinical manifestations comprise a variable degree of congenital hypothyroidism and goiter, and low to absent radioiodide uptake, as determined by thyroid scintigraphy. Hereditary molecular defects in NIS have been shown to cause ITD. Objective: Our objective was to perform molecular studies on NIS in a patient with congenital hypothyroidism presenting a clinical ITD phenotype. Design: The genomic DNA encoding NIS was sequenced, and an in vitro functional study of a newly identified NIS mutation was performed. Results: The analysis revealed the presence of an undescribed homozygous C to T transition at nucleotide -54 (-54C>T) located in the 5`-untranslated region in the NIS sequence. Functional studies in vitro demonstrated that the mutation was associated with a substantial decrease in iodide uptake when transfected into Cos-7 cells. The mutation severely impaired NIS protein expression, although NIS mRNA levels remained similar to those in cells transfected with wild-type NIS, suggesting a translational deficiency elicited by the mutation. Polysome profile analysis demonstrated reduced levels of polyribosomes-associated mutant NIS mRNA, consistent with reduced translation efficiency. Conclusions: We described a novel mutation in the 5`-untranslated region of the NIS gene in a newborn with congenital hypothyroidism bearing a clinical ITD phenotype. Functional evaluation of the molecular mechanism responsible for impaired NIS-mediated iodide concentration in thyroid cells indicated that the identified mutation reduces NIS translation efficiency with a subsequent decrease in protein expression and function. (J Clin Endocrinol Metab 96: E1100-E1107, 2011)Fondo Nacional de Ciencia y Tecnologia (FONCyT)Fondo Nacional de Ciencia y Tecnologia (FONCyT)Secretaria de Ciencia y Tecnologia de la Universidad Nacional de Cordoba (SeCyT)Secretaria de Ciencia y Tecnologia de la Universidad Nacional de Cordoba (SeCyT)Agencia Cordoba CienciaAgencia Cordoba CienciaFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Conselho Nacional de Pesquisa e Desenvolvimento (CNPq

Hypothyroidism in adult male rats alters posttranscriptional mechanisms of luteinizing hormone biosynthesis

BACKGROUND:\ud Studies in men are not consistent regarding the effects of thyroid hormone on the production of gonadotropins. In hypothyroidism consequent to diverse causes, an increase or no change in serum luteinizing hormone (LH) have been reported. The attempt to explain the mechanisms involved in this pathology using rats as an experimental model also seems to repeat this divergence, since hypothyroidism has been shown to induce hypogonadotropic hypogonadism, a hypergonadotropic state, or not to affect the basal levels of LH. Notably, the promoter region of the gene encoding the Lh beta subunit and GnRH (gonadotropin-releasing factor) does not contain a thyroid responsive element. Therefore, we investigated the hypothesis that, in male rats, posttranscriptional mechanisms of LH synthesis are altered in hypothyroidism. We also attempted to determine if hypothyroidism directly affects testicular function in male rats.\ud \ud METHODS:\ud Male Wistar rats, 60 days old, were thyroidectomized or sham-operated. After 20 days, they were decapitated, and the pituitaries were collected and analyzed for Lh mRNA, LH content, poly(A) tail length, and polysome profile. The testes were collected and analyzed for Lh receptor mRNA, LH receptor content, and histology using morphometric analyses. The testis, epididymis, seminal vesicle, and ventral prostate were weighed, and serum concentrations of LH, testosterone, thyrotropin (TSH), and triiodothyronine (T3) were measured.\ud \ud RESULTS:\ud Hypothyroidism was associated, in the pituitary, with an increase in Lh mRNA expression, a reduction in Lh mRNA poly(A) tail length, a reduction in the number of LH transcripts associated with polysomes. Pituitary LH was decreased but serum LH was increased from 102 to 543 pg/mL. Despite this, serum testosterone concentrations were decreased from 1.8 to 0.25 ng/mL. A decreased germinative epithelium height of the testes and a reduced weight of androgen-responsive tissues were observed (ventral prostrate: 74 vs. 23 mg/100 g body weight [BW]; seminal vesicle undrained: 280 vs. 70 mg/100 g BW; and seminal vesicle drained: 190 vs. 60 mg/100 g BW).\ud \ud CONCLUSIONS:\ud Hypothyroidism in adult male rats has dual effects on the pituitary testicular axis. It alters posttranscriptional mechanisms of LH synthesis and probably has a direct effect on testicular function. However, these data suggest the possibility that reduced LH bioactivity may account in part for impaired testicular function.CNPq, 305659/2009-0Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP, 2008/50977-2Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP, 2009/17822-8Conselho Nacional de Desenvolvimento Científico e Tecnológico - CNPq, 305659/2009-