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Research Paper

Sleep deprivation alters thyroid hormone economy in rats Nayana Coutinho Rodrigues, Nat´alia Santos da Cruz, Cristine de Paula Nascimento, Rodrigo Rodrigues da Conceic¸a˜o, Alba Cen´elia Matos da Silva, Emerson Lopes Olivares and Michelle Porto Marassi

Experimental Physiology

Multicenter Graduate Program in Physiological Sciences, Department of Physiological Sciences, Institute of Biology, Federal Rural University of Rio de Janeiro, Seropedica, Brazil

New Findings r What is the central question of this study? The relationship between the thyroid system and sleep deprivation has seldom been assessed in the literature, and mounting evidence exists that sleep disturbances influence human lifestyles. The aim of this study was to investigate the hypothalamic–pituitary–thyroid axis and thyroid hormone metabolism in sleep-deprived and sleep-restricted rats. r What is the main finding and its importance? Central hypothyroidism and high thyroxine (T4 ) to 3,5,3 -triiodothyronine (T3 ) activation in brown adipose tissue were observed following sleep deprivation. Sleep-restricted rats exhibited normal thyroid-stimulating hormone and T4 concentrations despite increased circulating T3 . Sleep recovery for 24 h did not normalize the high T3 concentrations, suggesting that high T3 is a powerful counterregulatory mechanism activated following sleep deprivation.

Modern life has shortened sleep time, and the consequences of sleep deprivation have been examined in both human subjects and animal models. As the relationship between thyroid function and sleep deprivation has not been fully investigated, the aim of this study was to assess the hypothalamic–pituitary–thyroid axis and thyroid hormone metabolism following paradoxical sleep deprivation (PSD) and sleep restriction (SR) in rats. The effects of a 24 h rebound period were also studied. Male Wistar rats (200–250 g, n = 10 per group) were subjected to sleep deprivation via the modified multiple platform method. Rats were assigned to the following seven groups: control, PSD for 24 or 96 h, 24 or 96 h of sleep deprivation with rebound (PSD24R and PSD96R), SR for 21 days (SR21) and SR21 with rebound (SR21R). Blood samples were collected to determine the 3,5,3 -triiodothyronine (T3 ), thyroxine (T4 ) and thyroid-stimulating hormone concentrations. Brown adipose tissue iodothyronine deiodinase type 2 (D2) activity was also evaluated. Body weight gain was dramatically reduced (by 50–100%) in all sleep-deprived and sleep-restricted rats; rebound restored this parameter in only the PSD24R group. The serum TSH and T4 concentrations decreased, whereas T3 increased in both the PSD24 and PSD96 groups compared with control animals (P < 0.05). Only PSD24R and PSD96R normalized T4 and thyroid-stimulating hormone concentrations, respectively, independently of the higher circulating T3 concentrations (20–30%) noted in all groups compared with control animals (P < 0.05). Brown adipose tissue D2 activity increased in the PSD 24 and 96 h groups (10 times), and PSD24R was more effective than PSD96R at restoring basal brown adipose tissue D2 activity. Our data suggest that thyroid hormone

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DOI: 10.1113/expphysiol.2014.083303

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metabolism adapts to sleep deprivation-induced hypothalamic–pituitary–thyroid alterations and increases T4 to T3 activation peripherally, thereby increasing circulating T3 in rats. (Received 24 September 2014; accepted after revision 1 December 2014; first published online 4 December 2014) ´ Corresponding author M. P. Marassi: Departamento de Ciˆencias Fisiologicas, Instituto de Biologia, Universidade Federal Rural do Rio de Janeiro BR 465, Km 07 Serop´edica, Rio de Janeiro 23890-000, Brasil. Email: [email protected]

Introduction Sleep disorders, a highly common medical issue that affects millions of people worldwide, result from modern lifestyle, health complications, medication side-effects and clinical disorders. Although sleep restriction and deprivation have been well studied (Tufik et al. 2009), little is known about possible changes in thyroid hormone economy following sleep disturbance. In mammals, sleep and waking represent two physiological states defined by electroencephalographic (EEG) patterns regulated by circadian cycles and homeostatic processes (Borb´ely, 1982). Sleep is composed of two stages, REM and non-REM sleep (rapid eye movement and non-rapid eye movement), which alternate cyclically during sleeping hours (Dement & Kleitman, 1957). During REM sleep, the information obtained during waking is reprocessed and integrated into existing neural templates. Modern life promotes significant reductions in this sleep phase (Tufik et al. 2009). In humans, Spiegel et al. (1999) demonstrated an increase in serum cortisol levels and sympathetic hyperactivity using a sleep-restriction protocol of 4 h per night over six nights. In animals, paradoxical sleep deprivation for 96 h resulted in other homeostatic disturbances, including decreased serum gonadal hormone levels (Andersen et al. 2004) and hypothermia (Seabra & Tufik, 1993). Thyroid hormones (THs) exert effects in virtually all tissues controlling metabolism and sympathetic activity (Bianco et al. 2002) and are influenced by other endocrine signals and compounds, such as cortisol (Tohei, 2004) and gonadal hormones (Marassi et al. 2007). The primary product of the thyroid gland is 3,5,3 ,5 -tetraiodothyronine (thyroxine or T4 ), a precursor molecule that is converted into 3,5,3 -triiodothyronine (T3 ), which effectively binds to nuclear thyroid hormone receptors and either mediates or represses thyroid hormone-dependent transcriptional activation (Gereben et al. 2008). The role played by the hypothalamic–pituitary–thyroid (HPT) axis in regulating T3 availability is complemented by the functions of the iodothyronine deiodinases, types 1 (D1), 2 (D2) and 3 (D3), enzymes that activate or inactivate T4 in a time- and tissue-specific fashion (Bianco et al. 2002). The hypothalamus releases thyrotrophin-releasing hormone (TRH) and stimulates the pituitary to synthetize and release thyroid-stimulating hormone (TSH), which

subsequently stimulates thyroid synthesis and secretion of THs. This system is controlled by a negative feedback mechanism established by high levels of circulating THs. Activation of the sodium–iodide (NIS) cotransporter is necessary for TH synthesis, as is the activity of thyroperoxidase enzyme (TPO). In a clinical study involving 11 healthy volunteers, Kessler et al. (2010) demonstrated that partial sleep restriction was accompanied by modest but statistically significant reductions in TSH and free T4 , detected primarily in female participants. Surprisingly, despite the recognized physiological role of thyroid hormones and the well-known influence of the circadian cycle on pulsatile TSH secretion (Brabant et al. 1990), only two studies have investigated thyroid function in sleep-deprivation models, to the best of our knowledge. Everson & Novack (2002) demonstrated that sleep deprivation for 15 or 21 days in rats decreased serum T4 levels and increased TRH mRNA expression, whereas serum TSH was not affected. Using the gyratory platform method, Balzano et al. (1990) observed reductions in plasma T4 concentrations and significant increases in D2 activity in the brown adipose tissue (BAT) of rats. Although these studies hint at sleep-deprivation -induced hypothyroidism, there is no research pertaining to the HPT axis, TH metabolism or activity of D2 in the BAT, a recognized site of energy homeostasis, in response to different sleep-deprivation protocols. Therefore, the aim of this study was to characterize serum thyroid hormones and TSH concentrations further in the settings of different sleep-deprivation protocols (sleep deprivation for 24 and 96 h and sleep restriction) as well as NIS and TPO activity in a sleep-deprivation protocol. We also tested whether a 24 h rebound period normalized any changes in these hormonal parameters.

Methods Animals

Male Wistar rats (200–250 g) from the Federal Rural University of Rio de Janeiro animal facility were used in this study. Throughout the study, the experimental room was kept at a constant temperature (22 ± 1°C), and a 12 h–12 h light–dark cycle (lights on at 07.00 h) was  used. Food (standard rat chow; Purina ) and water were R

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provided ad libitum. Animal handling and experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication no. 85-23, revised 1996). The experimental protocol was approved by the institutional committee of ethics and animal welfare (number of the protocol approved: 23083.006390/2010-34). Sleep-deprivation/restriction protocols

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for the D2 activity assay, and the same protocol was repeated for the NIS and TPO activity studies (n = 8 per group) in the PSD24 and PSD96 groups and in each rebound group. All animals were weighed at the beginning of the experiments, i.e. the day when the rats were placed into the tanks for the paradoxical sleep-deprivation or sleep-restriction protocol (basal body weight), at the beginning of the rebound experiments (when performed) and at the end of the experiment (final body weight). For the statistical propose of body weight gain, the final weight (day of killing) minus basal weight was considered.

The sleep-restriction (SR) and sleep-deprivation (SD) protocols were based on the modified multiple platform method. The paradoxical sleep-deprivation method used 10 narrow circular platforms (6.5 cm in diameter), protruding 1 cm above the surface in a 57 cm × 48 cm × 21 cm tank filled with water. Rats were placed on the platforms (five per tank) and could move around by leaping from one platform to another. When they reached the paradoxical sleep phase, they would fall into the water due to muscle atonia and awaken. The animals subjected to sleep restriction were removed from the tanks at 10.00 h and were allowed to sleep until 16.00 h, when they were again placed in the tanks; therefore, they were allowed 6 h of sleep per day.

Serum TSH levels were measured using a specific radioimmunoassay for rat TSH, which was obtained from the National Institute of Diabetes, Digestive and Kidney Diseases (Bethesda, MD, USA) and expressed in terms of reference preparation 3 (RP-3). Serum T3 and T4 were analysed via electrochemiluminescence (Clinical Laboratory of the Faculty of Pharmacy of the Federal University of Rio de Janeiro, Brazil) and expressed in nanograms per decilitre and micrograms per decilitre, respectively.

Groups

Iodothyronine deiodinase type 2 activity study

The rats were randomly assigned into the following five groups as part of the sleep-deprivation protocol: (i) control (n = 10) rats kept in the experimental room, with normal sleep patterns; (ii) paradoxical sleep deprivation for 24 h (PSD24, n = 12); (iii) PSD for 24 h, with a rebound period of 24 h (PSD24R, n = 12), which had the same protocol used for the PSD24 group but on the second day the animals were allowed to sleep; (iv) PSD for 96 h (PSD96, n = 10); and (v) PSD96 with a rebound period of 24 h (PSD96R, n = 12), which had the same protocol used for the PSD96 group but on the fifth day the animals were allowed to sleep. For the sleep-restriction protocol, the rats were randomly assigned into the following three groups: (i) control (n = 10) rats kept in the experimental room, with normal sleep patterns; (ii) sleep restriction for 21 days (SR21, n = 11), in which animals were allowed to sleep for 6 h a day (10.00–16.00 h); and (iii) SR21 with a sleep rebound of 24 h (SR21R, n = 15), which had the same protocol used for the S21 group but on 22nd day the animals were allowed to sleep. All animals were killed on the same day in the morning (between 07.00 and 09.00 h), and blood samples were collected to determine the T3 , T4 and TSH levels in animals from both the sleep-deprivation and the sleep-restriction protocols. Additionally, brown adipose tissue was excised

Iodothyronine deiodinase type 2 activity was assessed using a method published previously (Berry et al. 1991; Fortunato et al. 2006). The homogenate containing 50 mg of BAT was incubated in duplicate for 3 h at 37°C with 1 nM of 125 I-T4 , 1 mM optimal cutting temperature (OCT) compound and 20 mM DTT in 100 mM phosphate buffer containing 1 mM EDTA, in a final volume of 300 μl. Following incubation, the reaction was stopped by placing the tubes in an ice bath. Approximately 200 μl of fetal bovine serum was then added (Cultilab, Campinas, Sao Paulo, Brazil) and frozen in 100 μl of 50% trichloroacetic acid for protein precipitation. The tubes were shaken vigorously via vortex mixing for 2 min and centrifuged (11,963 g for 3 min, microfuge). Finally, 360 μl of supernatant was transferred to counting tubes, and radiation detection was performed using a Wizard scintillator (2470 WizardTM Wallac automatic gamma counter, Walthan, MA, USA). Enzyme activity was expressed in femtomoles of T4 per minute per milligram of protein.

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Serum TSH, T4 and T3 measurements

Sodium–iodide cotransporter and TPO activity assay

Sodium–iodide cotransporter activity was determined using a method published previously by Lima et al. (2006). To determine radioiodine uptake by the thyroid gland

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without assessing TPO activity, the animals received 100 μl of Na125 I (250,000 dpm) via I.P. injection; after 15 min, the animals were killed and their thyroid glands removed and weighed before being transferred to counting tubes for radiation detection by the Wizard scintillator (2470 WizardTM Wallac automatic gamma counter). Enzyme activity was expressed as the amount of 125 I injected per milligram of thyroid. To analyse TPO activity, the same protocols as those noted above were used, but the animals were killed 120 min following the I.P. injection of Na125 I (250,000 dpm). Enzyme activity was expressed as the amount of 125 I injected per milligram of thyroid. Statistical analysis

Results were expressed as the means ± SEM. Statistical analyses were performed using one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test, for T4 , T3 and D2 concentrations and NIS and TPO activity. The results for serum TSH, which did not have a normal distribution, were expressed as medians and minimal–maximal values and were analysed by non-parametric ANOVA (Kruskal–Wallis test) using  GraphPad Prism 4 (Graphpad Software, Inc., San Diego, CA, USA). The results were considered statistically significant when P < 0.05. R

Results Body weight gain

Figure 1A shows that the animals experienced significant decreases in body weight gain following sleep-deprivation periods of 24 and 96 h compared with control rats (25.2 ± 8.3 and −1.5 ± 17.5 versus 48.7 ± 11.6 g, respectively, P < 0.05). The rebound periods normalized body weight gain after 24 (29.5 ± 13.22 g, P > 0.05) but not after 96 h (−8.6 ± 12.9 g, P < 0.05) of sleep deprivation, compared with control animals. Figure 1B shows that sleep restriction for 21 days also decreased body weight gain (−1.0 ± 12.7 g, P < 0.05), and the rebound periods did not affect this change (−8.3 ± 11.5 g, P > 0.05), compared with the control group (48.7 ± 11.6 g). Serum T3 and T4 concentrations

Figure 2A shows that T3 increased in both the PSD24 (120.1 ± 5.57 ng dl−1 , P < 0.05) and PSD96 groups (116.9 ± 3.41 ng dl−1 , P < 0.05), and the rebound periods did not affect these changes (PSD24R, 108.0 ± 3.98 ng dl−1 and PSD96R, 117.8 ± 2.66 ng dl−1 ), compared with control animals (93.00 ± 2.16 ng dl−1 , P < 0.05). As shown in Fig. 2B, the sleep-restriction protocol also increased T3 levels (SR21 111.5 ± 2.93 ng dl−1 , P < 0.05),

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which were not affected by the rebound period (RS21R, 112.4 ± 2.52 ng dl−1 ), compared with control rats (93.00 ± 2.16 ng dl−1 , P < 0.05). Figure 3A shows that T4 decreased following paradoxical sleep deprivation for 24 (3.82 ± 0.15 μg dl−1 ) and 96 h (4.56 ± 0.31 μg dl−1 ) compared with control animals (5.30 ± 0.15 μg dl−1 , P < 0.05). The rebound period normalized T4 levels following 24 h of sleep deprivation (PSD24R, 5.53 ± 0.15 μg dl−1 versus control, 5.30 ± 0.15 μg dl−1 , P > 0.05); however, the rebound period did not affect the changes in serum T4 concentrations induced by 96 h of sleep deprivation (PSD96R, 4.59 ± 0.18 μg dl−1 versus control, 5.30 ± 0.15 μg dl−1 , P < 0.05). Serum T4 concentrations were not affected by the sleep-restriction methods used herein (control 5.30 ± 0.15 μg dl−1 , SR21 5.37 ± 0.23 μg dl−1 and RS21R 5.21 ± 0.15 μg dl−1 , P > 0.05). Serum TSH concentration

Serum TSH decreased in the PSD24 (0.61 ± 0.07 μg dl−1 ) and PSD96 groups (0.73 ± 0.14 μg dl−1 ) compared with control rats (1.69 ± 0.13 μg dl−1 , P < 0.05). Although low concentrations of TSH persisted in the PSD24R group (0.67 ± 0.12 μg dl−1 , P < 0.05), this parameter normalized in the PSD96R group (1.61 ± 0.10 μg dl−1 , P > 0.05) compared with control rats (1.69 ± 0.13 μg dl−1 ). These data travelled in opposite directions relative to the T4 data, as normal and lower serum thyroxine concentrations were noted in the PSD24R and PSD96R groups, respectively, compared with control animals (Fig. 4A). In other words, rebound periods following 24 h of sleep deprivation normalize T4 , but not TSH. However, rebounds following longer periods of sleep deprivation (96 h) do not normalize T4 , but return TSH to its normal range. Thyroid-stimulating hormone levels were not altered in response to the sleep-restriction protocol (P > 0.05 for SR21 and SR21R versus control, Fig. 4B). Brown adipose tissue D2 activity

To study the mechanisms underlying the alterations to thyroid hormone economy induced by the sleep-deprivation model used herein, an important thyroid hormone activation pathway was assessed. As shown in Fig. 5, BAT D2 activity increased significantly in the PSD24 [10.38 ± 3.96 fmol T4 min−1 (mg protein)−1 ] and PSD96 groups [11.79 ± 2.41 fmol T4 min−1 (mg protein)−1 ] compared with control animals [1.57 ± 0.63 fmol T4 min−1 (mg protein)−1 , P < 0.05]. These data suggest, at least in part, the low T4 and high T3 concentrations observed in these groups (Figs 2A and 3A).  C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

Brown adipose tissue D2 activity normalized fully in the PSD24 group [0.26 ± 0.12 fmol T4 min−1 (mg protein)−1 , P > 0.05] and partly normalized in the PSD96 group [5.95 ± 2.55 fmol T4 min−1 (mg protein)−1 ] compared with control rats [1.57 ± 0.63 fmol T4 min−1 (mg protein)−1 ]. The differences between the PSD24R and PSD96R groups may partly explain the normal and low T4 concentrations noted in the PSD24R and PSD96 groups, respectively. Sodium–iodide cotransporter and TPO activity

There were no differences in NIS activity in the sleep-deprived groups [PSD24 0.04 ± 0.008 125 I (mg thyroid)−1 and PSD96 0.05 ± 0.002 125 I (mg thyroid)−1 ] or the rebound groups [PSD24R 0.05 ± 0.004 125 I (mg

thyroid)−1 and PSD96R 0.05 ± 0.002 125 I (mg thyroid)−1 ] compared with control animals [0.05 ± 0.003 125 I (mg thyroid)−1 , P > 0.05]. Likewise, the TPO activity was not different among the groups (data not shown).

Discussion The most important findings of this study are that the sleep-deprivation model alters HPT physiology and thyroid hormone economy and also profoundly influences the extrathyroidal metabolism of iodothyronines. To the best of our knowledge, this is the first study to demonstrate that REM sleep plays a role in TSH secretion in rats. Based on our findings, REM sleep deprivation induces central hypothyroidism, which reduces TSH secretion

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Figure 1. Body weight gain (in grams) in the control, PSD24, PSD24R, PSD96 and PSD96R groups (A) and the control, SR21 and SR21R groups (B) Groups are as follows: control, paradoxical sleep deprivation (PSD) for 24 or 96 h, 24 or 96 h of sleep deprivation with rebound (PSD24R and PSD96R), sleep restriction (SR) for 21 days (SR21) and SR21 with rebound (SR21R). Means followed by different letters are significantly different (P < 0.05). Statistical analyses were performed using one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test.

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Figure 2. Serum 3,5,3 -triiodothyronine (T3 ) levels in the control, PSD24, PSD24R, PSD96 and PSD96R groups (A) and the control, SR21 and SR21R groups (B) Means followed by different letters are significantly different (P < 0.05). Statistical analyses were performed using one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test.

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and circulating T4 levels. Sleep-deprivation-induced hypothyroidism is accompanied by the powerful activation of D2 in BAT and other tissues not assessed in the present study, which subsequently results in high circulating T3 levels. The two previous studies that assessed the HPT axis following sleep deprivation in rats were undertaken by Everson & Nowak (2002) and Balzano et al. (1990), who also demonstrated reductions in serum T4 levels, but not in TSH, in sleep-deprived animals. In the study by Everson & Nowak (2002), the central hypothyroidism induced by total sleep deprivation was attributed to a lack of TRH secretion, which affected the TSH response to hypothyroxinaemia. In spite of the differences in our sleep-deprivation protocols (total versus REM-selective deprivation), we also failed to demonstrate a TSH response to the low T4 concentration in our REM

sleep deprivation model. In fact, TSH was reduced after our sleep-deprivation model in both the 24 and the 96 h groups. It is important to note that T3 was increased in our protocol; therefore, high T3 levels may also contribute to the inhibition of the HPT axis and partly explain the lack of a hypothyroxinaemia-induced TSH response. The accelerated D2 activity in BAT noted in the present study increased the systemic fractional conversion of T4 to T3 , which may have caused the elevated serum T3 and decreased serum T4 levels. We did not observe any alterations in TSH and T4 secretion in the rats subjected to the sleep-restriction protocol. Although we have no explanation for the differential HPT responses to our restriction and deprivation approaches, it appears that the type of protocol used may influence thyroid hormone physiology. Therefore, longer periods of sleep deprivation (21 days of

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Figure 3. Serum thyroxine (T4 ) levels in the control, PSD24, PSD24R, PSD96 and PSD96R groups (A) and the control, SR21 and SR21R groups (B) Means followed by different letters are significantly different (P < 0.05). Statistical analyses were performed using one-way ANOVA, followed by the Bonferroni multiple comparison post hoc test.

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sleep restriction) and daily periods of sleep (rats being allowed to sleep 6 h day−1 ) may induce physiological adaptations in the HPT axis to maintain serum T4 and TSH levels within the normal range, despite the high circulating T3 levels observed in these rats. Curiously, it has already been demonstrated that athyreotic patients often have a normal TSH and T4 and a highly heterogeneous capacity for T3 production after receiving oral levothyroxine (Gullo et al. 2011). The results of studies concerning the peripheral production of T3 by the iodothyronine deiodinases are contradictory. Using the gyratory platform method, Balzano et al. (1990) observed increased BAT D2 activity, which corroborates our results. Although we have no data to support the mechanism underlying the high levels of D2 activity observed our model, increased sympathetic activity, a powerful modulator of BAT D2 activity (Bianco & McAninch, 2013), may be one of the most important mechanisms involved. Indeed, Sgoifo et al. (2006) showed that subjecting rats to 48 h of sleep deprivation by placing them in slowly rotating wheels produced a tonic increase of heart rate together with an increased susceptibility to cardiac arrhythmias and higher sympathetic activity compared with control animals. Also, T3 upregulates the BAT D2 gene, thereby increasing BAT D2 activity (Hall et al. 2010). Therefore, high T3 levels may explain the BAT D2 activity noted in our model (Machado et al. 2005). Curiously, Balzano et al. (1990) did not observe any alterations in circulating T3 levels, and Everson & Nowak (2002) observed a decrease in serum T3 levels. Perhaps differences in sleep-deprivation methods and experimental protocols may explain these controversial

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data. Animals were selectively deprived of or restricted from receiving normal amounts of REM sleep in our model, whereas other models deprived animals of sleep altogether. Information regarding the correlation between thyroid function and acute or chronic stress has been published extensively in recent years. In spite of its recognized role in growth, differentiation and metabolism, the HPT axis has also been linked to psychiatric diseases (Helmreich et al. 2005), particularly in cases of HPT dysfunction. Many studies have suggested that the type of stressor, as well as its duration, is related to changes in thyroid function. Unfortunately, many of the results of these studies are inconclusive and contradictory. For example, while Takanuve et al. (1994) observed that stress reduced serum thyroid hormone levels, Cizza et al. (1996) observed the opposite. Stress induced by paradoxical sleep deprivation increases serum levels of corticosterone (Andersen et al. 2004, 2005; Machado et al. 2005). Therefore, although the modulatory effects of corticosterone on thyroid function are difficult to standardize, high levels of glucocorticoids may partly explain the low TSH and high BAT D2 and serum T3 levels observed in our stress model. Indeed, Kakucksa et al. (1995) previously demonstrated that administration of glucocorticoids inhibits the hypothalamic–pituitary axis, and cortisol has long been linked to increases in the numbers of D2 astrocytes in rats (Courtin et al. 1989). Of course, additional studies are necessary to provide a better understanding of the relationship between corticosterone and the thyroid gland noted in our model. Paradoxical sleep deprivation and sleep restriction increase sympathetic activity (Perry et al. 2011), thereby increasing metabolic rates and decreasing body weight gain (Machado et al. 2005). Thyroid hormone and noradrenaline signalling pathways are interconnected and affect each other, in addition to contributing to energy expendure (Bianco & McAninch, 2013); our study confirms these data and postulates that high circulating T3 levels may also be a mechanism underlying body weight loss induced by sleep deprivation in rats. Another important contribution of this project was the inclusion of a rebound period following the sleep-deprivation periods. This approach was used to determine whether a recovery period (24 h) mitigated the alterations to the thyroid system induced by acute and chronic sleep deprivation. We demonstrated that serum levels of T4 sharply decreased following sleep-deprivation periods of 24 and 96 h; the rebound period normalized T4 levels following only 24 h of sleep deprivation. These data suggest that 24 h of recovery (rebound) was sufficient to normalize TSH secretion, but not levels of circulating T4 following 96 h of sleep deprivation. Perhaps low TSH bioactivity may explain these data. Indeed, proper

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TSH glycosylation is necessary to achieve normal TSH bioactivity (Amir et al. 1987), a process that requires the interaction of TRH with its receptor on the thyrotroph (Baquedano et al. 2010). The requirement for TRH in this process is illustrated by the fact that patients with central hypothyroidism secondary to hypothalamic–pituitary dysfunction may exhibit normal or even slightly elevated levels of radioimmunoassayable but biologically impotent TSH in the presence of reduced free T4 (Baquedano et al. 2010). It is important to remember that sleep deprivation reduces TRH secretion in rats (Everson & Nowak, 2002). A rebound period of the same amount of time (24 h) did not result in normal TSH secretion following a shorter period of sleep deprivation (24 h). Curiously, normal T4 levels were observed in these rats. These data suggest a powerful acute desensitization of the HPT axis associated with robust peripheral adjustments in T4 content. Indeed, BAT D2 activity reached its lowest level during the rebound period following 24 h of sleep deprivation (40 times lower in the PSD24R group than in the PSD24 group). Decreased T4 clearance should not be ruled out as a possible explanation for the T4 normalization observed in this model. Future studies assessing not only D2 but also D1 and D3 activity are necessary to provide a better understanding of the mechanism underlying thyroid hormone metabolic adjustments that occurs during the rebound period following sleep deprivation in rats. The body weight reduction noted in sleep-deprived rats has already been well described (Suchecki et al. 2003; ´ Hipolide et al. 2006; Martins et al. 2010). Significant energy expenditure, central hypothyroidism notwithstanding, is the most plausible explanation for these findings, because high circulating T3 increases energy expenditure, thereby decreasing body weight (Bianco & McAninch, 2013). We have no explanation for the ability of the rebound period to normalize body weight gain and T4 only in the PSD24R group in spite of noting sustained increased T3 levels in both protocols. However, we observed that T3 generated from BAT D2 was more dramatically reduced in the PSD24R group (40-fold lower) than in the PSD6R group (2-fold lower) compared with their respective sleep-deprived groups (PSD24 and PSD96, respectively). Therefore, the striking reduction in local T3 signalling in target tissues (including BAT) noted in the PSD24R group may explain (at least partly) the body weight normalizations observed in this group. This is the first time that thyroid hormone physiology/metabolism has been assessed in selective REM-sleep-deprived rats. Our data suggest that the thyroid gland adapts to sleep deprivation-induced central hypothyroidism by increasing T4 to T3 activation peripherally (in brown adipose tissue and possibly other

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tissues), thereby increasing circulating T3 . When the sleep is restricted (not deprived), even over longer intervals (21 days), the HPT axis normalizes TSH and T4 secretion in spite of persistently increased levels of circulating T3 . The results of the rebound protocols, the protocols in which rats deprived of sleep were subsequently allowed to sleep for 24 h, indicate that this recovery period elicits different HPT responses depending on the length of deprivation (24 or 96 h). Rebound does not normalize persistently high T3 levels, which may be necessary to maintain normal T3 signalling, particularly in tissues characterized by low T4 to T3 conversion (low BAT D2 activity). Therefore, metabolism and temperature may be maintained within a range compatible with life in mammals subjected to homeostatic challenges. Further studies are necessary to clarify the mechanisms underlying the peripheral and central modulation of thyroid function in a rat model of sleep deprivation and restriction.

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Additional information Competing interests None declared.

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Author contributions

Acknowledgements

The experiments were performed in the Federal Rural University of Rio de Janeiro at the Behavioral laboratory, and the contribution of each author is as follows. Conception and design of the experiments: A.C.M.S., E.L.O. and M.P.M. Collection, analysis and interpretation of data: N.C.R., N.S.C., C.P.N., R.R.C., A.C.M.S., E.L.O. and M.P.M. Drafting the article or revising it critically for important intellectual content: N.C.R., N.S.C., C.P.N., R.R.C., A.C.M.S., E.L.O. and M.P.M. All authors approved the final version of the manuscript, all persons designated as authors qualify for authorship, and all those who qualify for authorship are listed.

The authors are grateful for the financial support of the National Council for Scientific and Technological Development (Conselho Nacional de Desenvolvimento Cient´ıfico e ´ Tecnologico, CNPq) and the Foundation for Research Support of the State of Rio de Janeiro (Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro, FAPERJ). Postgraduate students N.C.R., N.S.C. and C.P.N. received a scholarship from the Brazilian Federal Agency for the Support and Evaluation of Graduate Education (Coordenac¸a˜o de Aperfeic¸oamento de Pessoal de N´ıvel Superior, CAPES) during this study.

 C 2014 The Authors. Experimental Physiology  C 2014 The Physiological Society

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Sleep deprivation alters thyroid hormone economy in rats.

What is the central question of this study? The relationship between the thyroid system and sleep deprivation has seldom been assessed in the literatu...
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