Silvania da Silva Teixeira

The Acute Inhibitory Effect of Iodide Excess on Sodium/Iodide Symporter Expression and Activity Involves the PI3K/Akt Signaling Pathway

Iodide (I(-)) is an irreplaceable constituent of thyroid hormones and an important regulator of thyroid function, because high concentrations of I(-) down-regulate sodium/iodide symporter (NIS) expression and function. In thyrocytes, activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) cascade also inhibits NIS expression and function. Because I(-) excess and PI3K/Akt signaling pathway induce similar inhibitory effects on NIS expression, we aimed to study whether the PI3K/Akt cascade mediates the acute and rapid inhibitory effect of I(-) excess on NIS expression/activity. Here, we reported that the treatment of PCCl3 cells with I(-) excess increased Akt phosphorylation under normal or TSH/insulin-starving conditions. I(-) stimulated Akt phosphorylation in a PI3K-dependent manner, because the use of PI3K inhibitors (wortmannin or 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) abrogated the induction of I(-) effect. Moreover, I(-) inhibitory effect on NIS expression and function were abolished when the cells were previously treated with specific inhibitors of PI3K or Akt (Akt1/2 kinase inhibitor). Importantly, we also found that the effect of I(-) on NIS expression involved the generation of reactive oxygen species (ROS). Using the fluorogenic probes dihydroethidium and mitochondrial superoxide indicator (MitoSOX Red), we observed that I(-) excess increased ROS production in thyrocytes and determined that mitochondria were the source of anion superoxide. Furthermore, the ROS scavengers N-acetyl cysteine and 2-phenyl-1,2-benzisoselenazol-3-(2H)-one blocked the effect of I(-) on Akt phosphorylation. Overall, our data demonstrated the involvement of the PI3K/Akt signaling pathway as a novel mediator of the I(-)-induced thyroid autoregulation, linking the role of thyroid oxidative state to the Wolff-Chaikoff effect.

T3 Rapidly Increases SLC2A4 Gene Expression and GLUT4 Trafficking to the Plasma Membrane in Skeletal Muscle of Rat and Improves Glucose Homeostasis

BACKGROUND Glucose transporter 4 (GLUT4) is highly expressed in muscle and fat tissue, where triiodothyronine (T(3)) induces solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene transcription. T(3) was also shown to rapidly increase glucose uptake in myocytes exposed to cycloheximide, indicating that it might act nongenomically to regulate GLUT4 availability. We tested this hypothesis by evaluating, in thyroidectomized rats (Tx rats), the acute and/or chronic T(3) effects on GLUT4 mRNA expression and polyadenylation, protein content, and trafficking to the plasma membrane (PM) in skeletal muscle, as well as on blood glucose disappearance rate (kITT) after insulin administration. METHODS Rats were surgically thyroidectomized and treated with T(3) (0.3 to 100 μg/100 g body weight) from 10 minutes to 5 days, and killed thereafter. Sham-operated (SO) rats were used as controls. Total RNA was extracted from the skeletal muscles (soleus [SOL] and extensorum digitalis longus [EDL]) and subjected to Northern blotting analysis using rat GLUT4 cDNA probe. Total protein was extracted and subjected to specific centrifugations for subcellular fractionation, and PM as well as microsomal (M) fractions were subjected to Western blotting analysis, using anti-GLUT4 antiserum as a probe. GLUT4 mRNA polyadenylation was examined by a rapid amplification of cDNA ends-poly(A) test (RACE-PAT). RESULTS Thyroidectomy reduced skeletal muscle GLUT4 mRNA, mRNA poly(A) tail length, protein content, and trafficking to the PM, as well as the kITT. The acute T(3) treatment rapidly (30 minutes) increased all these parameters compared with Tx rats. The 5-day T(3) treatment increased GLUT4 mRNA and protein expression, and restored GLUT4 trafficking to the PM and kITT to SO values. CONCLUSIONS The results presented here show for the first time that, in parallel to its transcriptional action on the SLC2A4 gene, T(3) exerts a rapid post-transcriptional effect on GLUT4 mRNA polyadenylation, which might increase transcript stability and translation efficiency, leading to the increased GLUT4 content and availability to skeletal muscle, as well as on GLUT4 translocation to the PM, improving the insulin sensitivity, as shown by the kITT.

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 T(3), 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.

Beta hydroxy beta methylbutyrate supplementation impairs peripheral insulin sensitivity in healthy sedentary Wistar rats

Investigate, in healthy sedentary rats, the potential mechanisms involved on the effects of beta hydroxy beta methylbutyrate (HMB) supplementation upon the glycaemic homeostasis, by evaluating the insulin sensitivity in liver, skeletal muscle, and white adipose tissue.

3,5-diiodothyronine (3,5-T2) reduces blood glucose independently of insulin sensitization in obese mice

Thyroid hormones regulate metabolic response. While triiodothyronine (T3) is usually considered to be the active form of thyroid hormone, one form of diiodothyronine (3,5‐T2) exerts T3‐like effects on energy consumption and lipid metabolism. 3,5‐T2 also improves glucose tolerance in rats and 3,5‐T2 levels correlate with fasting glucose in humans. Presently, however, little is known about mechanisms of 3,5‐T2 effects on glucose metabolism. Here, we set out to compare effects of T3, 3,5‐T2 and another form of T2 (3,3‐T2) in a mouse model of diet‐induced obesity and determined effects of T3 and 3,5‐T2 on markers of classical insulin sensitization to understand how diiodothyronines influence blood glucose.

Thyroid hormone treatment decreases hepatic glucose production and renal reabsorption of glucose in alloxan‐induced diabetic Wistar rats

The thyroid hormone (TH) plays an important role in glucose metabolism. Recently, we showed that the TH improves glycemia control by decreasing cytokines expression in the adipose tissue and skeletal muscle of alloxan‐induced diabetic rats, which were also shown to present primary hypothyroidism. In this context, this study aims to investigate whether the chronic treatment of diabetic rats with T3 could affect other tissues that are involved in the control of glucose homeostasis, as the liver and kidney. Adult Male Wistar rats were divided into nondiabetic, diabetic, and diabetic treated with T3 (1.5 μg/100 g BW for 4 weeks). Diabetes was induced by alloxan monohydrate (150 mg/kg, BW, i.p.). Animals showing fasting blood glucose levels greater than 250 mg/dL were selected for the study. After treatment, we measured the blood glucose, serum T3, T4, TSH, and insulin concentration, hepatic glucose production by liver perfusion, liver PEPCK, GAPDH, and pAKT expression, as well as urine glucose concentration and renal expression of SGLT2 and GLUT2. T3 reduced blood glucose, hepatic glucose production, liver PEPCK, GAPDH, and pAKT content and the renal expression of SGLT2 and increased glycosuria. Results suggest that the decreased hepatic glucose output and increased glucose excretion induced by T3 treatment are important mechanisms that contribute to reduce serum concentration of glucose, accounting for the improvement of glucose homeostasis control in diabetic rats.

Thyroid hormone improves insulin signaling and reduces the activation of neurodegenerative pathway in the hippocampus of diabetic adult male rats

Aims: Diabetes mellitus (DM) and impairments of glucose metabolism and insulin resistance in the brain have been suggested as a likely etiology of Alzheimers disease (AD). Studies have shown that thyroid hormones (THs) improve insulin sensitivity in DM rats and act as mediators of the plasticity of the nervous system altering behavior and cognitive function. Based on these findings, this study aimed to evaluate the effects of diabetes and triiodothyronine (T3) treatment upon proteins associated with DM and AD in the central nervous system. Main methods: Euglycemic and Diabetic (alloxan‐induced) male Wistar rats were daily treated with T3 (1.5 &mgr;g/100 g body weight) or vehicle (saline) for a 4‐week period and subdivided into the following groups: euglycemic treated with saline (Control = C); diabetic treated with saline (Diabetic = D); euglycemic treated with T3 (T3); diabetic treated with T3 (DT3). The expression of insulin signaling, neurodegenerative and neuron survival markers was evaluated in the hippocampus by immunoblotting, ELISA, and RT‐PCR. Key findings: T3 treatment decreased glycemia, restored the insulin signaling and reduced the activation of glycogen synthase kinase 3 (GSK3) and tau proteins content in the hippocampus of diabetic rats. Significance: The present data provide evidence that T3 treatment of diabetic rats is able to improve insulin sensitivity and reduce the activation of the neurodegenerative pathway in the brain, which might provide neuroprotection in this experimental model.

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.