Skip to main content
NCBI home page
Endocrinology logoLink to Endocrinology
. 2015 Jul 27;156(10):3842–3852. doi: 10.1210/en.2015-1246

Thyroid Hormone Signaling in Male Mouse Skeletal Muscle Is Largely Independent of D2 in Myocytes

Joao P Werneck-de-Castro 1, Tatiana L Fonseca 1, Daniele L Ignacio 1, Gustavo W Fernandes 1, Cristina M Andrade-Feraud 1, Lattoya J Lartey 1, Marcelo B Ribeiro 1, Miriam O Ribeiro 1, Balazs Gereben 1, Antonio C Bianco 1,✉
PMCID: PMC4588812  PMID: 26214036

Abstract

The type 2 deiodinase (D2) activates the prohormone T4 to T3. D2 is expressed in skeletal muscle (SKM), and its global inactivation (GLOB-D2KO mice) reportedly leads to skeletal muscle hypothyroidism and impaired differentiation. Here floxed Dio2 mice were crossed with mice expressing Cre-recombinase under the myosin light chain 1f (cre-MLC) to disrupt D2 expression in the late developmental stages of skeletal myocytes (SKM-D2KO). This led to a loss of approximately 50% in D2 activity in neonatal and adult SKM-D2KO skeletal muscle and about 75% in isolated SKM-D2KO myocytes. To test the impact of Dio2 disruption, we measured soleus T3 content and found it to be normal. We also looked at the expression of T3-responsive genes in skeletal muscle, ie, myosin heavy chain I, α-actin, myosin light chain, tropomyosin, and serca 1 and 2, which was preserved in neonatal SKM-D2KO hindlimb muscles, at a time that coincides with a peak of D2 activity in control animals. In adult soleus the baseline level of D2 activity was about 6-fold lower, and in the SKM-D2KO soleus, the expression of only one of five T3-responsive genes was reduced. Despite this, adult SKM-D2KO animals performed indistinguishably from controls on a treadmill test, running for approximately 16 minutes and reached a speed of about 23 m/min; muscle strength was about 0.3 mN/m·g body weight in SKM-D2KO and control ankle muscles. In conclusion, there are multiple sources of D2 in the mouse SKM, and its role is limited in postnatal skeletal muscle fibers.

A flow of the active thyroid hormone T3 steadily enters virtually all vertebrate cells through membrane transporters. Once inside the cells, T3 diffuses to the nucleus and interacts with thyroid hormone receptors (TRα or TRβ) to modulate the expression of specific sets of thyroid hormone-responsive genes, thus promoting T3-dependent biological effects. Tissues contain different combinations of TRα and TRβ as well as other coregulators of the T3-TR complex. Therefore, the effects of T3 depend on these combinations and are obviously highly specific, individualized for each cell type (1).

The presence of deiodinases in T3-responsive cells adds another mechanism through which thyroid hormone action can be customized on a cell-specific fashion but in this case at a prereceptor level (2). Deiodinases are homodimeric selenoenzymes that have a single transmembrane segment connected to a cytosolic globular domain containing the active center embedded in a thioredoxin-like fold (3, 4). The type 2 deiodinase (D2) catalyzes the conversion of the prohormone T4 into T3. Thus, its presence in cells strengthens the flow of T3 molecules, reaching the nucleus with the additional T3 produced locally. In contrast, the type 3 deiodinase (D3) attenuates the flow of T3 to the nucleus because it inactivates T3 to T2 and prevents T4 use by inactivation to reverse T3 (5).

The expression of D2 and D3 can change rapidly in response to a number of developmental, metabolic, and disease cues through different signaling pathways. Because the expression of these enzymes can be turned on or off in discrete groups of cells, most of the time their actions do not affect circulating thyroid hormone levels, which are tightly controlled via the TRH/TSH/thyroid axis. For example, D2 expression in brown adipose tissue stimulates T3-responsive genes such as Ucp1 and Pgc1a, without elevating serum T3 levels (2, 6). In contrast, ectopic D3 expression in the heart and brain during ischemia or hypoxia lifts the T3-dependent transcriptional footprint in these organs, in what can be seen as an adaptive mechanism to the disease state (7–10).

Thyroid hormone affects a number of processes in the skeletal muscle. This is explained by the relatively large number of T3-responsive genes in this tissue, such as genes involved in muscle development and contractility, calcium homeostasis, and muscle metabolism (11). T4-to-T3 conversion in skeletal muscle was first observed in perfused rat hindlimb (12). Subsequently, D2 expression and activity were found in human skeletal muscle (13) and in primary cultures of human (14) and mouse skeletal muscle (15) as well as human rhabdomyosarcoma cells (16). In fact, D2 expression increases markedly in primary cultures of myocytes transitioning to myotubes (15) as well as differentiating C2C12 cells (17). These observations raised the possibility that, like brown adipose tissue (BAT), D2 in skeletal muscle fiber modulates local thyroid hormone action.

Subsequent studies revealed that, as opposed to cultured cells, D2 activity in biopsy samples of skeletal muscle from multiple species including humans is remarkably low, eg, 3 orders of magnitude lower than seen in other D2-expressing tissues (15, 18–20). Nevertheless, in a mouse model of global Dio2 inactivation (GLOB-D2KO), there is impaired muscle regeneration after cardiotoxin-induced injury attributed to a reduction in T3 signaling (17). Cultures of skeletal myocytes obtained from this mouse exhibited a phenotype of excessive proliferation and impaired differentiation (17), which is reminiscent of the phenotype exhibited by differentiating brown preadipocytes obtained from the same mouse line (GLOB-D2KO) (6). Understandably, these results were attributed to the presence of D2 in skeletal muscle fiber. However, unlike other D2-expressing tissue, the very low D2 activity in skeletal muscle requires an understanding of the contribution of other cells types within the tissue. In fact, other potential sources of low levels of D2 in skeletal muscle include fibroblasts (21, 22), endothelium (23), smooth muscle (24), and ectopic BAT mixed with muscle fibers (25, 26), the content of which is positively correlated with energy expenditure and protection against diet-induced obesity in mice (25). Thus, GLOB-D2KO mouse might be limited as a model to study the role played by D2 in skeletal muscle fiber and also because global Dio2 inactivation impacts adaptive thermogenesis and overall sympathetic activity (27, 28), which could also independently affect gene expression in muscle.

The availability of a mouse with skeletal muscle-specific (SKM-D2KO) Dio2 inactivation provides an opportunity for a better understanding of the role played by D2 in the skeletal muscle fiber (29). This animal model exhibits Dio2 disruption in mature muscle fibers, thus avoiding interference during the early phases of muscle development. Here we report that in contrast to the severe phenotype observed in GLOB-D2KO skeletal muscle, the SKM-D2KO mouse exhibits normal muscle fiber architectural structure, exercise capacity, muscle strength, and only mild signs of tissue hypothyroidism. Thus, thyroid hormone signaling in the adult skeletal muscle is largely independent of D2 in muscle fibers.

Materials and Methods

Animals

All experimental procedures were planned following the American Thyroid Association guide to investigating thyroid hormone economy and action in rodent and cell models (30) and approved by the local Institutional Animal Care and Use Committee at Miami University and Rush University Medical Center. To obtain a mouse that lacks D2 activity in all tissues (GLOB-D2KO) and WT littermates, heterozygous mice for D2KO allele were mated. Our recently established floxed D2 (Dio2Flx) mice (31) were crossed with transgenic mice expressing Cre-recombinase under myosin light chain 1f (cre-MLC) (29) to eliminate D2 activity myocytes (SKM-D2KO). Myosin light chain (MLC) is expressed during the late phase of skeletal muscle differentiation (32, 33), and the recombinase activity of the cre-MLC mouse is specific and expected in all myonuclei (33, 34). To eliminate Dio2 expression in muscle and brown adipocytes precursor cells, floxed D2 mice were also crossed with the cre-recombinase expression driven by the myogenic regulator factor-5 (MYF5) (35). MYF5 is one of the muscle regulatory factors that determines myogenesis (36). All animals used in our experiments were hemizygous for the cre transgene expression, and the genetic background was C57/B6. Newborn (12–24 h of age) or male adult mice (9–14 wk of age) were used in our studies. Animals were kept on standard chow diet (3.1 kcal/g) (2918 Teklan Global Protein rodent diet) at room temperature (22°C), with a 12-hour dark, 12-hour light cycle starting at 6:00 am and housed in standard plastic cages with four to five mice per cage. Cre-MLC littermates were used as controls.

Hypothyroidism and T4 replacement

Hypothyroidism was induced by an iodine-deficient diet with 0.15% propylthiouracil (PTU; TD 95125; Harlan Teklad) and 0.05% of methimazole (MMI) in drinking water for 3 or 6 weeks. In the T4 replacement study, mice received a single injection (5 or 10 μg per 100 g of body weight, sc) of T4 and were killed 5 hours later.

Maximum exercise capacity test

Mice were acclimatized to the treadmill (Columbus Instruments) during 5–6 consecutive days by running 5–10 minutes per day at 5–10 m/min. The test started at 10 m/min, and the speed increased 2 m/min every 2 minutes until exhaustion. Maximal speeds were recorded when animals ran at least 75% of the stage (37). All the tests were performed by personnel blind to the genotype.

Physiological evaluation of muscle strength

Under isoflurane anesthesia the right leg was shaved and the electrode positioned through a sc incision made in the posterior area of the leg; this allowed for the stimulation of plantar flexors, eg, soleus and gastrocnemius muscles. Animals were placed in supine position with the knee joint immobilized while flexed at 90 degrees while the ankle was flexed at 90 degrees. The foot and part of the ankle were attached to a platform coupled to a force transducer. The electric current applied to the muscle was progressively increased until the force developed plateaued at maximum level, which was then used to create tetanus at 100 Hz for 2 seconds. Three tetanic stimulations were performed at 90-second intervals using the DMC software (Aurora Scientific; version 5.420) and the data analyzed with DMA software (Aurora Scientific; version 5.220). On average, the overall muscle function was calculated with data obtained from three tetanic torques (muscle torque was normalized by body weight).

Deiodinase assays

Skeletal muscle (SKM) samples were sonicated in phosphate-EDTA buffer containing 10 mM dithiothreitol (DTT), 0.25 M sucrose, and protease inhibitor cocktail (Roche). Protein was measured by the Bio-Rad protein assay solution (Bio-Rad Laboratories), and 200 μg was incubated for 3 hours at 37°C in the presence of 20 mM DTT, 1 mM PTU, 10 nM T3, 0.1 nM T4, and 200K cpm 125I-T4 (PerkinElmer Life and Analytical Sciences, Inc; number NEX111H500UC). Assays were stopped with the addition of horse serum and 50% trichloroacetic acid and free 125I counted on the 2470 automatic γ-counter Wizard2 (PerkinElmer) as described previously (38). Neonatal measurements were performed in all hindlimb skeletal muscles combined. In the adult male mice, soleus, tibialis anterior, and gastrocnemius muscles were used. D2 activity was also measured in BAT (150 μg of protein, 3 h, 0.5 nM T4), brain cortex (100 μg, 3 h, 0.1 nM T4), and pituitary (20 μg, 1 h, 0.1 nM T4) in the presence of 1 mM PTU, 20 mM DTT, and 150 K of 125I-T4. Blank measurements (spontaneous deiodination) for each sample were performed through enzyme saturation with 100 nM of T4, and all D2 activity results are expressed in femtomoles of T4 per milligram of protein per minute. Using tissues from GLOB-D2KO mice as background resulted in a similar estimation of spontaneous deiodination (data not shown). To determine the T4 fate and validate our D2 assay, SKM and BAT reactions were stopped by the addition of methanol and the products of deiodination were resolved by UPLC (ACQUITY; Waters Corp) after incubation with the same conditions described above. Fractions were automatically processed through a Flow Scintillation Analyzer Radiomatic 610TR (PerkinElmer) for radiometry as described. SKM crude microsomal protein was isolated as previously described (19).

Muscle histology and cross-sectional area determination

Skeletal muscle histology and cross-sectional fiber area were assessed in soleus muscle after 10-μm sections were stained with hematoxylin and eosin using the NIS Elements imaging software (Nikon). At least 100 muscle fibers were measured.

Tissue T3 content

Mice were anesthetized with ketamine and xilazine and perfused with PBS containing heparin through a needle placed in the left ventricle. Tissues were snap frozen in liquid nitrogen and stored at −80°C. T3 was extracted from soleus muscle using a method described previously (39), and T3 content was measured by a RIA as previously detailed (40). Recovery was monitored by the addition of 125I-T3 before tissue extraction.

Culture of murine primary skeletal muscle cells

Primary skeletal muscle cells of Cre-MLC and SKM-D2KO mice (3 wk of age) were obtained as previously described (41). Briefly, hindlimb muscles were digested in collagenase 0.2%, followed by dispase 2.4 U/mL−1 and tripsin (0.1%) digestion. After passing thought a 70-mm cell strainer, cells were plated in collagen-coated flasks in DMEM supplemented with 20% fetal bovine serum and expanded twice as they reached 70% confluence. Cells were treated with 1 μM forskolin (FSK; Sigma-Aldrich) and 1 μM MG132 (Calbiochem) or an appropriate volume of vehicle dimethylsulfoxide for 6 hours. The former stimulates Dio2 gene transcription and the latter prevents D2 protein degradation by inhibiting proteasome pathway. Cells were harvested, sonicated, and 100 μg of total sonicate protein processed for measurement of D2 activity for 3 hours. To evaluate myotube formation, cells were differentiated for 3 days in DMEM containing 2% horse serum. Fibroblasts were isolated after trypsinized cultured cells were plated and myocytes removed with the supernatant 30 minutes later (34). DNA recombination in primary myocytes was performed using the AccuStart II GelTrack PCR SuperMix (Quanta Biosciences) as recommended by the manufacturer. Primer sequence is provided in Supplemental Table 1.

Gene expression analysis

Total RNA was extracted using RNeasy kits (QIAGEN), according to the manufacturer's instructions. The extracted RNA was quantified with a NanoDrop spectrophotometer, and 0.5–1.0 μg total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Genes of interest were measured by RT-PCR (StepOnePlus real time PCR system; Applied Bioscience) using SYBR Green Supermix (Quanta Biosciences). Standard curves were performed for all gene expression analysis and consisted of four to five points of serially diluted cDNA. The coefficient of correlation was greater than 0.98 for all curves, and the amplication efficiency ranged between 80% and 110%. Amplicon specificity was assessed by the melting curve. Cyclophilin A or RNA polymerase II was used as internal control gene, and no significant changes in gene expression of either gene were observed between groups. All pairs of primers used in the present studies are provided in Supplemental Table 1.

Statistical analysis

All data are expressed as mean ± SEM. A Student's t test was used to compare two groups. Significance was set as P < .05 to reject the null hypothesis. Bonferonni's correction for multiple comparisons in skeletal muscle gene expression studies (see Figure 6, B and C) was applied. In this case, significance was set as P < .002 to reject the null hypothesis.

Figure 6.

Figure 6.

Open in a new tab

Skeletal muscle T3 content and mRNA levels in neonatal and adult SKM-D2KO mice. A, T3 content in soleus muscle of SKM-D2KO and control mice. T3 was determined by RIA after tissue extraction of iodothyronines. B, Relative skeletal muscle-related genes mRNA levels in neonatal hindlimb muscles measured by quantitative RT-PCR and using cyclo-A mRNA levels as internal control. C, This was the same as in panel B except that adult soleus muscle was used. Sample size is shown in parentheses for each group. Dashed bars represent systemic hypothyroid animals. Significance was set at P < .002 after Bonferroni's correction for multiple comparisons (two comparisons for each 11 genes). Hypothyroidism was induced by feeding on a low iodine diet containing 0.15% PTU and on water containing 0.05% MMI for 6 weeks. Values are the mean ± SEM of the indicated number of animals. ***, P < .002 vs controls.

Results

D2 activity in skeletal muscle homogenates decreases with age and is regulated by thyroid hormone

Measuring D2 in adult skeletal muscle is challenging because the catalytic levels are markedly lower as compared with other tissues. Thus, our first studies aimed at establishing a reliable method for measuring D2 activity in skeletal muscle homogenates using GLOB-D2KO as a negative control. D2 activity in skeletal muscle decreases with age (Figure 1A). Whereas after birth the activity is 0.02 ± 0.008 fmol/mg · min T4 in hindlimb muscles, during adulthood the activity in soleus drops to 0.002 ± 0.001 fmol/mg · min T4 in the soleus muscle (Figure 1A) of wild-type (WT) mice. As expected, GLOB-D2KO mice exhibit undetectable levels of D2 activity in neonatal calf muscle and adult soleus (Figure 1A). The reliability of the D2 assay was verified by UPLC with production of equimolar amounts of 125I and 125I-T3 (Supplemental Figure 1, A–D), comparable with BAT control samples (Supplemental Figure 1, E–H). Soleus D2 activity in the adult WT animal is approximately 2 orders of magnitude below those levels observed in BAT (Figure 1B).

Figure 1.

Figure 1.

Open in a new tab

Skeletal muscle and brown adipose tissue D2 activity in GLOB-D2KO and WT littermate mice. A, D2 activity in neonatal (hindlimb muscles; n = 3–6) and adult skeletal muscle (soleus; n = 3–5) of GLOB-D2KO and littermate WT mice. B, D2 activity in BAT (n = 3–4) of GLOB-D2KO and littermate control mice. C, D2 activity in slow-twitch red soleus (SOL), tibialis anterior (TA), and fast-twitch white gastrocnemius (WG) (n = 3–4) of WT mice. D, D2 activity in microsomes isolated and assayed as in (18) from BAT, SOL, and WG of WT mice (n = 3). Values are the mean ± SEM of the indicated number of animals. *, P < .05; **, P < .01; ***, P < .001 vs controls.

D2 activity is higher in red muscles such as soleus (19). Indeed, red oxidative slow-twitch soleus muscle displays greater D2 activity compared with tibialis anterior muscle (mixed with red and white muscle fibers) (Figure 1C). We failed to measure D2 activity in white fast-twitch gastrocnemius (WG) muscle using tissue homogenates (Figure 1C). However, when crude microsomes were used (19, 42), D2 catalytic activity was measurable (Figure 1D), which is not surprising, given that D2 is an estrogen receptor-resident protein (43).

Next, we tested whether D2 activity in skeletal muscle would be accelerated by systemic hypothyroidism, such as reported for other tissues. Accordingly, D2 activity in skeletal muscle increased approximately 6.6-fold in hypothyroid WT animals and was reduced 5 hours after a single injection of high doses of T4 (Figure 2A). A similar induction in D2 activity was observed in the BAT of the same animals (Figure 2B). Notably, a longer period of hypothyroidism (6 wk) did not increase D2 activity further in neither soleus muscle nor BAT (Supplemental Figure 2, A and B).

Figure 2.

Figure 2.

Open in a new tab

Influence of systemic T4 levels in D2 activity in soleus muscle and BAT. A, D2 activity in euthyroid, hypothyroid (Hypo), and hypothyroid treated with T4 (Hypo + T4) adult soleus muscle of GLOB-D2KO and WT littermates. B, This is the same as in panel A except that BAT samples were used. Hypothyroidism was induced by feeding on a low-iodine diet containing 0.15% PTU and on water containing 0.05% MMI for 3 weeks. T4 treatment consisted in one single injection (5 or 10 μg per 100 g of body weight, sc) 5 hours before the animals were killed. Values are the mean ± SEM of the indicated number of animals in parenthesis. **, P < .01, ***, P < .001 vs WT; ###, P < .001 vs euthyroid WT mice (fold induction is indicated in parentheses).

Dio2 inactivation in skeletal myocytes (SKM-D2KO mouse) reveals additional sources of D2 in skeletal muscle

The SKM-D2KO mouse is systemically euthyroid (Figure 3A), grows normally (29), and exhibits normal D2 activities in nontargeted tissues (Figure 3, B–D). Cerebral cortex D3 and liver D1 activities are normal in these animals (Figure 3, E and F). Inactivation of Dio2 in myocytes decreased D2 mRNA and activity by 40%–50% in neonatal hindlimb and adult soleus muscle (Figure 3, G and H). In contrast, neither cre expression nor flox insertion alone affected skeletal muscle D2 activity (Figure 4A). In systemically hypothyroid mice, D2 activity increased markedly in both control and SKM-D2KO animals, albeit D2 activity was approximately 50% in SKM-D2KO mice as compared with control littermates (Figure 4B). To further explore why only 50% of the D2 activity was reduced in the SKM-D2KO skeletal muscle, we isolated and cultured cells from hindlimb muscles to obtain myocyte-enriched or fibroblast-enriched populations. Notably, the drop in D2 mRNA levels observed in primary cultures of SKM-D2KO skeletal myocytes reached approximately 70%–80% when compared with control cells (Figure 5A). This difference remained, even after cells were treated with FSK (Figure 5B) or with FSK and MG132 (Figure 5B). Notably, muscle fibroblasts obtained from SKM-D2KO mice express as much D2 mRNA as those from cre-MLC animals (Figure 5A). To ensure that cre-mediated DNA recombination occurred in SKM-D2KO myocytes, we used PCR to amplify a segment of the Dio2 gene that includes the 5′ p-flox sequence. In the absence of recombination an approximately 2-kbp PCR product is observed (Figure 5C), whereas after recombination, the PCR product measures approximately 500 bp (Figure 5D). As expected, there was no recombination in the myocytes expressing only cre recombinase, but in SKM-D2KO myocytes the recombination reached almost 100% (Figure 5E).

Figure 3.

Figure 3.

Open in a new tab

Dio2 expression and D2 activity in skeletal muscle of SKM-D2KO. A, Serum TSH, T3, and T4 of SKM-D2KO (n = 5–7) and control mice (n = 5). B, D2 activity in BAT of SKM-D2KO (n = 5) and control mice (n = 11). C, This is the same as in panel B except that brain cortex samples were used. D, This is the same as in panel B except that pituitary samples were used. E, D3 activity in brain cortex of SKM-D2KO (n = 5) and control (n = 6) mice. F, D1 activity in liver of the same animals are as in panel A. G, relative Dio2 mRNA levels in neonatal (hindlimb; n = 3–10) and adult skeletal muscle (soleus; n = 4–5) as measured by quantitative RT-PCR and using cyclo-A or RNA polymerase II mRNA levels as internal control and H, D2 activity in neonatal and adult soleus of SKM-D2KO and control littermates. SKM-D2KO indicates Dio2 inactivation in skeletal muscle. Values are normalized by respective littermate controls. Values are the mean ± SEM of the indicated number of animals. *, P < .05, **, P < .01, ***, P < .001 vs control.

Figure 4.

Figure 4.

Open in a new tab

D2 activity in skeletal muscle of WT, floxed D2 (Flox), and Cre-myosin light chain (Cre) expressing mice of SKM-D2KO strain and effects of hypothyroidism. A, D2 activity in neonatal (hindlimb) and adult (soleus) skeletal muscle of WT, Cre, and Flox mice (four to seven mice). B, D2 activity in euthyroid (n = 4) adult soleus of SKM-D2KO and control and hypothyroid mice (n = 4–6); hypothyroidism was induced by feeding on a low-iodine diet containing 0.15% PTU and on water containing 0.05% MMI for 6 weeks. Values are the mean ± SEM of the indicated number of animals. *, P < .05, ***, P < .001 vs respective control.

Figure 5.

Figure 5.

Open in a new tab

Primary myocytes of SKM-D2KO mice. A, Dio2 mRNA levels in primary cultured myocytes or muscle fibroblasts (n = 3) of SKM-D2KO (black bars) and cre-MLC (white bars) hindlimb. Fibroblasts were obtained by plating muscle cultured cells for 30 minutes. B, Dio2 mRNA levels of primary myocytes treated with FSK (10 μM) or D2 activity after 6 hours of treatment with FSK and MG132 (a proteasome inhibitor, 1 μM). Numbers on top of black bars denote expression or activity relative to cre-MLC control. All mRNA results are normalized by myocytes of control mice in panel A. C, Illustration of the floxed Dio2 gene showing the loxP sites flanking the selenocysteine (Se) insertion site. Sense and antisense primers locations to determine DNA recombination are shown. Black bar denotes the PCR product size (∼2000 bp). CDS, coding sequence. D, Floxed Dio2 gene after cre-mediated recombination. Note the small PCR product (∼500b p) produced after DNA recombination. E, Evaluation of DNA recombination by PCR in primary cultured myocytes of Cre-MLC (lanes 2 and 4) and SKM-D2KO (lanes 3 and 5). In lane 5 the reaction was performed with no DNA. DNA ladder was added lane 1 (100–15 000 kb). Cre-MLC (F) and SKM-D2KO (G) myoblasts were differentiated for 3 days in 2% horse serum. Note the elongated myotubes in both groups. Images were taken at ×10 magnification. Values are the mean ± SEM of the indicated number of animals. ***, P < .001.

To further test whether there is SKM Dio2 expression outside myocytes, we crossed the floxed-D2 mouse with the Cre-MYF5 animal. Myf5 is a key transcription factor that affects both myogenesis (36) and brown adipogenesis (35). Notably, Dio2 expression in BAT fell to undetected levels (∼2%) in both neonatal and adult MYF5-D2KO mice, indicating that Myf5-driven Cre recombinase is highly effective in the flox-D2 mouse (Supplemental Figure 3A). Nonetheless, substantial amounts (∼30%–40%) of Dio2 mRNA were still detected in MYF5-D2KO skeletal muscle (Supplemental Figure 3B).

Obviously some of the observations in D2 expression could be due to differences in the skeletal muscle differentiation as reported for the GLOB-D2KO mouse (17). To test this possibility, we differentiated both cre-MLC and SKM-D2KO skeletal muscle cells for 3 days in DMEM supplemented with 2% horse serum and cells fused into myotubes at similar rates (Figure 5, F and G); contracting myotubes were present in both cell types (Supplemental Figure 4, A and B).

Dio2 inactivation in skeletal myocytes preserves T3 content and only mildly disrupts thyroid hormone signaling

The SKM-D2KO constitutes an ideal model to define the role played by D2 in thyroid hormone signaling, given that cre-MLC is expressed later in myocyte development (32). This was first approached by measuring soleus T3 levels, a reliable index for thyroid hormone signaling (30). Notably, no differences in T3 concentration were observed between cre-MLC and SKM-D2KO soleus (Figure 6A). To gain further insight into thyroid hormone signaling, we measured the expression of a number of skeletal muscle genes known to be responsive to T3 (11, 44), similarly to what was done in the GLOB-D2KO mouse (17). Remarkably, no differences in the expression of these T3-responsive genes, ie, MyoD, Mhc1, Mhc2, Mhc7, and Serca1–2 were found in SKM-D2KO neonatal muscles (Figure 6B). Among the T3-sensitive genes, only Serca1 expression was affected in adult SKM-D2KO soleus (Figure 6C). In addition, Myf5 expression decreased in adult SKM-KO. As a reference it is notable that systemic hypothyroidism resulted in a marked decrease in the expression of T3-responsive genes in skeletal muscle (Figure 6C).

SKM-D2KO mice exhibit normal exercise capacity and muscle strength

In an attempt to extend our analysis of the SKM-D2KO skeletal muscle, SKM-D2KO animals were further tested by measuring exercise capacity during a treadmill test. The test starts at a speed of 10 m/min and is progressively increased by 2 m/min every 2 minutes up until exhaustion. SKM-D2KO animals ran for approximately 16 minutes and reached a speed of about 23 m/min, indistinguishable from littermate control animals (Figure 7A). Because exercise involves several factors such as substrate mobilization, cardiovascular adjustments, and oxygen delivery, we next evaluated muscle strength using an in situ set-up. Again, no differences were observed in the maximal developed force between SKM-D2KO and control littermates (Figure 7B). We also did not observe differences in the cross-sectional muscle fiber area (Figure 7, C–E) when comparing SKM-D2KO and littermates controls.

Figure 7.

Figure 7.

Open in a new tab

Treadmill maximal exercise capacity, muscle strength, and cross-sectional area of SKM-D2KO mice. A, Maximal exercise time and speed achieved during the treadmill test. The test starts at a speed of 10 m/min and progressively increased by 2 m/min every 2 minutes up to exhaustion (n = 7–9). B, Muscle tetanic force normalized by body weight. C, Soleus mean cross-sectional area assessed in 10-μm sections were stained with hematoxylin and eosin in SKM-D2KO mouse (n = 3). Representative soleus cross-section of control littermate (D) and SKM-D2KO mice (E) are shown. Bar scale, 50 μm. Values are the mean ± SEM of the indicated number of animals. ***, P < .001.

Discussion

The present studies reveal that disruption of the D2 pathway in skeletal muscle fibers using two cre-expressing animal models (early or late during myocyte development) only decreases D2 mRNA and activity by approximately 40%–50% (Figure 3, G and H, and Supplemental 3). The decrease in D2 expression was substantially greater in cell cultures enriched with SKM-D2KO myocytes (70%–80%), suggesting that other sources of D2 exist in the skeletal myocyte, eg, skeletal muscle fibroblasts (Figure 5, A and B). Whereas it is anticipated that multiple cell types in any tissue express D2, this is particularly relevant in the skeletal muscle because of the very low level of expression exhibited by the muscle fibers. This is in contrast to the brain, BAT, or pituitary gland, in which the other sources of D2 are insignificant in the face of D2 levels that are several orders of magnitude higher. At the same time, disruption of D2 in skeletal muscle fibers did not decrease T3 content (Figure 6A) in soleus muscle neither affected the expression of typical T3-responsive genes in neonatal hindlimb muscle (Figure 6B) and only reduced Serca1 expression by 25% in the adult soleus (Figure 6C). These data support a limited D2 role in skeletal muscle. This is in contrast to the finding of substantial changes in the expression of T3-responsive genes in systemically hypothyroid mice (Figure 6C). The decrease in the T3 signal was mild, and it impaired neither the exercise capacity as assessed through a treadmill test (Figure 7A) nor the in situ muscle strength test (Figure 7B); the muscle cross-sectional area was also preserved in SKM-D2KO (Figure 7, C and F).

An important aspect of studies involving D2 expression in skeletal muscle is the ability to accurately measure catalytic enzyme activity (5). For example, reported D2 activity in mouse skeletal muscle homogenates varies from undetectable (15) to 0.02 fmol/mg·min T4 high levels (17). The present studies used a sensitive in vitro assay for D2 activity that measured equimolar amounts of T3 and iodide, which ensures that iodide release faithfully represents T3 production (Figure 3). Using this assay we observed that neonatal skeletal muscle D2 activity is much higher than in adult animals (Figure 1A) and that soleus D2 activity (slow-twitch muscle) is about double of that found in tibialis anterioris (fast-twitch muscle) (Figure 1C). Moreover, soleus D2 activity was found to be about 5-fold elevated by hypothyroidism and suppressed by T4 administration (Figure 2A), which are the homeostatic hallmarks of D2 expression in other tissues (Figure 2B). We have also verified that D2-specific activity in skeletal muscle can be increased orders of magnitude by assaying isolated microssomes, again with greater activity present in slow-twitch vs fast-twitch muscle (Figure 1D).

The reported data obtained in GLOB-D2KO mice (17) suggest that D2 plays a major role in regulating thyroid hormone signaling in this tissue. However, by specifically knocking out D2 in the skeletal muscle fibers, we documented that D2's role is mostly likely during development (17), only playing a minor role in adults as assessed by four typical T3-responsive genes (Figure 6B). Obviously it is conceivable that our strategy did not eliminate all D2 expression in the skeletal muscle fiber and thus its limited impact on gene expression and function. This, however, is unlikely, given that the myosin light chain 1f (cre-MLC) promoter has been widely and successfully used to specifically knockout genes in skeletal muscle (33, 34, 45, 46). Histological studies indicate that approximately45% of the nuclei within skeletal muscle tissue are contained within muscle fibers and the remaining are associated with blood vessels, Schwann cells, satellite cells, adipose cells, and fibroblasts (47). Thus, it is expected that MLC cre-driven inactivation of floxed genes takes place in myonuclei of skeletal muscles but not in other cell types within the skeletal muscle (33, 34, 45, 46). Indeed, the decrease in Dio2 mRNA levels and activity was greater in cultures enriched with myocytes (Figure 5A). Using a PCR-based strategy, we demonstrated that cre-mediated Dio2 recombination occurs in virtually 100% of the SKM-D2KO myocytes (Figure 5D). Further support of these studies was obtained from the analysis of the MYF5-D2KO mouse (Supplemental Figure 3, A and B).

MLC is expressed during the late phase of skeletal muscle differentiation (32), avoiding the possible interfering disruption of T3 signaling during skeletal muscle development (17). In this respect, we confirmed herein that cells isolated from SKM-D2KO mice are able to form myotubes (Figure 5, F and G), which is in contrast to the absence of myotubes in differentiating GLOB-D2KO myocytes (17).

The fact that only approximately half of muscle D2 originates from skeletal myocytes points to a different cell type(s) within skeletal muscle expressing D2. Fibroblasts, endothelial cells, smooth muscle cells, white and brown adipocytes (BAT) constitute potential sources of D2 within the skeletal muscle. For example, here we found that skeletal muscle fibroblasts express D2 that is not affected in the SKM-D2KO mouse (Figure 5B) (41). In addition, BAT can be found intermingled with muscle fibers (25), opening the possibility that D2-expressing BAT cells could contribute to muscle D2 expression. Future studies targeting D2 in white adipose tissue and BAT should reveal their specific contributions to skeletal muscle D2.

In conclusion, D2 expression in skeletal muscle contributes only mildly to thyroid hormone signaling in skeletal muscle fiber, as opposed to tissues such as brain and BAT.

Acknowledgments

We are grateful to Dr Carlos Moraes (University of Miami) for kindly providing the Cre-MLC mouse used in this study and Dr Maria de Jesus Obregon (Spain) for providing the T3 antibody and the invaluable expertise to measure the T3 content in the skeletal muscle.

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 65055 (to A.C.B.), the European Community's Seventh Framework Program FP7/2007–2013, Grant 259772 (to B.G.), Brazilian National Research Council Grant CNPq-202189/2011-2 (to J.P.W.d.C.), and American Thyroid Association Grant M1301627 (to J.P.W.d.C.).

The funders had no role in the study design, the data collection and analysis, the decision to publish, or the preparation of the manuscript.

Disclosure Summary: The authors have nothing to disclose.

Footnotes

Abbreviations:
BAT
brown adipose tissue
D2
type 2 deiodinase
D3
type 3 deiodinase
DTT
dithiothreitol
FSK
forskolin
MLC
myosin light chain
MMI
methimazole
MYF5
myogenic regulator factor-5
PTU
propylthiouracil
SKM
skeletal muscle
TR
thyroid hormone receptor
WT
wild type.

References

  • 1. Brent GA. Mechanisms of thyroid hormone action. J Clin Invest. 2012;122:3035–3043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2. Gereben B, Zavacki AM, Ribich S, et al. Cellular and molecular basis of deiodinase-regulated thyroid hormone signaling. Endocr Rev. 2008;29:898–938. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3. Callebaut I, Curcio-Morelli C, Mornon JP, et al. The iodothyronine selenodeiodinases are thioredoxin-fold family proteins containing a glycoside hydrolase clan GH-A-like structure. J Biol Chem. 2003;278:36887–36896. [DOI] [PubMed] [Google Scholar]
  • 4. Sagar GD, Gereben B, Callebaut I, et al. The thyroid hormone-inactivating deiodinase functions as a homodimer. Mol Endocrinol. 2008;22:1382–1393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5. Arrojo EDR, Fonseca TL, Werneck-de-Castro JP, Bianco AC. Role of the type 2 iodothyronine deiodinase (D2) in the control of thyroid hormone signaling. Biochim Biophys Acta. 2013;1830:3956–3964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6. Hall JA, Ribich S, Christoffolete MA, et al. Absence of thyroid hormone activation during development underlies a permanent defect in adaptive thermogenesis. Endocrinology. 2010;151:4573–4582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7. Jo S, Kallo I, Bardoczi Z, et al. Neuronal hypoxia induces hsp40-mediated nuclear import of type 3 deiodinase as an adaptive mechanism to reduce cellular metabolism. J Neurosci. 2012;32:8491–8500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8. Freitas BC, Gereben B, Castillo M, et al. Paracrine signaling by glial cell-derived triiodothyronine activates neuronal gene expression in the rodent brain and human cells. J Clin Invest. 2010;120:2206–2217. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9. Simonides WS, Mulcahey MA, Redout EM, et al. Hypoxia-inducible factor induces local thyroid hormone inactivation during hypoxic-ischemic disease in rats. J Clin Invest. 2008;118:975–983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 10. Olivares EL, Marassi MP, Fortunato RS, et al. Thyroid function disturbance and type 3 iodothyronine deiodinase induction after myocardial infarction in rats a time course study. Endocrinology. 2007;148:4786–4792. [DOI] [PubMed] [Google Scholar]
  • 11. Simonides WS, van Hardeveld C. Thyroid hormone as a determinant of metabolic and contractile phenotype of skeletal muscle. Thyroid. 2008;18:205–216. [DOI] [PubMed] [Google Scholar]
  • 12. van Hardeveld C, Kassenaar AA. Thyroid hormone uptake and T4 derived T3 formation in different skeletal muscle types of normal and hyperthyroid rats. Acta Endocrinol (Copenhagen). 1978;88:306–320. [DOI] [PubMed] [Google Scholar]
  • 13. Salvatore D, Bartha T, Harney JW, Larsen PR. Molecular biological and biochemical characterization of the human type 2 selenodeiodinase. Endocrinology. 1996;137:3308–3315. [DOI] [PubMed] [Google Scholar]
  • 14. Hosoi Y, Murakami M, Mizuma H, Ogiwara T, Imamura M, Mori M. Expression and regulation of type II iodothyronine deiodinase in cultured human skeletal muscle cells. J Clin Endocrinol Metab. 1999;84:3293–3300. [DOI] [PubMed] [Google Scholar]
  • 15. Grozovsky R, Ribich S, Rosene ML, et al. Type 2 deiodinase expression is induced by peroxisomal proliferator-activated receptor-γ agonists in skeletal myocytes. Endocrinology. 2009;150:1976–1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16. da-Silva WS, Harney JW, Kim BW, et al. The small polyphenolic molecule kaempferol increases cellular energy expenditure and thyroid hormone activation. Diabetes. 2007;56:767–776. [DOI] [PubMed] [Google Scholar]
  • 17. Dentice M, Marsili A, Ambrosio R, et al. The FoxO3/type 2 deiodinase pathway is required for normal mouse myogenesis and muscle regeneration. J Clin Invest. 2011;120:4021–4030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18. Mebis L, Langouche L, Visser TJ, Van den Berghe G. The type II iodothyronine deiodinase is up-regulated in skeletal muscle during prolonged critical illness. J Clin Endocrinol Metab. 2007;92:3330–3333. [DOI] [PubMed] [Google Scholar]
  • 19. Marsili A, Ramadan W, Harney JW, et al. Type 2 iodothyronine deiodinase levels are higher in slow-twitch than fast-twitch mouse skeletal muscle and are increased in hypothyroidism. Endocrinology. 2010;151:5952–5960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20. Heemstra KA, Soeters MR, Fliers E, et al. Type 2 iodothyronine deiodinase in skeletal muscle: effects of hypothyroidism and fasting. J Clin Endocrinol Metab. 2009;94:2144–2150. [DOI] [PubMed] [Google Scholar]
  • 21. Pruvost V, Valentin S, Cheynel I, Vigouroux E, Bezine MF. 5′-Deiodinase activity in cultured glial and fibroblastic cells from the cerebella of newborn rats. Horm Metab Res. 1999;31:591–596. [DOI] [PubMed] [Google Scholar]
  • 22. Dumitrescu AM, Liao XH, Abdullah MS, et al. Mutations in SECISBP2 result in abnormal thyroid hormone metabolism. Nat Genet. 2005;37:1247–1252. [DOI] [PubMed] [Google Scholar]
  • 23. Sabatino L, Lubrano V, Balzan S, Kusmic C, Del Turco S, Iervasi G. Thyroid hormone deiodinases D1, D2, and D3 are expressed in human endothelial dermal microvascular line: effects of thyroid hormones. Mol Cell Biochem. 2015;399:87–94. [DOI] [PubMed] [Google Scholar]
  • 24. Mizuma H, Murakami M, Mori M. Thyroid hormone activation in human vascular smooth muscle cells: expression of type II iodothyronine deiodinase. Circ Res. 2001;88:313–318. [DOI] [PubMed] [Google Scholar]
  • 25. Almind K, Manieri M, Sivitz WI, Cinti S, Kahn CR. Ectopic brown adipose tissue in muscle provides a mechanism for differences in risk of metabolic syndrome in mice. Proc Natl Acad Sci USA. 2007;104:2366–2371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26. Crisan M, Casteilla L, Lehr L, et al. A reservoir of brown adipocyte progenitors in human skeletal muscle. Stem Cells. 2008;26:2425–2433. [DOI] [PubMed] [Google Scholar]
  • 27. Castillo M, Hall JA, Correa-Medina M, et al. Disruption of thyroid hormone activation in type 2 deiodinase knockout mice causes obesity with glucose intolerance and liver steatosis only at thermoneutrality. Diabetes. 2011;60:1082–1089. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28. Christoffolete MA, Linardi CC, de Jesus L, et al. Mice with targeted disruption of the Dio2 gene have cold-induced overexpression of the uncoupling protein 1 gene but fail to increase brown adipose tissue lipogenesis and adaptive thermogenesis. Diabetes. 2004;53:577–584. [DOI] [PubMed] [Google Scholar]
  • 29. Fonseca TL, Werneck-De-Castro JP, Castillo M, et al. Tissue-specific inactivation of type 2 deiodinase reveals multilevel control of fatty acid oxidation by thyroid hormone in the mouse. Diabetes. 2014;63:1594–1604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30. Bianco AC, Anderson G, Forrest D, et al. American Thyroid Association guide to investigating thyroid hormone economy and action in rodent and cell models. Thyroid. 2014;24:88–168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. Fonseca TL, Correa-Medina M, Campos MP, et al. Coordination of hypothalamic and pituitary T3 production regulates TSH expression. J Clin Invest. 2013;123:1492–1500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. Schiaffino S, Reggiani C. Fiber types in mammalian skeletal muscles. Physiol Rev. 2011;91:1447–1531. [DOI] [PubMed] [Google Scholar]
  • 33. Bothe GW, Haspel JA, Smith CL, Wiener HH, Burden SJ. Selective expression of Cre recombinase in skeletal muscle fibers. Genesis. 2000;26:165–166. [PubMed] [Google Scholar]
  • 34. Lee SJ, Huynh TV, Lee YS, et al. Role of satellite cells versus myofibers in muscle hypertrophy induced by inhibition of the myostatin/activin signaling pathway. Proc Natl Acad Sci USA. 2012;109:E2353–E2360. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35. Seale P, Bjork B, Yang W, et al. PRDM16 controls a brown fat/skeletal muscle switch. Nature. 2008;454:961–967. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36. Comai G, Sambasivan R, Gopalakrishnan S, Tajbakhsh S. Variations in the efficiency of lineage marking and ablation confound distinctions between myogenic cell populations. Dev Cell. 2014;31:654–667. [DOI] [PubMed] [Google Scholar]
  • 37. Neto RA, de Souza Dos Santos MC, Rangel IF, et al. Decreased serum T3 after an exercise session is independent of glucocorticoid peak. Horm Metab Res. 2013;45(12):893–899. [DOI] [PubMed] [Google Scholar]
  • 38. Christoffolete MA, Ribeiro R, Singru P, et al. Atypical expression of type 2 iodothyronine deiodinase in thyrotrophs explains the thyroxine-mediated pituitary thyrotropin feedback mechanism. Endocrinology. 2006;147:1735–1743. [DOI] [PubMed] [Google Scholar]
  • 39. Morreale de Escobar G, Pastor R, Obregon MJ, Escobar del Rey F. Effects of maternal hypothyroidism on the weight and thyroid hormone content of rat embryonic tissues, before and after onset of fetal thyroid function. Endocrinology. 1985;117:1890–1900. [DOI] [PubMed] [Google Scholar]
  • 40. Ferrara AM, Liao XH, Gil-Ibanez P, et al. Changes in thyroid status during perinatal development of MCT8-deficient male mice. Endocrinology. 2013;154:2533–2541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41. Gharaibeh B, Lu A, Tebbets J, et al. Isolation of a slowly adhering cell fraction containing stem cells from murine skeletal muscle by the preplate technique. Nat Protoc. 2008;3:1501–1509. [DOI] [PubMed] [Google Scholar]
  • 42. Ramadan W, Marsili A, Huang S, Larsen PR, Silva JE. Type-2 iodothyronine 5′deiodinase in skeletal muscle of C57BL/6 mice. I. Identity, subcellular localization, and characterization. Endocrinology. 2011;152:3082–3092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43. Baqui MM, Gereben B, Harney JW, Larsen PR, Bianco AC. Distinct subcellular localization of transiently expressed types 1 and 2 iodothyronine deiodinases as determined by immunofluorescence confocal microscopy. Endocrinology. 2000;141:4309–4312. [DOI] [PubMed] [Google Scholar]
  • 44. Visser WE, Heemstra KA, Swagemakers SM, et al. Physiological thyroid hormone levels regulate numerous skeletal muscle transcripts. J Clin Endocrinol Metab. 2009;94:3487–3496. [DOI] [PubMed] [Google Scholar]
  • 45. Diaz F, Thomas CK, Garcia S, Hernandez D, Moraes CT. Mice lacking COX10 in skeletal muscle recapitulate the phenotype of progressive mitochondrial myopathies associated with cytochrome c oxidase deficiency. Hum Mol Genet. 2005;14:2737–2748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46. McPherron AC, Huynh TV, Lee SJ. Redundancy of myostatin and growth/differentiation factor 11 function. BMC Dev Biol. 2009;9:24. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47. Schmalbruch H, Hellhammer U. The number of nuclei in adult rat muscles with special reference to satellite cells. Anat Rec. 1977;189:169–175. [DOI] [PubMed] [Google Scholar]

Articles from Endocrinology are provided here courtesy of The Endocrine Society

RESOURCES