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Logo of jbcThe Journal of Biological Chemistry
 
J Biol Chem. 2015 December 18; 290(51): 30551–30561.
Published online 2015 October 23. doi:  10.1074/jbc.M115.665505
PMCID: PMC4683275

Coupling between Nutrient Availability and Thyroid Hormone Activation*

Abstract

The activity of the thyroid gland is stimulated by food availability via leptin-induced thyrotropin-releasing hormone/thyroid-stimulating hormone expression. Here we show that food availability also stimulates thyroid hormone activation by accelerating the conversion of thyroxine to triiodothyronine via type 2 deiodinase in mouse skeletal muscle and in a cell model transitioning from 0.1 to 10% FBS. The underlying mechanism is transcriptional derepression of DIO2 through the mTORC2 pathway as defined in rictor knockdown cells. In cells kept in 0.1% FBS, there is DIO2 inhibition via FOXO1 binding to the DIO2 promoter. Repression of DIO2 by FOXO1 was confirmed using its specific inhibitor AS1842856 or adenoviral infection of constitutively active FOXO1. ChIP studies indicate that 4 h after 10% FBS-containing medium, FOXO1 binding markedly decreases, and the DIO2 promoter is activated. Studies in the insulin receptor FOXO1 KO mouse indicate that insulin is a key signaling molecule in this process. We conclude that FOXO1 represses DIO2 during fasting and that derepression occurs via nutritional activation of the PI3K-mTORC2-Akt pathway.

Keywords: insulin, mTOR complex (mTORC), signal transduction, signaling, thyroid hormone, FOXO1, type 2 deiodinase

Introduction

In mammals, body energy expenditure is gradually decreased with caloric restriction as well as prolonged starvation. This is an adaptive response driven by the hypothalamus that includes behavioral as well as metabolic modifications, such as increased sleep, diminished motor activity, and core temperature (1). During fasting, the hormonal milieu is dominated by low levels of insulin and thyroid hormones, which has been proposed as an explanation for the slowdown in weight loss after a few days of caloric restriction (2, 3). Notably, thyroidal activity is restored promptly upon refeeding, as seen in patients recovering from anorexia nervosa (4). In such patients, weight gain and an increase in serum T32 are closely associated with acceleration in energy expenditure (4). These observations indicate that the set point at which the hypothalamic-pituitary-thyroid activity is maintained is affected by nutrient availability, with the lowest (default) levels of activity seen during caloric restriction. Leptin is the key molecule signaling food intake and the availability of energy substrates, which defines the hypothalamic-pituitary-thyroid set point by stimulating secretion of thyrotropin-releasing hormone and thyroid-stimulating hormone and hence thyroidal activity (5, 6).

In humans, the thyroid gland contributes with about 20% of the daily T3 production, and the residual 80% is contributed by two deiodinases that convert T4 to T3 outside the thyroid parenchyma (i.e. D1 and D2, with the latter playing the major role) (7). In vivo kinetic studies in rats indicate that serum T3 levels fall during fasting also as a result of decreased thyroidal T3 secretion (8). In addition, there is also accelerated thyroid hormone inactivation via D3 expression in multiple tissues as well as accelerated sulfation and glucuronidation in the liver (9, 10). However, it is less clear whether the extrathyroidal T3 production is reduced as well. Whereas hepatic D1 activity is reduced during caloric restriction in rodents (11), this seems to be a consequence rather than a cause of the low serum T3, given that Dio1 is highly responsive to T3 (8, 12). On the other hand, D2 is normally stimulated during hypothyroidism (7), and the fact that it is reduced by fasting in the pituitary gland and brown adipose tissue (BAT) (10) indicates a role in the fall of serum T3. D2 is an endoplasmic reticulum-resident protein that increases intracellular T3 concentration and the expression of thyroid hormone-dependent genes (7, 13). For example, approximately half of the T3 present in the brain and in the BAT is originated locally via D2 activity; D2 inactivation in both tissues has been shown to dampen thyroid hormone signaling (7). Therefore, control of the D2 pathway by caloric intake could potentially modulate thyroid hormone signaling by affecting intracellular T3 concentration as well as circulating T3 levels.

D2 expression is controlled by transcriptional (i.e. cAMP, FOXO3, and NF-κB) (14, 15), translational (i.e. ER stress) (16), and post-translational mechanisms, such as ubiquitination and proteasomal degradation (17). In addition, D2 activity in rat BAT is up-regulated by growth factors such as IGF-1 and the multifunctional protein insulin (18, 19), which promotes cellular glucose uptake and growth by acting through various nutrient-sensing pathways, such as the PI3K-mTOR signaling pathway. Mammalian target of rapamycin (mTOR) is a signaling pathway that functions as a regulator of translational initiation for cellular metabolism, growth, survival, proliferation, and protein synthesis (20, 21). mTOR, a 289-kDa Ser/Thr kinase, serves as the catalytic core of two distinct multicomponent complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (22). Numerous studies have linked mTORC1 to protein synthesis and growth (23) and to the downstream activation of a myriad of metabolic regulatory genes, including HIF-1α and SREBP1/2 (24). In addition, it has been determined that mTORC2 phosphorylates the AGC kinases (25) and mediates cell survival and proliferation through phosphorylation of its downstream effector, Akt (26).

The observation that insulin stimulates D2 activity in rat brown adipocytes (19) and that insulin sensitizers stimulate D2 expression in cultures of skeletal myocytes (27) indicate that the nutritional state of an organism can also affect thyroid hormone signaling by acting at the target cell level. The mechanism behind D2 stimulation by insulin remain unknown, despite evidence that an indirect effect via the adrenergic receptors could play a role (28). In the present study, we have examined how nutrient availability and insulin stimulate D2 activity in mice and cell models. We have found that insulin signals through PI3K-mTORC2-Akt to relieve FOXO1-mediated repression of DIO2, indicating that nutrient availability controls serum levels of thyroid hormones not only by modulating the hypothalamic-pituitary-thyroid set point but also by regulating extrathyroidal T3 production. Given that serum T3 is a major determinant of energy expenditure (29), this explains how the cellular metabolic rate as defined by T3 signaling is kept at a level that is commensurate to the availability of energy substrates.

Experimental Procedures

Reagents and Antibodies

Outer ring-labeled [125I]T4 (specific activity 4400 Ci/mmol; PerkinElmer Life Sciences) was purified on LH-20 columns before use; fetal bovine serum (FBS) was from Atlanta Biologicals (Flowery Branch, GA); cyclohexamide, insulin, IGF-1, LY294002, wortmannin, GSK609693, rapamycin, and PP242 were from Sigma-Aldrich; AS1842856 was from EMD Millipore (Billerica, MA); α-phospho-S6rp (Ser-235/236), α-phospho-S6rp (Ser-240/244), α-total S6rp, α-phospho-Akt (Ser-473), α-phospho-Akt (Thr-308), α-pan-Akt, α-phospho-4EBP1, α-total 4EBP, α-total rictor, α-total raptor, α-phospho-FOXO1 (Ser-256), and α-total FOXO1 were from Cell Signaling Technology (Boston, MA); GAPDH antibody was from GeneTex (Irvine, CA); α-FKHR (H-128) was from Santa Cruz Biotechnology, Inc. (Dallas, TX).

Cell Culture

MSTO-211H cells were from American Type Culture Collection (Manassas, VA) and cultured in humidified atmosphere with 5% CO2 at 37 °C, in RPMI 1640 from CellGro (Manassas, VA) supplemented with 10% FBS and 100 nm sodium selenite (16, 30). As indicated, cells were serum-starved for 48 h in 0.1% FBS medium prior to re-exposure to 10% FBS medium, insulin stimulation, or IGF-1 stimulation.

Deiodinase Assays

After harvesting, tissues and MSTO-211H cells were resuspended in Dulbecco's PBS containing 1 mm EDTA (PE), 0.25 m sucrose, and 10 mm dithiothreitol (DTT) and sonicated, and cell lysates were analyzed for protein concentration via Bradford assay (Bio-Rad). D2 assays were performed as described previously in duplicate (27).

Gene Expression Analysis

RNA was extracted from MSTO-211H cells or animal tissue using the RNAqueous Micro Kit (Life Technologies Inc.) or RNeasy Mini Kit (Qiagen), respectively. RNA was quantified with a NanoDrop and reverse transcribed using High Capacity cDNA (Applied Biosystems) or the First Strand cDNA for RT-PCR (AMV) Kit (Roche Applied Science). Genes of interest were measured by qPCR (Bio-Rad iCycler iQ real-time PCR detection system) using the iQ SYBR Green Supermix (Bio-Rad) or qPCR (Applied Biosystems Step One Plus Real-Time PCR System) using the SYBR Green FastMix ROX (Quanta Biosciences). Relative quantitation was using the standard curve method and the iCycler or Step One Plus software.

Western Blot Analysis

Cells/tissues were lysed in 0.25 m sucrose PE containing 10 mm DTT. The lysates were diluted with 4× sample loading buffer (Invitrogen), and 5–25 μg of total protein were run on 4–12% NuPAGE BisTris gels (Life Technologies, Inc.). Samples were transferred to Immobilon-FL PVDF transfer membrane (Millipore, Billerica, MA) and probed with antibodies as indicated at a 1:1000 dilution overnight. Fluorescent labeled secondary antibodies (LI-COR Biosciences, Lincoln, NE) were used at 1:2500 for 1 h. All blots were imaged using the LI-COR Odyssey instrument per the manufacturer's instructions.

T4 to T3 Conversion in Intact Cells

The production of T3 from outer ring-labeled T4 in intact cells was analyzed by measuring 125I in the medium as described elsewhere (31) except that the assay was stopped 12 h after the addition of [125I]T4, and the free T4 concentration was 30 pm.

Lentivirus-mediated shRNA Knockdown of Rictor and Raptor

Rictor and raptor knockdown were established by transduction with GIPZ lentiviral shRNAmir vectors in MSTO-211H cells using GIPZ lentiviral shRNAmir for non-silencing control (NSC), human rictor (clone ID V2LHS_120392 and clone ID V3LHS_367492), or human raptor (clone ID V3LHS_636800) from Thermo Scientific (Lafayette, CO). For generation of stable knockdown cell lines, MSTO-211H cells were plated at 1.5 × 105 cells/well and transduced with lentiviral particles at a multiplicity of infection of ~23 and ~15 for rictor, respectively, and a multiplicity of infection of ~13 for raptor and diluted in 1 ml of serum-free RPMI containing 8 μg/ml Polybrene (Sigma-Aldrich). After 6 h, 1 ml of complete RPMI was added to each well (6-well plates). 72 h later, the transfection mixture was replaced with complete medium containing puromycin (1 μg/ml) to select for shRNA-expressing cells. Stable cell lines were generated after puromycin selection for 7 days.

Adenovirus-mediated Transduction of WT and Constitutively Active (CA) FOXO1

Adenoviral particles expressing WT and CA FOXO1 as described (32) were used to generate transient transfections for WT and CA FOXO1 in MSTO-211H. Cells were plated at 2.5 × 105 cells/well in 6-well dishes and transduced with adenoviral particles of concentration 2.0 × 1010 pfu/ml and 1.8 × 1010 pfu/ml, respectively, that were diluted in 1 ml of serum-free RPMI. Complete medium containing 10% FBS was added after 6 h, and the medium was changed again after 24 h. At 48 h, cells were refed or stimulated with insulin for the experimental procedure.

Animal Studies

All studies performed were approved by the institutional animal care and use committee of the University of Miami in compliance with National Institutes of Health Standards. Male, 3-month-old C57BL/6J mice purchased from Jackson Laboratory were housed at room temperature (22 °C) on a 12-h dark/light cycle. Mice were randomly divided into three groups (control, fasting, refeeding) and acclimatized for 5 days. Animals were fed ad libitum with a standard chow diet (3.5 kcal/g, 28.8% protein, 58.5% carbohydrate, 12.7% fat; 5010 LabDiet laboratory autoclavable rodent diet; PMI Nutrition, Richmond, IN) and water. When indicated, food was withdrawn at 21:00 h for the fasting and refeeding groups. After 36 h, fasted mice were sacrificed by asphyxiation in a CO2 chamber. Mice from the refeeding group were weighed, refed for 8 h, and sacrificed. Soleus muscle and cortex were rapidly dissected and frozen in liquid nitrogen and stored at −80 °C until analysis. As indicated, freely feeding 11-week-old male insulin receptor (IR) floxed, liver-specific IR knock-out (LIRKO), and IR/FOXO1 double knock-out (LIRFKO) mice (33) were used as well. They were sacrificed by decapitation following brief sedation with isofluorane.

Blood Biochemistry

Blood was collected by cardiac puncture, and plasma was stored at −80 °C until analysis. Insulin was measured using mouse insulin ELISA (Mercodia, AB). Serum T3 and T4 were measured as described previously (34).

Chromatin Immunoprecipitation

ChIP was performed using the EZ-ChIP chromatin immunoprecipitation kit (EMD Millipore) according to the manufacturer's guidelines. Primers for RT-qPCR (Applied Biosystems Step One Plus Real-Time PCR System) were designed for predicted FOXO1 binding sites in the DIO2 promoter region (forward, 5′-CTGTCTGGAGGAACTTGGATTT-3′; reverse, 5′-CCATGACTCCAACCCTTTGT-3′), and qPCR was performed using the SYBR Green FastMix ROX (Quanta Biosciences).

Statistical Analysis

All data were analyzed using PRISM software (GraphPad Software) and expressed as mean ± S.E. Student's t test was used to compare two groups. One-way analysis of variance was used to compare more than two groups. Significance was held at p < 0.05 (two-tailed).

Results

Fasting Lowers Soleus Dio2 mRNA Expression Levels and Activity in Mice

Fasting for 36 h caused a marked loss in body weight (Fig. 1A), a drop in serum insulin levels (Fig. 1B), and insulin signaling in skeletal muscle (Fig. 1C). This was associated with an ~80% reduction in soleus Dio2 mRNA levels (Fig. 1D) and D2 activity (Fig. 1E). The decrease in D2 expression and activity was tissue-specific given that cerebral cortex Dio2 mRNA remained stable throughout the fasting period (Fig. 1F). This led to a ~45% reduction in serum T3 levels (0.56 ± 0.11 versus 0.29 ± 0.06 ng/ml; n = 4; p < 0.01), whereas serum T4 remained largely unaffected (59 ± 12 versus 53 ± 10 ng/ml; n = 4; not significant). Refeeding for 8 h normalized body weight (Fig. 1A), increased serum insulin levels by about 100-fold (Fig. 1B), increased insulin signaling (Fig. 1C), elevated Dio2 mRNA (Fig. 1, G and H) and activity levels (Fig. 1I), normalized serum T3 (0.48 ± 0.09 ng/ml; n = 4), and did not affect serum T4 (54 ± 13 ng/ml; n = 4).

FIGURE 1.
Fasting inhibits Dio2 expression levels and activity in the soleus muscle of mice. A, change in body weight of control, fasted, and refed mice measured before and after the experimental period; n = 7; ***, p < 0.001. B, serum insulin levels measured ...

Fresh Serum Induces D2 Expression in a Cell Model

Given that D2 activity is near background level in skeletal muscle cell lines (27), the role played by fasting and refeeding on DIO2 expression was next modeled using MSTO-211H cells, a cell line that endogenously expresses readily measurable D2 activity (30). In this setting, keeping the cells for 48 h in 0.1% FBS caused a progressive reduction in DIO2 mRNA and activity to approximately half of the baseline values seen in cells cultured in 10% FBS (Fig. 2outf;F2, A and B). Notably, transition to 10% FBS resulted in a ~3.5-fold increase in D2 activity (Fig. 2C) that followed a ~2-fold increase in D2 mRNA levels (Fig. 2D); both subsided and returned to baseline levels by 24 h (Fig. 2, C and D). The increases in D2 activity (Fig. 2E) and typical serum-dependent downstream targets (Fig. 2F) were dependent on the amount of FBS contained in the fresh medium. D2 is known for having a post-translational regulation via ubiquitination and proteasomal degradation, explaining its relatively short activity half-life of less than 1 h (35). Thus, we tested whether the surge in D2 activity that follows refeeding could also involve the ubiquitin-proteasomal mechanism. D2 activity half-life was measured by exposing cells in 10% FBS to cyclohexamide, but at no time was there a significant effect on D2 activity half-life (Fig. 2, G–J).

FIGURE 2.
Nutrient availability increases D2 activity via transcriptional activation of DIO2 in MSTO-211H cells. A and B, DIO2 mRNA expression levels (A) and D2 activity (B) measured during 48 h of starvation in 0.1% FBS; n = 3; ***, p < 0.001 versus 0 ...

PI3K-dependent Pathways Stimulate D2 Activity

Serum/FBS contains a number of growth factors that could potentially induce D2 expression during refeeding, mostly acting via PI3K. Thus, we used insulin and observed a concentration-dependent increase in D2 activity of ~50% after 8 h (Fig. 2K). We also observed a time-dependent increase in D2 activity following the stimulation of serum-starved MSTO-211H cells with 50 ng/ml IGF-1 (Fig. 2L). The acceleration in D2 activity increased thyroid hormone activation, given that T3 production was accelerated 4–5-fold after the addition of IGF-1 or insulin (Fig. 2M). Next, different strategies were used to dissect the molecular pathway that mediates the insulin/IGF-1 stimulation of DIO2 expression. First, we inhibited PI3K using LY294002, a well known reversible inhibitor of PI3K phosphorylation. Following the standard 48 h starvation period in 0.1% FBS, MSTO-211H cells were treated with 50 μm LY294002 and either stimulated with 10% FBS (Fig. 3A) or exposed to insulin (Fig. 3B). Notably, inhibition of PI3K abrogated the increase in D2 activity at all time points studied, which coincided with the inhibition of phosphorylation of the downstream targets Akt and S6rp (Fig. 3, A and B). Similar results were observed when 0.1–1 μm wortmannin was used to inhibit PI3K signaling (Fig. 3C, a and b). Next, we looked further down the pathway and used GSK690693, an inhibitor of all three Akt isoforms (36, 37) to examine the involvement of Akt in this mechanism. MSTO-211H cells that had been starved for 48 h were pretreated for 1 h with 10 nm GSK690693 (37) and subsequently exposed to insulin. Again, insulin induction of D2 activity was prevented at all time points tested throughout a 24 h time period (Fig. 3D). Western blot analysis of Akt phosphorylation shows overstimulation of the Akt pathway, a feature commonly observed when small molecule Akt inhibitors are used (36, 37). This was coupled with inhibition of S6rp phosphorylation (Fig. 3D), indicating successful Akt inhibition.

FIGURE 3.
Inhibition of PI3K signaling using LY294002 and wortmannin or of Akt signaling using GSK690693 prevents the insulin-induced increase in D2 activity in MSTO-211H cells. A, top, D2 activity following 48 h of starvation in 0.1% FBS prior to the addition ...

Inhibition of mTOR Pathway Prevents FBS and Insulin-mediated D2 Stimulation

We next used PP242, a selective ATP-competitive inhibitor of both mTORC1 and mTORC2 (38, 39) to test the involvement of the mTOR signaling pathway in the D2 stimulation by FBS or insulin. Treating cells with 2 μm PP242 prevented the surge in D2 activity that follows 10% FBS (Fig. 4A) or insulin stimulation (Fig. 4B). The effectiveness of PP242 in blocking both the mTORC1 and mTORC2 signaling pathways was documented by Western blot analysis of downstream targets of mTORC1 (S6rp and 4EBP1) and of mTORC2 (Akt) (Fig. 4, A and B). Next, we used rapamycin, an inhibitor of mTORC1 (40), to further investigate the involvement of the mTOR pathway. Inhibition of mTORC1 delayed but did not prevent the surge in D2 activity caused by 10% FBS (Fig. 4C). Inhibitory selectivity was documented by Western blot analysis of phosphorylated and total S6rp, a downstream marker of mTORC1. Indeed, there was reduction of mTORC1 signaling after 4 h of rapamycin treatment, whereas phosphorylation of Akt via mTORC2 was not inhibited (Fig. 4D). Similar to D2 activity, we also found a significant increase of DIO2 mRNA levels at various time points over a 24 h period (Fig. 4E). Studies with insulin led to similar observations (Fig. 4F). As further confirmation, we used GIPZ lentiviral shRNA vectors to knockdown raptor (regulatory-associated protein of mTOR), a component of the mTORC1 complex. Despite successful raptor knockdown in these cells, both 10% FBS (Fig. 5A) and the addition of insulin (Fig. 5B) stimulated D2 activity. Altogether, these data suggest that D2 induction by 10% FBS or exposure to insulin is mediated by an mTORC2-dependent pathway.

FIGURE 4.
Inhibition of mTOR signaling using PP242 prevents FBS- and insulin-mediated D2 stimulation; however, inhibition of mTORC1 signaling using rapamycin has no effect on the insulin-induced increase in D2 activity in MSTO-211H cells. A, top, D2 activity following ...
FIGURE 5.
Inhibition of mTORC2 prohibits the insulin-induced increase in D2 activity in MSTO-211H cells. A, top, D2 activity measured in MSTO-211H cells stably expressing GIPZ NSC or GIPZ-raptor-shRNAmir A lentiviral vector; cells were serum-starved in 0.1% FBS ...

To assess the role played by mTORC2, we next used GIPZ lentiviral shRNA vectors to knock down an essential component of the mTORC2 complex, rictor (rapamycin-insensitive component of mTOR) in MSTO-211H cells. Rictor is a core unit of the mTORC2 complex, and its reduction disrupts mTORC2 complex assembly (41). Stably expressing cells were starved for 48 h prior to the addition of 10% FBS (Fig. 5C) or insulin stimulation (Fig. 5D). Inhibition of mTORC2 significantly inhibits the surge in D2 activity at all time points tested, peaking at a 4-fold reduction by 12 h in both conditions as compared with NSCs. The successful knockdown of rictor was documented by Western blot. These data show the dependence of induction of D2 activity on the mTORC2 pathway.

FOXO1 Transcriptionally Represses DIO2

While looking for the mTORC2/Akt-dependent downstream targets that could activate DIO2, we serendipitously observed that Dio2 mRNA levels were low in a mouse with liver-specific inactivation of the insulin receptor (LIRKO) and that Dio2 mRNA levels were restored when this mouse was crossed with a mouse harboring a liver-specific disruption of FOXO1 (LIRFKO) (Fig. 6A). This observation, along with previous studies indicating that FOXO3 interacts with the Dio2 gene (15), suggested that FOXO1 may be the key molecule mediating the effects of nutrient status and insulin signaling on Dio2. This suspicion was strengthened by the observation that there was synchronized stimulation of D2 activity and FOXO1 phosphorylation after serum-starved MSTO-211H cells were exposed to 10% FBS (Fig. 6, B and C). Subsequently, the specific FOXO1 inhibitor AS1842856, which prevents Ser-256 phosphorylation, was added to cells during 0.1% FBS starvation (42). Whereas 0.1% FBS resulted in the expected loss of D2 activity, treatment with AS1842856 not only reversed this but increased D2 activity above baseline levels (Fig. 6D). Similar findings were observed in cells that had been starved in 0.1% FBS for 48 h and then treated with AS1842856 (Fig. 6E). Taken together, these results indicate that loss of D2 activity associated with fasting is mediated by FOXO1.

FIGURE 6.
The phosphorylation of FOXO1 or its inhibition using AS1842856 is correlated with an increase in D2 activity, whereas constitutive activation of FOXO1 abrogates the insulin and FBS-induced stimulation in D2 activity in MSTO-211H cells. A, the relative ...

To further explore the role of FOXO1 in regulating D2 expression, MSTO-211H cells were infected with WT or CA FOXO1-expressing adenovirus and processed through the 0.1%/10% FBS or insulin stimulation protocols. Remarkably, expression of the CA FOXO1 protein prevents all of the induction of DIO2 mRNA (Fig. 6, F and G) and most of the D2 activity (Fig. 6, H and I) caused by 10% FBS and insulin stimulation. This indicates that DIO2 is a probable direct target of FOXO1 and that 10% FBS/insulin acts by relieving FOXO1 gene repression. To demonstrate the binding of FOXO1 to predicted binding sites in the DIO2 promoter region (Fig. 6J), ChIP analysis was performed in MSTO-211H cells in 0.1% FBS and 10% FBS. There is specific binding of FOXO1 to a site in the DIO2 promoter located −570 bp from the transcription start site that is abrogated in refed cells (Fig. 6J).

Discussion

The present studies employed a combination of animal and cell models to establish that nutritional availability affects thyroid hormone activation via induction of D2. This enzyme contributes to about two-thirds of the circulating T3 in humans (about one-third in rodents) and at least one-half of the T3 present in murine brain and BAT (7). D2 stimulation by 10% FBS is transcriptional and involves insulin and IGF-1 signaling through PI3K-mTORC2-Akt to relieve FOXO1-mediated DIO2 repression (Fig. 7). These data provide a mechanistic explanation for the observation in humans that fasting is associated with a reduction in skeletal muscle D2 activity that is partially prevented by insulin administration (43). The present findings constitute a new aspect of the overarching homeostatic role played by the hypothalamic-pituitary-thyroid axis to adjust thyroid hormone activation to the appropriate level of energy substrates; thyroidal activity and serum T3 levels are kept at a minimum (default) during fasting or severe caloric restriction, and both increase as food becomes available.

FIGURE 7.
Proposed mechanism of insulin/IGF-1/FBS-mediated stimulation of D2. Insulin, IGF-1, 10% FBS stimulates D2 transcription and activity via the PI3K-mTORC2-Akt signaling pathway. The inhibitory phosphorylation of FOXO1 by Akt relieves its transcriptional ...

T3 receptors (TRs) are ligand-dependent transcription factors that control tissue-specific sets of T3-responsive genes. TRs are bound to the promoter of T3-responsive genes, and, as opposed to most other receptors, unoccupied TRs actively inhibit transcription, given their affinity for corepressors. Upon binding of T3, TRs gain affinity for coactivators and trigger T3-dependent gene transcription. Thus, the role of T3 is not only to promote expression of T3-dependent genes but also to relieve TR-mediated gene repression (44). The present observation that fasting shuts down DIO2 expression (and T3 production) via shifting the balance between PI3K-mTORC2-Akt and FOXO1 highlights the importance of unoccupied TRs.

D2 expression in the cerebral cortex was not modified by fasting or refeeding (Fig. 1F), indicating that Dio2 regulation by nutrient availability is not universal, probably occurring in tissues where the metabolic pathways are responsive to T3 and insulin (e.g. BAT and skeletal muscle) (7). D2-generated T3 in BAT enhances local thyroid hormone signaling and the expression of Ucp1 and Pgc-1α, both critical for thermogenesis (45). A local role for D2-generated T3 in skeletal muscle is less clear. The fact that skeletal muscle physiology is T3-sensitive (46) sets the stage for a potential D2 contribution, such as during development and response to injury (47). Nevertheless, selective D2 inactivation in the mouse skeletal muscle failed to identify a metabolic phenotype, and serum T3 was preserved due to increased thyroidal T3 production (48, 49). The present findings are particularly relevant because the thyroid gland does not adjust T3 production during fasting (8), and serum T3 drops also as a consequence of FOXO1-mediated DIO2 inhibition.

In addition, the present findings are clinically relevant because there are ~10 million hypothyroid individuals in the United States alone who depend >80% on the D2 pathway to convert levothyroxine to T3. Because these patients are hypothyroid, they lack the ability to compensate for FOXO1-mediated DIO2 suppression during fasting. Thus, serum T3 levels in these individuals could fluctuate substantially with caloric intake. In many aspects, the balance between PI3K-mTORC2-Akt and FOXO1 signaling should provide nutritional input and fine tuning to the regulation of circulating T3 levels and T3-dependent processes.

The data from the LIRKO and LIRFKO mice indicate that insulin is probably a key molecule mediating Dio2 regulation (Fig. 6A), known to provide nutritional input through the PI3K-mTOR signaling pathway in many other systems (50). In the cell model, both insulin and IGF-1 stimulate D2 (Fig. 2, K and L), indicating that this is an insulin/IGF-1-mediated mechanism. mTOR, which is downstream of insulin/IGF-1 signaling, also plays a central role in defining the balance between anabolic and catabolic processes (51). Downstream targets of mTORC2 include the AGC kinases (i.e. Akt, SGK, and PKCα, which function to promote cell survival, metabolism, and actin organization) (41). In turn, Akt directly phosphorylates FOXO1 (25, 52), a nuclear transcription factor that modulates multiple sets of genes in a tissue-specific manner (53, 54).

A functional FOXO3 binding site is located in the 5′-UTR of the human DIO2, positively regulating DIO2 expression in developing skeletal muscle (15). Although proteins in the FOXO family can potentially bind to the same DNA cis-elements, the FOXO1 binding site identified in the present studies is within the DIO2 promoter, close to the transcription start site. Binding of FOXO1 to this site (Fig. 6J) suppresses DIO2 gene expression.

In conclusion, the present study reveals that insulin/IGF-1 transcriptionally up-regulates DIO2 expression and activity via signaling through the PI3K-mTORC2-Akt pathway, relieving FOXO1-mediated DIO2 inhibition. D2 is a major contributor to plasma T3 levels in rodents and in humans and is the predominant pathway producing T3 in hypothyroid individuals treated with levothyroxine. Therefore, the PI3K-mTORC2-Akt pathway is likely to have major metabolic consequences because it provides a coupling mechanism between nutrient availability and thyroid hormone activation.

Author Contributions

L. J. L. conducted experiments, analyzed and interpreted the data, and prepared the manuscript. J. P. W. and I. O. conducted experiments and analyzed data. T. G. U. created the LIRKO and LIRFKO mice, directed the experiments with these animals, and reviewed/edited the manuscript. A. C. B. directed the studies, interpreted the data, and prepared the manuscript along with L. J. L.

Acknowledgments

We are grateful to John Harney for pointing out that D2 activity in cultured cells increases after a change in the medium; we thank Balázs Gereben for valuable discussions regarding the manuscript; we thank Manish Tandon for assistance with the ChIP experiments.

*This work was supported in part by National Institutes of Health, NIDDK, Grant DK65055 and by a grant from the Department of Veterans Affairs Merit Review Program (to T. G. U.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

2The abbreviations used are:

T3
triiodothyronine
T4
thyroxine
TR
T3 receptor
D1
D2, and D3, type 1, 2, and 3 deiodinase, respectively
CA
constitutively active
BAT
brown adipose tissue
mTOR
mammalian target of rapamycin
MTORC1 and -2
mTOR complex 1 and 2, respectively
qPCR
quantitative PCR
BisTris
2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol
NSC
non-silencing control
IR
insulin receptor
LIRKO
liver-specific IR knock-out
LIRFKO
IR/FOXO1 double knock-out
IGF
insulin-like growth factor.

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