T 3 Rapidly Increases SLC2A4 Gene Expression and GLUT4 Trafficking to the Plasma Membrane in Skeletal Muscle of Rat and Improves Glucose Homeostasis

Abstract

Background:

Glucose transporter 4 (GLUT4) is highly expressed in muscle and fat tissue, where triiodothyronine (T₃) induces solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene transcription. T₃ 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 T₃ 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 T₃ (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 T₃ treatment rapidly (30 minutes) increased all these parameters compared with Tx rats. The 5-day T₃ 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, T₃ 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.

Introduction

T riiodothyronine (T₃) classically acts in regulating gene expression by binding to high-affinity thyroid hormone receptors (TRs) that recognize specific response elements (TREs) in the promoter region of T₃-target genes, a mechanism by which T₃ causes its biological effects (1).

In contrast, a number of the effects of T₃ and T₄ occur very rapidly and are unaffected by gene transcription and protein synthesis inhibitors, indicating that they also act nongenomically (1). These nongenomic actions continue to be reported. Though their mechanisms are still not well understood, it appears that some involve activation of protein kinase signaling pathways (1).

Skeletal muscle is an important target for T₃, which regulates the transcription rate of several genes that code for contractile and other proteins that are important for energy metabolism, such as glucose transporter 4 (GLUT4) (2 –5). GLUT4 is upregulated by T₃; is highly expressed in skeletal muscle, heart, and adipose tissues; and plays an important role in whole body glucose homeostasis (5,6). It is stored in intracellular vesicle membranes, and can be acutely translocated to and inserted in the plasma membrane (PM) by insulin stimulation and muscle contraction, allowing glucose uptake to increase (7).

The high GLUT4 expression in skeletal muscle and its regulation by T₃ make this tissue and this hormone particularly important for the control of glycemic homeostasis (6 –8). Long-term T₃ administration has been shown to stimulate GLUT4 expression and glucose transport in rat skeletal muscle, which might contribute to the increased peripheral glucose utilization observed in hyperthyroidism (5).

Torrance et al. have reported a specific T₃ receptor binding site within the T₃-responsive region of the solute carrier family 2 facilitated glucose transporter member 4 (SLC2A4) gene, but this binding site had a relatively low affinity for the T₃ receptor (9). The role of this binding with regard to the ability of T₃ to stimulate GLUT4 mRNA does not seem to explain our preliminary results that T₃ administration increases GLUT4 mRNA content in rat skeletal muscles in only 30 minutes (10). This would not be expected if the effect of T₃ were mediated by a low-affinity T₃-responsive region of the SLC2A4 gene, and the rapidity of the response favors the hypothesis that a nongenomic mechanism was involved. Similarly, Segal demonstrated that T₃ increased glucose uptake by freshly isolated rat heart slices in vitro, as well as by several rat tissues in vivo, in only a few minutes (11,12). In the studies of Segal (11,12), evidence was provided that these effects were mediated by a nongenomic pathway, as not only the onset of action was rapid, but also that the effects occurred in the presence of the protein synthesis inhibitor cycloheximide. More recently, Gordon et al. obtained evidence that T₃ stimulated the uptake of 2-deoxy-D-glucose into L6 cells by a nongenomic pathway (13).

One of the recognized nongenomic actions of T₄ and T₃ is the induction of rapid remodeling of actin filaments (2,14), which is required for GLUT4 translocation into the PM and glucose uptake (15), and is supposedly involved in the control of mRNA stability (16). mRNA stability is dependent on the poly(A) tail length of mRNA transcripts, a step that has been shown to be regulated by T₃ (17). These data further support a significant role for T₃ in the control of glucose uptake through its well-known transcriptional effects on the SLC2A4 gene expression and indicate that there are novel aspects yet to be defined, involving nongenomic actions of T₃ on GLUT4 expression and trafficking to the PM.

Considering that our preliminary studies have shown that T₃ administration increased GLUT4 mRNA content in rat skeletal muscles in only 30 minutes, which strongly indicates that this effect is mediated by nongenomic mechanisms, in the current study, we attempted to evaluate whether this effect results from an increase in GLUT4 mRNA stability, which could lead to an increase in GLUT4 protein content. Furthermore, we have investigated whether acute T₃ administration induces GLUT4 translocation to the PM, augmenting blood glucose decay rate.

Materials and Methods

Animals and treatments

Male Wistar rats weighing 200–250 g were obtained from our own breeding colony and maintained on rat chow and tap water ad libitum. They were housed in a room kept at a constant temperature (23°C±2°C) and on a 12-hour-light/12-hour-dark (lights on at 0700 hours) schedule. The animals were made hypothyroid by surgical thyroidectomy, after being deeply anesthetized with ketamine and xylazine (100 and 10 mg/kg body weight [BW], intraperitoneal [i.p.], respectively) (18), and received 0.03% methylmercaptoimidazole (Sigma Chemical Co., St. Louis, MO) plus 4.5 mM CaCl₂ (Sigma Chemical Co.) in drinking water during 21 days, before being treated with T₃ (19).

Several groups of rats were studied. The control group comprised rats that were subjected to sham thyroidectomy, referred to as the SO group. The Tx group included rats that had been thyroidectomized 21 days before they were sacrificed and studied. To evaluate responses to chronic T₃ administration, a group of thyroidectomized rats (Tx rats) were treated with T₃, 5.0 μg/100 g BW, i.p., divided in two doses, for 5 days. They were referred to as the T₃-chronic group. To evaluate rapid-onset responses to T₃ administration, Tx rats were treated with three different doses of T₃ and sacrificed 10 to 30 minutes after T₃ administration. One group of Tx rats were treated with supra-physiological doses of T₃ (100 μg/100 g BW, intravenous [i.v.]). These rats were divided into three subgroups. These subgroups were sacrificed 10, 20, and 30 minutes after T₃ administration and are referred to as the T₃ 10′, T₃ 20′, and T₃ 30′ subgroups, respectively. Another group of Tx rats were treated with T₃, 0.3 μg/100 g BW, and sacrificed 30 minutes later. These are referred to as the T₃ 0.3 subgroup. Another group of Tx rats were treated with T₃, 1.5 μg/100 g BW, i.v., and sacrificed 30 minutes later. These are referred to as the T₃ 1.5 subgroup.

After being anesthetized, the rats were weighed and treated with T₃ for 10 to 30 minutes; then, they had their slow-oxidative (soleus [SOL]) and fast-glycolytic (extensorum digitalis longus [EDL]) skeletal muscles excised. They were subsequently killed by decapitation, and trunk blood was collected for the measurement of total T₃ serum concentrations by radioimmunoassay (RIA; Diagnostic Products Corporation, Llamberis, United Kingdom). The heart was removed for ventricular weight (VW) measurement.

The experimental protocol conformed with ethics principles in animal research adopted by the Brazilian College of Animal Experimentation and was approved by the local Ethics Committee for Animal Research (#16/04). The effectiveness of the treatments in the different experimental groups was evaluated by determining the T₃ serum levels by RIA, using a standard curve prepared by adding different concentrations of T₃ in iodothyronine-free rat serum and by the ratio between the VW and BW (4,14,20).

Evaluation of SLC2A4 gene expression

The GLUT4 mRNA and protein expression in SOL and EDL muscles was evaluated by Northern and Western blot, respectively.

Northern blot analysis

Total RNA was extracted by the guanidinium thiocyanate–phenol–chloroform method, electrophoresed, transferred to a nylon membrane, and subjected to Northern blotting analysis, as described (21 –24). The results were expressed as mean±standard error of mean (SEM) of GLUT4 mRNA/18S rRNA ratio.

Western blot analysis

The SOL and EDL muscles were homogenized in 10 mM Trizma, 1 mM EDTA, and 250 mM sacarose buffer. The homogenate was subjected to specific centrifugations for subcellular fractionation, in agreement with Yonemitsu et al. (25), with little modifications (18). Immunoblotting of the tissue protein extracts was performed using anti-GLUT4 antiserum (1:3000 Polyclonal Rabbit Raised Anti-GLUT4, AB1346; Chemicon International, Inc., Temecula, CA) in 8% BSA/PBS solution, according to the manufacturer's recommendations. Immunoblotting of SOL protein extracts was also performed using anti-Na⁺/K⁺ ATPase antiserum (1:5000 Polyclonal Rabbit Raised Anti-Na⁺/K⁺ ATPase; Upstate Biotechnology, Lake Placid, NY) as a control of subcellular fractionation procedure.

The band detection was performed using secondary peroxidase-conjugated antibody (1:10,000; Santa Cruz Biotechnology, Santa Cruz, CA) and the Enhanced Chemiluminescence kit (Amersham Biosciences, Buckinghamshire, United Kingdom). Blots were analyzed with Scion Image (Scion Corp., Frederick, MD). Results were expressed as arbitrary units (AU).

Considering the total protein yield of each subcellular membrane fraction (PM and M) and the respective tissue weight, PM, and M GLUT4, protein per gram of tissue was calculated (AU/g tissue); the sum of these data was considered as the total GLUT4 content per gram of tissue. The GLUT4 translocation index (%) was then calculated as indicated: ([100 × PM GLUT4]/[PM GLUT4+M GLUT4]) (18).

Evaluation of GLUT4 mRNA poly(A) tail length

Measurements of the GLUT4 and Cyclophilin mRNA poly(A) tail length from SOL and EDL muscles of SO, Tx, and T₃ 30′ (100 μg/100 g BW, i.v.) groups were performed by a rapid amplification of cDNA ends–poly(A) test (RACE-PAT), according to a standard protocol (24,26). Amplicon sizes were estimated by densitometry and compared with a 100-bp DNA ladder (Invitrogen Life Technologies) using the ImageMaster 1D-Pharmacia Biotech SW software (Pharmacia Biotech). The top of the smear indicated the longest amplified fragment, which represents the poly(A) tail size plus 100 bases upstream, according to the GLUT4 primer used. Cyclophilin was used as an internal control.

ITT and blood glucose disappearance rate determination

Rats were deprived of food for about 2 hours, anesthetized, and received an i.v. injection of regular insulin (0.75 U/kg BW, i.v.). Glucose blood levels were measured in samples obtained from the tail vein using a glucometer (Precision QIR^®; Mediense Products) at 0 (T0), 4 (T4), 8 (T8), 12 (T12), and 16 (T16) minutes after insulin injection (23). The values obtained were used to calculate the kITT using the linear regression of the neperian logarithm of the blood glucose values obtained from 0 to 16 minutes with ITT (23).

Statistical analysis

The data were subjected to one-way analysis of variance, followed by the Student–Newman–Keuls post-test, and were expressed as means±SEM. Differences were considered significant at p<0.05.

Results

The effectiveness of thyroidectomy and T₃ treatment of all groups was checked by determining the serum T₃ concentrations. This was at least one-third lower (0.13±0.01 vs. 0.34±0.07 ng/mL, p<0.001) and two–fold higher (0.73±0.10 vs. 0.34±0.07 ng/mL, p<0.001) in T₃-treated Tx rats, respectively, than those of the SO group. This was also indirectly monitored by checking the ratio between VW and BW. This was decreased (0.23±0.01 vs. 0.27±0.02, p=0.05) in Tx rats and increased (0.4±0.02 vs. 0.27±0.02, p<0.001) in T₃-chronic rats when compared with the SO group. These data confirm the induction of hypo- and hyperthyroid states in our experimental groups.

Effect of thyroid state on GLUT4 mRNA and protein expression

The data presented in the Figure 1 were obtained from experiments that aimed at characterizing a rapid-onset (30 minutes) action of T₃ (T₃ 30′ [100 μg/100 g] vs. Tx and SO groups), supposedly triggered by a nongenomic mechanism, and compare it with the chronic (5 days) actions of T₃, recognized as genomic (Tx and T₃ 5 days [5 μg/100 g] vs. SO groups) on GLUT4 mRNA and protein expression in SOL and EDL muscles. A and B represent SOL, while C and D represent the EDL muscles.

FIG. 1.

Northern blot analysis of (A) glucose transporter 4 (GLUT4) mRNA of soleus (SOL) and (C) extensorum digitalis longus (EDL) muscles of (1) sham-operated (SO) rats; (2) thyroidectomized (Tx) rats; (3) Tx rats treated with 100 μg of triiodothyronine (T₃)/100 g BW, intravenous [i.v.], and killed 30 minutes thereafter (T₃ 30′); and (4) Tx rats treated with 5 μg of T₃/100 g BW, intraperitoneal [i.p.], for a 5-day period (T₃ 5d). At the top, typical autoradiograms of GLUT4 and 18S rRNA transcripts of one experiment in a total of four experiments are shown. At the bottom, the quantitative representation of hybridization of GLUT4 and 18S rRNA transcripts ratio obtained by densitometric analysis of the exposed films, in arbitrary units (AU), is depicted. Results are expressed as mean ± standard error of the mean (SEM) of GLUT4 mRNA/18S rRNA ratios. Western blot analysis of GLUT4 protein in (B) plasma membrane (PM) and microsomal (M) fractions of SOL and (D) EDL muscles. Equal amounts of protein were subjected to the electrophoresis and immunoblotted, as described. At the top, typical autoradiograms of GLUT4 protein in PM and M samples of one experiment in a total of four experiments are shown. The graphic analysis representative of the sum of GLUT4 present in PM and M fractions (PM + M = total GLUT4) is shown at the bottom of the figure. (E) Typical autoradiograms of Na⁺/K⁺ ATPase protein in PM and M fractions of SOL of SO rats; Tx rats; Tx rats treated with 100 μg of T₃/100 g BW, i.v., and killed 30 minutes thereafter (T₃ 30′); and Tx rats treated with 5 μg of T₃/100 g BW, i.p., for a 5-day period (T₃ 5d). Results are expressed as mean ± SEM of GLUT4/g of tissue, in arbitrary units (AU). *p < 0.01 versus SO, ^# p < 0.05 versus Tx; ^@ p < 0.05 versus T₃ 30′ (n = 11 per group).

A significant reduction of GLUT4 mRNA content was observed in Tx rats in both muscles versus SO rats, and an increase after T₃ treatment (30 minutes and 5 days) versus Tx (Fig. 1A, C). However, while in the SOL muscle the GLUT4 mRNA content was restored to values in the SO group after acute T₃ treatment, and rose above them in chronic T₃-treated rats (Fig. 1A), in the EDL muscle, the GLUT4 mRNA increase did not achieve SO values, after either T₃ treatment schedules (Fig. 1C).

The data regarding total GLUT4 protein expression (M+PM) in SOL and EDL muscle of Tx rats subjected to the same T₃ treatment schedules are presented in Figure 1B and D, respectively. Thyroidectomy caused a significant reduction of total GLUT4 protein content in both SOL and EDL muscles versus the SO group. However, 30 minutes after T₃ treatment (100 μg/100 g BW, i.v.; T₃ 30′ group), the SOL and EDL muscle total GLUT4 content increased versus the Tx group, although remaining lower than values in the SO group. When Tx rats were subjected to chronic T₃ treatment (T₃ 5d group), the SOL and EDL total GLUT4 content was higher than the T₃ 30′ group values, but only the SOL muscles (Fig. 1B) achieved the values in the SO group. Figure 1E shows the immunoblotting of SOL muscle protein extracts using anti-Na⁺/K⁺ ATPase antiserum. This was performed to determine the efficiency of the subcellular fractionation method employed (PM and M). As anticipated, Na⁺/K⁺ ATPase was present mostly in the putative PM fractions of the rat SOL muscle in all groups studied (SO, Tx, T₃ 30′ and T₃ 5d), confirming the methods for preparing the PM fractions.

T₃ effect on GLUT4 mRNA poly(A) tail length

The analyses of GLUT4 (left side) and cyclophilin (right side) mRNA poly(A) tail length carried out in SOL and EDL muscles of SO, Tx, and T₃ 30′ rats are presented in Figure 2A and B, respectively. The polymerase chain reaction-amplified mRNAs appeared as a smear indicating that there were a mixture of GLUT4 mRNA with different poly(A) tail lengths. Thyroidectomy reduced the SOL and EDL GLUT4 mRNA poly(A) tail length by ∼100 adenylate residues and acute T₃ treatment promptly increased it, although only the SOL of T₃-treated Tx rats reached values in the SO group. Cyclophilin mRNA poly(A) tail length was not altered by these treatments.

FIG. 2.

GLUT4 and cyclophilin mRNA poly(A) tail length analyzed by rapid amplification of cDNA ends–poly(A) test (RACE-PAT) assay, in (A) SOL and (B) EDL muscle from SO rats, Tx rats, and Tx rats treated with 100 μg of T₃/100 g BW, i.v., and killed 30 minutes thereafter (T₃ 30′). In each panel, the smearing pattern of ethidium bromide-stained polymerase chain reaction products obtained by RACE-PAT is shown at the top, in which GLUT4 and cyclophilin mRNA poly(A) tail length are shown on the left and right, respectively. The maximal size of the amplicons, corresponding to the top of the smear of each sample (in base pairs [bp]), is shown at the bottom. Results are expressed as mean ± SEM of six rats per group. *p < 0.05 versus all groups, ^# p < 0.01 versus SO.

Time–course and dose–response studies of the T₃ effects on GLUT4 protein expression

The GLUT4 protein expression of SOL and EDL muscles excised from Tx rats subjected to (i) 100 μg of T₃/100 g BW, i.v., and killed 10, 20, and 30 minutes thereafter, or (ii) 0.3 and 1.5 μg of T₃/100 g BW, i.v., and killed 30 minutes later is shown in Figure 3, in which 3A and B represent the time–course study, and 3C and D, the dose–response study. Thyroidectomy caused a significant reduction of total GLUT4 protein content in both muscles. However, 30 minutes after T₃ treatment (100 μg/100 g BW, i.v.; T₃ 30′ group), the SOL and EDL muscle total GLUT4 content increased, as shown in Figure 3A1, and B1, even though remaining lower than the values in SO rats. The GLUT4 content also increased in EDL muscle when Tx rats were killed 20 minutes after treatment with the same dose of T₃ (T₃ 20′ group; Fig. 3B1). No changes of GLUT4 content in SOL and EDL were detected when the animals were killed 10 minutes after T₃ treatment (T₃ 10′ group) or when Tx rats were treated with physiological doses of T₃ and killed 30 minutes later (T₃ 0.3 group; Fig. 3C1, D1). However, the administration of T₃ in a dose five times higher than the physiological one led to an increase of GLUT4, which was restricted to SOL muscle (T₃ 1.5 group; Fig. 3C1). The amounts of GLUT4 measured in the PM and M fraction in SOL and EDL muscles of rats subjected to the experimental conditions just indicated are presented in the same Figure 3. This shows a significant decrease of the SOL and EDL PM GLUT4 content in the Tx group versus the SO group. Figure 3A2 and A3 shows that T₃ acutely increased PM GLUT4 and decreased M GLUT4 content in SOL muscle, in 20 minutes (T₃ 20′ group), an effect that was observed in EDL muscle only 30 minutes after T₃ treatment (T₃ 30′ group; Fig. 3B2, B3). No changes were detected in PM GLUT4 after T₃ treatment for 10 minutes. Figure 3C2 and D2 shows that whereas the amount of PM GLUT4 increases, the M GLUT4 decreases (Fig. 3C3, D3) in SOL and EDL muscle after treating Tx rats, with 1.5 μg of T₃/100 g, for 30 minutes. These alterations in PM and M GLUT4 content were also observed in Tx rats treated with physiological doses (0.3 μg/100 g BW), but only in the EDL muscle.

FIG. 3.

Western blot analysis of GLUT4 protein in (A, C) PM and M of SOL and (B, D) EDL muscles. Equal amounts of protein were subjected to electrophoresis and immunoblotted, as described. At the top, typical autoradiograms of GLUT4 protein in PM and M samples of one experiment, in a total of two experiments, are shown. The graphic of total GLUT4 is representative of the sum of GLUT4 present in PM and M fractions (PM + M = total GLUT4). Results are expressed as mean ± SEM of GLUT4/g of tissue, in AU. In (A) and (B), the blots are represented as follows: (1) SO rats (SO); (2) Tx rats (Tx); and Tx rats treated with 100 μg of T₃/100 g BW, i.v., and killed (3) 10 minutes (T₃ 10′), (4) 20 minutes (T₃ 20′), or (5) 30 minutes (T₃ 30′) thereafter. In (C) and (D), the blots are represented as follows: (1) SO rats (SO); (2) Tx rats (Tx); and Tx rats treated with (3) 0.3 μg of T₃/100 g BW, i.v. (T₃ 0.3), or (4) 1.5 μg of T₃/100 g BW, i.v. (T₃ 1.5), and killed 30 minutes thereafter. *p < 0.05 versus SO, ^# p < 0.05 versus Tx (n = 10 per group).

T₃ effect on GLUT4 translocation index

The translocations of GLUT4 measured as the percentage of total GLUT4 present in the PM in SOL and EDL muscles of rats are presented in Figure 4 (A–C). The figure shows that there was a significant decrease of GLUT4 (%) in the PM in SOL and EDL muscles in the Tx group versus the SO group. Figure 4A, which compares the acute (100 μg/100 g, BW, i.v., 30 minutes) versus the chronic (5 μg/100 g, BW, i.p., 5 days) effects of T₃, shows that the T₃ treatment increased the SOL and EDL PM GLUT4 content (%), but only the latter schedule restored the percentage of GLUT4 in PM to control (SO group) values. Figure 4B shows that acute T₃ treatment led to an increase of PM GLUT4 content in SOL muscle within 20 minutes (T₃ 20′ group), an effect that was observed in EDL muscle only 30 minutes after T₃ treatment (T₃ 30′ group). No changes were detected in PM GLUT4 content 10 minutes after T₃ treatment. Figure 4C shows that the percentage of PM GLUT4 increases in SOL and EDL muscle after treating Tx rats for 30 minutes with 1.5 μg of T₃/100 g. The administration of physiological doses of T₃ (0.3 μg/100 g BW) induced this alteration only in EDL muscle.

FIG. 4.

Graphic representation of the amount (%) of total GLUT4 protein detected in the PM (Western blotting) of SOL and EDL muscles at the moment of tissue harvesting. Results are expressed as mean ± SEM of GLUT4 content in PM (%). (A) SO rats (SO); Tx rats (Tx); Tx rats treated with 100 μg of T₃/100 g BW, i.v., and killed 30 minutes thereafter (T₃ 30′); and Tx rats treated with 5 μg of T₃/100 g BW, i.p., for a 5-day period (T₃ 5d). (B) SO rats (SO); Tx rats (Tx); and Tx rats treated with 100 μg of T₃/100 g BW, i.v., and killed 10 minutes (T₃ 10′), 20 minutes (T₃ 20′), or 30 minutes (T₃ 30′) thereafter. (C) SO rats (SO); Tx rats (Tx); and Tx rats treated with 0.3 μg of T₃/100 g BW, i.v. (T₃ 0.3), or 1.5 μg of T₃/100 g BW, i.v. (T₃ 1.5), and killed 30 minutes thereafter. *p < 0.05 versus SO and ^# p < 0.05 versus Tx (n = 10 per group).

T₃ effect on insulin sensitivity

The blood glucose decay rates (%/min) of control (SO), Tx, and rats treated with T₃ acutely or chronically, after insulin challenge (kITT), are shown in Figure 5. kITT was decreased in Tx rats. Acute and chronic T₃ treatment increased kITT; however, only the Tx rats subjected to chronic T₃ treatment achieved the SO kITT values.

FIG. 5.

Effect of T₃ treatment on insulin sensitivity. Glucose decay rate (kITT; %/min) was evaluated by means of insulin tolerance test (ITT) in SO rats (SO); Tx rats (Tx); Tx rats treated with 100 μg of T₃/100 g BW, i.v., and killed 30 minutes thereafter (T₃ 30′); and Tx rats treated with 5 μg of T₃/100 g BW, i.p., for a 5-day period (T₃ 5d). Results are expressed by mean ± SEM. *p < 0.05 versus SO, ^# p < 0.05 versus Tx (n = 10 per group).

Discussion

Our data indicate that GLUT4 mRNA and protein content, and total GLUT4 content in the PM fraction are reduced in skeletal muscle of hypothyroid rats and that these changes can be reversed by T₃. This supports the concept that thyroid hormones participate in the maintenance of glucose homeostasis.

The increased GLUT4 mRNA and protein content in the T₃-chronic group is likely consistent with previous data that show a transcriptional action of T₃ upregulating the SLC2A4 gene expression (5,27).

In studies to evaluate rapid-onset responses to T₃ administration, the increase of GLUT4 mRNA and protein content, observed as soon as 30 minutes after T₃ administration, was a novel and unexpected finding. This does not appear to be related to effects of T₃ and its receptor on the TRE present in the promoter region of the SLC2A4 gene; this TRE has been characterized as having a low affinity for TR binding (9).

It is supposed that higher-affinity TREs are under active repression by unoccupied heterodimers and/or homodimers and are highly responsive to thyroid hormone. As a consequence, they would be extremely sensitive to changes of T₃ availability. In contrast, low-affinity TRE(s) would be less sensitive to changes of T₃ availability; nevertheless, under certain conditions, they are up-regulated to some degree, and they may also predominate in genes that need to be constitutively expressed, such as the SLC2A4 gene (9). Therefore, the rapid increase of GLUT4 mRNA and protein expression 30 minutes after T₃ administration strongly indicates that, rather than an induction of the SLC2A4 gene transcription rate, the acute T₃ treatment may have increased GLUT4 mRNA stability and/or half-life, unless these effects were mediated by a high-affinity TRE that has not yet been identified in the SLC2A4 gene. However, the rapidity of the T₃ effects would not account for its interaction with high- or low-affinity TRE, which favors the hypothesis that they resulted from a nongenomic action of T₃.

The stability of an mRNA is directly related to its poly(A) tail length, with a longer poly(A) tail often being associated with enhanced translation efficiency (24,28). This is related to the fact that the mRNA poly(A) tail binds the Poly(A)-binding protein (PABP), which interacts with an α-CP protein, forming an α-complex that inhibits poly(A) deadenylation (29). It is known that the progressive addition of 15–20 adenylate residues to the poly(A) tail increases the number of PABP monomers bound, which progressively increases the translation response. PABP also associates with translation initiation factors, favoring the interaction of the poly(A) tail with the mRNA 5′-cap structure (30,31), thus playing an important role in translation initiation.

It was significant to note that in the current study the length of the poly(A) of GLUT4 mRNA in Tx rats was ∼100 adenylate residues shorter than that of SO rats and that it increased to the length of the SO rats GLUT4 mRNA length 30 minutes after treatment. This finding favors an increase of GLUT4 mRNA stability, and could explain the rapid increase of GLUT4 mRNA and protein content after acute T₃ treatment. It also reinforces the hypothesis that T₃ has a nongenomic effect in enhancing GLUT4 protein content. Changes in mRNA polyadenylation are post-transcriptional, and the changes noted should enhance intracellular synthesis of GLUT4. Our results are consistent with previous studies showing positive effects of T₃ on the poly(A) tail length of a number of other proteins (32,33,28).

Other evidence for a nongenomic effect of T₃ is that the relatively low amount of total GLUT4 in the PM fraction of Tx rats increased as soon as 20 minutes after the acute treatment with 100 μg of T₃/100 g BW in SOL muscle, and in 30 minutes in both SOL and EDL muscles. An increase of PM GLUT4 was also observed in EDL muscle 30 minutes after Tx rats had been treated with physiological doses of T₃ (0.3 μg of T₃/100 g BW) and in both muscles 30 minutes after they had been treated with doses of T₃ 1.5 μg of T₃/100 g BW.

GLUT4 translocation and insertion at the surface of insulin-responsive cells involves several steps in which many proteins are activated or inactivated by phosphorylation. The translocation step requires the activation of intracellular signaling pathways initiated by insulin interaction with its receptors at the PM, and further activation of PI3 kinase and protein kinase B (AKT/PKB) pathway. Additionally, PI3-kinase-independent signals involving c-Cbl associated protein, Cbl, and the GTPase TC10 contribute to GLUT4 translocation in adipocytes (7) and skeletal muscle cells (34). After translocation, GLUT4 needs to be inserted into the PM to allow the glucose uptake. Some studies have suggested that this protein needs to be activated, to ensure full stimulation of glucose uptake (35). In skeletal muscle, GLUT4 translocation is also triggered by muscular contraction, which determines the activation of AMP-activated protein kinase (AMPK) pathway and of pathways that involve the calcium-activated calmodulin-dependent protein kinase (CaMK) and/or protein kinase C (PKC) (36).

Considering that acute T₃ treatment caused a rapid increase of PM GLUT4, it might be inferred that one of these pathways could have been activated by T₃. Such an action remains hypothetical. It is interesting to note, however, that T₃ increases calcium and 2-deoxyglucose uptake in several tissues of the rat, including diaphragm and fat (37). Increased intracellular calcium could activate CaMK and/or PKC pathways, leading to GLUT4 mobilization to the PM (36). In addition, D'Arezzo et al. have reported that rapid nongenomic effects of T₃ on intracellular pH of L-6 myoblasts are mediated by intracellular calcium mobilization and kinase pathways (38).

Our current study also showed that SOL was more responsive to the effects of T₃ on SLC2A4 gene expression and GLUT4 mobilization to the PM than the EDL muscle. This could be secondary to the high GLUT4 expression/availability in slow oxidative muscles, in comparison to fast glycolytic ones (39). With regard to the effects of T₃ that occur after its chronic administration, this might be related to the fact that T₃ nuclear receptors are highly expressed in oxidative muscles, which might explain the higher GLUT4 mRNA response to chronic T₃ treatment in SOL than EDL muscles (40). Moeller et al. have reported that some thyroid hormone-mediated changes in gene expression can be initiated by cytosolic action of the TRβ through the PI3K pathway (41). Therefore, another consideration is whether this action, which is nongenomic, even though it involves a classic T₃ receptor, might mediate some of the effects of T₃ on GLUT4 translocation in skeletal muscle.

Even though the current study showed a novel nongenomic effect of T₃ on GLUT4 mobilization to the PM, we are aware of the fact that the amount of GLUT4 protein appears to be the major determinant of the muscle's ability to respond to insulin (39). In fact, Figure 5 shows in Tx rats a reduced amount of GLUT4 on skeletal muscle and a decreased response to insulin stimulus, findings that have been reported in animal models (42,43) and hypothyroid patients (44,45). It also shows a re-establishment of the insulin response, in rats under 5-day T₃ treatment, a procedure that increased the GLUT4 content, as described (5,27). Moreover, the acute T₃ administration also improved the Tx rat response to insulin stimulus, a condition in which total GLUT4 was increased, probably as a result of the rapid nongenomic effect of T₃ in increasing GLUT4 mRNA poly(A) tail length, which is known to be involved in mRNA stability and translation rate, as already noted (Fig. 2). However, the increased PM GLUT4 content in the Tx rat skeletal muscles detected 30 minutes after T₃ administration, in doses five times higher than the physiological one, even in the absence of insulin stimulus (Fig. 3), is a convincing evidence that intracellular signaling pathways responsible for GLUT4 mobilization to the PM were nongenomically turned on by T₃.

Even though there is a slight increase in GLUT4 at the PM fraction in response to acute T₃ administration, the impact on glucose uptake may be significant, as depicted by the kITT shown in Figure 5, because skeletal muscle represents 40% of the total body mass. However, we cannot assure that docked GLUT4-containing vesicles were actually fused to the PM. The increased blood glucose decay rate in T₃-treated rats indicates this possibility. However, we cannot rule out the hypothesis that T₃ could be activating GLUT4 molecules already present in the PM, in parallel to the effects on GLUT4 trafficking to the PM, which would improve glucose uptake, as already reported for insulin activity (35).

In conclusion, the present results confirm the transcriptional action of T₃ on the SLC2A4 gene, and reveal a rapid, T₃-mediated post-transcriptional effect on GLUT4 mRNA polyadenylation. This is likely to increase GLUT4 mRNA stability and translational efficiency. T₃ also rapidly increases GLUT4 trafficking to the PM. Thus, T₃ has effects that should rapidly improve insulin sensitivity in muscles. Studies are required to determine the intracellular pathways that mediate the rapid effects of T₃ on GLUT4 mRNA polyadenylation and translocation of GLUT4 to the PM.

Footnotes

Acknowledgments

The authors are grateful to Leonice L. Poyares for excellent technical assistance and to Dr. Luiz R. G. Britto for reviewing the article.

Disclosure Statement

No competing financial interests exist.

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T 3 Rapidly Increases SLC2A4 Gene Expression and GLUT4 Trafficking to the Plasma Membrane in Skeletal Muscle of Rat and Improves Glucose Homeostasis

Abstract

Background:

Methods:

Results:

Conclusions:

Introduction

Materials and Methods

Animals and treatments

Evaluation of SLC2A4 gene expression

Northern blot analysis

Western blot analysis

Evaluation of GLUT4 mRNA poly(A) tail length

ITT and blood glucose disappearance rate determination

Statistical analysis

Results

Effect of thyroid state on GLUT4 mRNA and protein expression

T3 effect on GLUT4 mRNA poly(A) tail length

Time–course and dose–response studies of the T3 effects on GLUT4 protein expression

T3 effect on GLUT4 translocation index

T3 effect on insulin sensitivity

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

T₃ effect on GLUT4 mRNA poly(A) tail length

Time–course and dose–response studies of the T₃ effects on GLUT4 protein expression

T₃ effect on GLUT4 translocation index

T₃ effect on insulin sensitivity