Abstract
Sirtuin 1 (SIRT1), a NAD+-dependent deacetylase, has been connected to beneficial effects elicited by calorie restriction. Physiological adaptation to starvation requires higher activity of SIRT1 and also the suppression of thyroid hormone (TH) action to achieve energy conservation. Here, we tested the hypothesis that those two events are correlated and that TH may be a regulator of SIRT1 expression. Forty-eight-hour fasting mice exhibited reduced serum TH and increased SIRT1 protein content in liver and brown adipose tissue (BAT), and physiological thyroxine replacement prevented or attenuated the increment of SIRT1 in liver and BAT of fasted mice. Hypothyroid mice exhibited increased liver SIRT1 protein, while hyperthyroid ones showed decreased SIRT1 in liver and BAT. In the liver, decreased protein is accompanied by reduced SIRT1 activity and no alteration in its mRNA. Hyperthyroid and hypothyroid mice exhibited increases and decreases in food intake and body weight gain respectively. Food-restricted hyperthyroid animals (pair-fed to euthyroid group) exhibited liver and BAT SIRT1 protein levels intermediary between euthyroid and hyperthyroid mice fed ad libitum. Mice with TH resistance at the liver presented increased hepatic SIRT1 protein and activity, with no alteration in Sirt1 mRNA. These results suggest that TH decreases SIRT1 protein, directly and indirectly, via food ingestion control and, in the liver, this reduction involves TRβ. The SIRT1 reduction induced by TH has important implication to integrated metabolic responses to fasting, as the increase in SIRT1 protein requires the fasting-associated suppression of TH serum levels.
Introduction
Calorie deprivation has been proved to extend life span and reduce the rate of aging in several organisms, from yeast to rodents and primates (Weindruch & Walford 1982, Lin et al. 2000, Colman et al. 2009). It was described in some species that the mechanism by which calorie restriction promotes extended life span was dependent on silent information regulator 2 (Sirtuin 1 (Sir2)) proteins (Lin et al. 2000, Rogina & Helfand 2004). However, a recent report described that the apparent life span extension effect in both Caenorhabditis elegans and Drosophila was independent of Sir2 (Burnett et al. 2011). In mammals, a similar role has been postulated for the Sir2 homolog, SIRT1, because of important regulatory actions on metabolism (Allard et al. 2008, Boily et al. 2008, Redman et al. 2011).
The SIRT1 protein is a NAD+-dependent deacetylase that removes acetyl groups from acetyllysine-modified proteins, such as histone (Vaquero et al. 2004) and non-histone proteins, like nuclear transcription factors (Vaziri et al. 2001, Nemoto et al. 2005, Li et al. 2007) and also cytosolic enzymes (Hallows et al. 2006, Mattagajasingh et al. 2007, Akieda-Asai et al. 2010). The deacetylation of histones provokes repression of gene transcription, whereas SIRT1 action on transcription factors and enzymes results in modulation of their functions. As the deacetylation mechanism requires NAD+, the enzyme activity is influenced by cytosolic NAD+/NADH ratio perturbations (Tanny & Moazed 2001), connecting SIRT1 activity to cellular metabolism.
SIRT1 is expressed in several tissues, such as liver, adipose tissues, muscle, pancreas, pituitary, and hypothalamus. Tissue-specific deletion or overexpression of SIRT1, pharmacological activators of SIRT1, and in vitro approaches have been used to clarify the roles of SIRT1 in each cell type. SIRT1 has been associated with decreased white fat depot accumulation (Picard et al. 2004), increased hepatic and muscular lipid catabolism (Feige et al. 2008, Purushotham et al. 2009), increased gluconeogenesis (Rodgers & Puigserver 2007), and improvement of insulin sensitivity (Sun et al. 2007, Feige et al. 2008, Pfluger et al. 2008). These punctual contributions are extremely valuable to understand the essential role of SIRT1 in adaptation to calorie restriction and fasting, conditions that are able to trigger SIRT1 protein expression in various tissues (Cohen et al. 2004, Kanfi et al. 2008), by still unknown mechanisms. On the contrary, consumption of an excessive high-fat diet is generally accompanied by reduced SIRT1 levels (Deng et al. 2007), and, if SIRT1 expression or activity is restored, adverse effects induced by high-fat diet ingestion are attenuated (Baur et al. 2006, Feige et al. 2008, Pfluger et al. 2008). However, at this point in time, our knowledge on SIRT1 protein expression regulation is limited.
Similar to SIRT1, thyroid hormones (THs) are major regulators of fuel metabolism (Yen 2001). Tri-iodothyronine (T3) acts mainly through nuclear TH receptors (TR), which modulate target genes' expression (Lazar 2003). Nuclear proteins also participate in this process, such as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a cofactor involved in both TH and SIRT1 effects (Rodgers et al. 2005, Attia et al. 2010). Beyond that, confronting SIRT1 and TH actions, we will come across common targets, especially in lipid metabolism (Ness et al. 1990, Rodgers et al. 2005, Hashimoto et al. 2006, Li et al. 2007).
Moreover, TH is a major determinant of the basal metabolic rate of cells, accelerating metabolic processes that lead to increases in energy expenditure (Kim 2008). It is very well established that physiological adaptation to fasting involves the suppression of TH production, as a mechanism to conserve energy (Boelen et al. 2008). In addition, recent studies have shown the fasting-induced rise in SIRT1 expression in several tissues (Rodgers et al. 2005, Kanfi et al. 2008). Therefore, the existence of common metabolic routes regulated by TH and SIRT1, together with the inverse correlation of these factors' variation during calorie deprivation, led us to investigate whether SIRT1 expression is regulated by TH in mice and the possible interactions of TH and SIRT1 during energy deprivation.
Materials and methods
Animal care and treatments
All procedures were approved by Ethics Committee on Animal Care of the Health Sciences Center, Federal University of Rio de Janeiro (protocol: IBCCF 120). The regulation followed by the local committee is endorsed by a federal Brazilian law and is in accordance with the guidelines published in ‘ARRIVE’ ‘Care and Use of Animals’. Animals were maintained in a controlled temperature room, with 12 h light:12 h darkness cycles (lights on from 0700 till 1900 h), and experiments were performed when male mice (mixed background strain 129Svj/C57BL/6) reached ∼3 months of age. Mice were killed by decapitation after CO2 sedation, and liver and brown adipose tissue (BAT) tissues were excised and stored at −70 °C until total RNA and protein extraction procedures. Serum was obtained from trunk blood and kept frozen at −20 °C for measurements of hormones and cholesterol.
Fasting and thyroxine reposition
The fasting protocol consisted of submitting the animals to 48-h starvation. Some animals were only exposed to fasting, while others also received s.c. injections of thyroxine (T4 – reposition dose: 0.75 μg/100 g body weight per day, Sigma; for 2 days, starting 8 h after food withdrawal). Fasting mice and T4-replaced fasting mice were compared to mice fed chow ad libitum, and all animals that did not receive T4 reposition were submitted to saline injections to mimic stress.
Hypo- and hyperthyroidism models
Mice were rendered hypo- or hyperthyroid with specific protocols. Hypothyroidism was induced by feeding animals a diet containing 0.15% 5-propyl-2-thiouracil (PTU, Sigma) for 28 days and hyperthyroidism was induced by daily s.c. injections of T3 (supraphysiological dose: 50 μg/100 g body weight per day, Sigma) for 14 days.
Hyperthyroidism and food restriction
A pair-fed protocol was performed using euthyroid and hyperthyroid mice. Serum TH excess promotes increased food intake, thus one group of hyperthyroid mice was pair-fed to euthyroid ones, named hyperthyroid-restricted. And, for comparison, one group of euthyroid animals was submitted to the same percentage of food restriction as the hyperthyroid-restricted group and called euthyroid restricted. Hyperthyroidism was induced by daily s.c. injections of T3 (50 μg/100 g body weight per day) for 13 days, which started simultaneously to the pair-fed protocol. The amount of food offered for the hyperthyroid group was the same as the mean food intake of the euthyroid group on the previous day. Mean food ingestion of euthyroid and hyperthyroid mice was significantly different from the fifth day on T3 treatment.
Transgenic TRβΔ337T model
In addition, we analyzed a transgenic mice model bearing a dominant negative mutation (Δ337T) in TRβ, described earlier (Hashimoto et al. 2006). The mutant TRβ is unable to interact with T3 but can bind to DNA and to nuclear cofactors. As TRβ participates actively in the hormonal feedback regulation, these mice are resistant to TH and exhibit elevated serum T3 and T4. Homozygous mice used in this study were acquired by heterozygous mating pairs, and the genotype was determined by analysis of DNA from the tail employing PCR, using the following primers: 5′-ATGGGGAAATGGCAGTGAGAG (match), 5′-ATGGGGAAATGGCAGTGACAC (mismatch) and 3′-AGCACACTCACCTGAAGACAT (out) (Santiago et al. 2011). Direct comparisons were made with littermate controls (WT).
Sirt1 mRNA and protein analysis
Total RNA was isolated from liver using Trizol reagent (Invitrogen Corp.). Subsequently, 1 μg of total RNA was submitted to reverse transcription using Superscript III kit (Invitrogen Corp.). Products were amplified on Applied Biosystems 7500 Real-Time PCR System (Life Technologies Co.) using iQ SYBR Green Supermix. Cycle parameters were 50 °C for 2 min and 95 °C for 10 min, followed by 44 cycles at 95 °C for 15 s, 53 °C for 30 s, and 72 °C for 45 s.
The intron spanning primers used in this manuscript have been described earlier by our own group and by others (Rodgers et al. 2005, Akamine et al. 2007, Machado et al. 2009, Paula et al. 2010, Faustino et al. 2011, 2012, Santiago et al. 2011). Sirt1 primers: forward, 5′-CAG-GTT-GCA-GGA-ATC-CAA-A-3′; and reverse, 5′-CAA-ATC-AGG-CAA-GAT-GCT-GT-3′ (Rodgers et al. 2005). 36B4 (Rplp0, Genbank
Efficiency of each reaction was calculated using a serial dilution and varied from 94% for SIRT1 to 98% for 36B4, and products' purity was confirmed by agarose gel analysis. Both primer sets were verified to provide a single peak on a melting curve analysis. Relative mRNA expression was calculated by comparing the PCR cycle threshold (Ct) between the groups, after correcting for 36B4 using the ΔΔCt method (Livak & Schmittgen 2001). Results are expressed relative to values of control group, which was set to 1.
Protein expression was determined by western blotting. Liver and BAT samples were homogenized in lyses buffer (pH 6.4; 50 mM HEPES, 1 mM MgCl2, 10 nM EDTA, and 1% Triton X) with protease inhibitor cocktail Complete (Roche/DSM Nutritional). Thirty micrograms of total protein extract of each sample were resolved by SDS–PAGE on 10% gel and transferred onto a polyvinylidene difluoride membrane (Westran; Whatman, Maidstone, Kent, UK). The membrane was blocked with 5% nonfat dry milk (Molico; Nestle, São Paulo, SP, Brazil) and incubated overnight at room temperature with anti-SIRT1 polyclonal antibody (1:500 dilution; Santa Cruz Biotechnology) and anti-cyclophilin antibody (1:70 000 dilution for liver and 1:6000 for BAT, Affinity Bioreagents, Golden, CO, USA), used as internal control. Membranes were then washed and incubated with peroxidase-labeled anti-rabbit IgG antibody (1:7500 dilution; Amersham Biosciences) for 3 h at room temperature. All blots were then washed and incubated with a luminogen detection reagent (ECL; Amersham Biosciences) for further exposure on an autoradiograph film (Kodak). Protein bands were evaluated by densitometry using the software Kodak 1D3.5. The membranes were stained with Rouge Ponceau and submitted to densitometry analysis. Densities of protein bands were normalized for that of cyclophilin and also for that of Ponceau staining and they were expressed in relation to control groups. Both methods of loading correction gave the same results.
SIRT1 activity
SIRT1 activity was measured in liver samples, processed to obtain nuclear extract as described earlier (Setsuie et al. 2009). Briefly, frozen livers were ground into powder using liquid nitrogen and suspended in lysis buffer (10 mM Tris–HCl, pH 7.5; 10 mM NaCl; 15 mM MgCl2; 250 mM sucrose; 0.5% Nonidet P-40; and 0.1 mM EGTA) in the absence of protease inhibitor. This preparation was submitted to a sucrose cushion (30% sucrose; 10 mM Tris–HCl, pH 7.5; 10 mM NaCl; and 3 mM MgCl2) and centrifuged at 1300 g for 10 min. The pellet was washed once (10 mM Tris–HCl, pH 7.5) and suspended in extraction buffer (50 mM HEPES-KOH, pH 7.5; 420 mM NaCl; 0.5 mM EDTA; 0.1 mM EGTA; and 10% glycerol). The suspension was centrifuged at 15 000 g for 10 min, and supernatant was stored in −70 °C. Forty micrograms of nuclear extracts were used for Sirt1 activity analysis using commercial kit CycLex SIRT1/Sir2 Deacetylase Fluorometric Assay (MBL International, Woburn, MA, USA), following manufacturer's recommendations. The assay was based on SIRT1 ability to deacetylate a fluorometric substrate, in the presence of NAD+, and the reaction product was detected using a fluorescence plate reader (Viktor X2: excitation, 355 nm; emission, 460 nm). Measurements were recorded every 2 min, and the 30-min record was used for comparisons.
Serum measurements
Serum T3 and T4 concentrations were determined using a commercial RIA kit (ICN Pharmaceuticals, Costa Mesa, CA, USA) according to the manufacturer's instructions. Serum TSH was measured using a specific mouse TSH RIA, with reagents acquired from the National Hormone and Pituitary Program (Torrance, CA, USA), as detailed previously (Oliveira et al. 2006). The minimum assay detection value was 25 ng/dl for T3, 1 μg/dl for T4, and 30 ng/ml for TSH. The samples that were compared were analyzed in the same assay and the intra-assay variation was <6%. Serum cholesterol was determined using commercial kit (Applied Biosystems) following the recommendations of the manufacturer.
Statistical analyses
Results are presented as mean±s.e.m. One-way ANOVA, followed by the Student–Newman–Keuls multiple comparisons test, was used for comparisons between three or more groups. The Student's t-test was used for comparisons between two groups (data from the mutant TR mice). For TSH analysis, nonparametric tests were employed: Kruskal–Wallis or Mann–Whitney test. The analysis was performed using the software GraphPad Prism (GraphPad Software, Inc., San Diego, CA, USA). Differences were considered to be significant at P<0.05.
Results
Increased SIRT1 expression induced by fasting depends on TH suppression
As fasting is associated with suppression of TH secretion and to increased SIRT1 protein expression, we wondered about a possible relationship between these two events. Expectedly, 48 h-fasted mice presented lower serum TH levels related to fed animals, T4 concentration was below detection level, and serum T3 was reduced by 26% (Fig. 1D, P<0.01). As shown in Fig. 1A, compared with fed mice, SIRT1 protein content was 21% higher in the liver of 48-h-fasted mice (P<0.005), whereas T4 replacement in physiological dose prevented the fasting-induced increment in SIRT1 protein levels. SIRT1 activity mirrored the protein expression profile, being increased by 28% in fasted animals (Fig. 1C, P<0.01) and similar to fed mice in the liver of fasted mice replaced with T4. These results indicate that hepatic SIRT1 protein expression induced by fasting is dependent on the decline of serum TH. In BAT, fasting promoted a 2.6-fold increase in SIRT1 expression (Fig. 1B, P<0.01), and T4-replaced fasting mice presented intermediary values of SIRT1 expression, between fed and fasted groups, being statistically different from both. Thus, BAT SIRT1 protein regulation in fasting is partly dependent on reduction of TH levels, but other mechanisms may contribute.
TH negatively regulates SIRT1 protein expression and activity
To further investigate the role of TH in the regulation of SIRT1 expression, we studied hypothyroid and hyperthyroid mice. As depicted in Table 1, serum concentrations of TH and TSH, as well as the evaluated physiological parameters, were consistent with pharmacological modification of thyroid status. In hypothyroid mice, total serum T3 was reduced by 52%, T4 concentration was undetectable, and TSH levels were 81-fold higher than in euthyroid group. T3-treated mice exhibited T3 concentrations about three times higher than the euthyroid group, while T4 and TSH were below the detection limit. As expected, serum cholesterol levels were increased in hypothyroid mice and decreased in hyperthyroid ones. Food intake was diminished in hypothyroidism (∼33%) and augmented in hyperthyroidism (∼25%) in relation to the euthyroid group. The body weight gain during treatment was about 25% for hyperthyroid, 10% for euthyroid mice, and 0.01% for hypothyroid group.
Physiological parameters of hypo-, eu-, and hyper-thyroid mice. Data are presented as mean±s.e.m.
HYPO | HYPO EU | HYPER | P value | |
---|---|---|---|---|
T3 (ng/dl) | 56.74±5.66a | 107.8±4.32 | 352.8±35.96a | <0.001a |
T4 (μg/dl) | ND | 1.55±0.15 | ND | – |
TSH (ng/ml) | 8.713±10a | 107.5±10.7 | – | <0.0001a |
Cholesterol (mg/dl) | 141.50±3.63a | 127.30±3.63 | 98.17±4.85a | <0.0001a |
Food intake (g/day) | 4.03±0.19a | 6.11±0.22 | 7.62±0.60a | <0.001a |
Body weight variation (g) | −0.03±0.65a | 2.34±0.84 | 5.88±0.33a | <0.0001a |
HYPO, hypothyroid; HYPO EU, euthyroid; HYPER, hyperthyroid.
vs euthyroid. n=8–9 mice per group.
SIRT1 protein content in liver and BAT of hypo- and hyperthyroid mice are depicted in Fig. 2. Liver SIRT1 protein content was increased in hypothyroid and decreased in hyperthyroid mice related to euthyroid ones (Fig. 2A, P<0.0001). SIRT1 expression was diminished in BAT of hyperthyroid animals when compared with euthyroid and hypothyroid groups (Fig. 2D, P<0.01). Therefore, the protein expression of SIRT1 was downregulated by TH in tissues such as liver and BAT and upregulated by TH deficiency in the liver. As hepatic SIRT1 expression was differently altered in the absence and excess of TH, we also verified a significant negative correlation between SIRT1 content in the liver and serum T3 levels (data not shown, r=−0.78, P<0.001). Furthermore, as shown in Fig. 2B, liver SIRT1 activity mirrored the protein content, being significantly higher in hypothyroid than in hyperthyroid mice (P<0.05).
To have some insight into the mechanism underlying the regulation of SIRT1 expression by TH, we proceeded with the investigation at the mRNA level. Liver Sirt1 mRNA was not significantly modified by hypo- or hyperthyroidism (Fig. 2C).
Both TH level alterations and their secondary effect on food ingestion have a role in SIRT1 regulation
Hyperthyroidism and hypothyroidism were associated with increased and decreased food intake and body weight respectively (Table 1). Therefore, to evaluate the participation of an indirect effect of ingestion and weight gain variations on SIRT1 expression, we studied pair-fed groups. Mice were submitted to daily saline or T3 injections simultaneously to pair-fed protocol. As depicted in Fig. 3C, euthyroid-restricted mice exhibited decreased serum T3 (P<0.005), while T4 concentration reduction did not reach statistical significance. As expected, serum T3 was increased and T4 levels were undetectable in hyperthyroid (T3-treated) mice and food restriction did not alter the hormonal profile of hyperthyroid mice. The hyperthyroid mice's food ingestion was about 40% higher than that of euthyroid ones (Fig. 3B, P<0.0001). The hyperthyroid-restricted group received the same amount of chow as the euthyroid mice, and euthyroid-restricted group was submitted to the same percentage of chow restriction (40%). Hyperthyroid mice gained weight from the beginning till the end of the treatment (11% increase), while food restriction in hyperthyroid and euthyroid mice induced a similar weight loss of about 18 and 13% respectively (Fig. 3A, P<0.0001 vs free-feeding controls). Euthyroid-restricted mice exhibited no alteration in liver SIRT1 content in relation to euthyroid free-fed mice. As expected, liver and BAT SIRT1 expression was decreased in hyperthyroid mice with free access to food (Fig. 3D and E, P<0.01). However, hyperthyroid animals pair-fed to euthyroid ones presented intermediary expression between hyperthyroid and euthyroid groups, not being statistically different from any of them. These data indicate that TH-induced increases in food ingestion and body weight are only partly responsible for the SIRT1 protein expression regulation observed in hyperthyroid mice.
SIRT1 regulation by TH involves TRβ
Considering that TRβ is the major TR isoform expressed in the liver, with an essential role in the regulation of hepatic metabolism, we reasoned that TRβ might participate in SIRT1 regulation. Therefore, we employed an animal model of transgenic mice that possesses the germline mutation TRβΔ337T, which abrogates the ability of the receptor to bind T3, leading to resistance to TH action. Because of the resistance to TH at hypothalamus and pituitary, these animals have high serum TH (Fig. 4D, P<0.0001), but even so, the liver protein content of SIRT1 in homozygous mutant mice has the same pattern as in hypothyroid animals, being 26% increased in comparison with WT mice (Fig. 4A, P<0.05). SIRT1 activity was also 40% augmented in homozygous mice (Fig. 4B, P<0.05) and, at the mRNA level, Sirt1 expression was similar between groups (Fig. 4C). These results are consistent with the hypothesis that SIRT1 protein expression is downregulated by TH in the liver by a mechanism involving TRβ.
Discussion
SIRT1 and THs are major regulators of the metabolism in response to energy availability. This study shows that increased TH levels in mice reduce SIRT1 protein expression in liver and BAT and that fasting-induced suppression of TH axis plays a major role in the concomitant elevation of SIRT1.
The physiological relevance of the negative regulation of SIRT1 protein abundance by TH can be envisaged by the results on experiments in fasting mice. Nutritional limitation is accompanied by activation of specific metabolic pathways, which ensure adaptation and survival to these conditions. We observed that 48-h-fasting induced decreased T3 and T4 levels and increased hepatic SIRT1 content. Other reports in the literature have already described, independently, fasting-induced decreased TH levels (Diano et al. 1998, Hashimoto et al. 2001) and hepatic SIRT1 content increment (Rodgers et al. 2005, Kanfi et al. 2008). Reduced TH concentration serves to conserve energy, as these hormones are important stimulators of basal metabolism and thermogenesis (Klieverik et al. 2009). Simultaneously, increased SIRT1 activity and decreased TH action avoid metabolic processes that consume ATP, such as lipogenesis and other anabolic pathways (Oppenheimer et al. 1991, Picard et al. 2004). Concomitantly, SIRT1 increases gluconeogenesis rates and mobilizes other energy sources, avoiding hypoglycemia (Rodgers & Puigserver 2007). Here, we demonstrated that TH replacement in a fasting condition prevented SIRT1 protein increment observed in liver of fasted mice. This result indicates that TH suppression during fasting is required for the upregulation of SIRT1 in the liver. We also observed the same regulatory pattern for BAT, another important metabolic tissue, which SIRT1 expression response to fasting was attenuated by TH replacement. To the best of our knowledge, there is no previous study of SIRT1 protein in BAT during fasting. It is possible that SIRT1 in BAT, similar to its action in white adipose tissue, may induce lipid oxidation pathways. Therefore, the data suggest that the suppression of thyroid function during fasting is responsible in great part for the induction of SIRT1 expression in this condition.
In support to this hypothesis, our study shows that excess or deficiency of TH modifies SIRT1 expression in mice. Hyperthyroid mice exhibited lower SIRT1 protein content in liver and BAT, while hypothyroid mice presented higher SIRT1 protein expression in the liver, suggesting that TH regulation of SIRT1 protein expression exhibits tissue specificities. In the liver, even though the magnitude of the variation was small, it was observed, consistently, in the classical pattern of a negative TH-regulated target protein: decreased protein expression in the presence of TH excess and increased protein content in TH deficiency. However, the mechanism underlying the regulation of SIRT1 protein content by TH is not clear, and the unchanged steady levels of Sirt1 mRNA do not support a transcriptional mechanism. Other reports had demonstrated altered levels of the protein in the presence of unchanged Sirt1 mRNA expression (Kanfi et al. 2008, Cakir et al. 2009), and post-transcriptional and post-translational mechanisms have been shown to be involved (Zschoernig & Mahlknecht 2008, Gao et al. 2011). However, in our study, we are not able to exclude the possibility that Sirt1 mRNA level might have preceded the significant increase in SIRT1 protein, as we did the analysis just by the end of the hypo- and hyperthyroidism induction protocols.
TH controls body weight gain by stimulating food ingestion and energy expenditure (Kong et al. 2004, Klieverik et al. 2009). In rodents, hypothyroidism is frequently associated with decreased food intake and lower body weight gain, whereas the opposite phenotype is observed in moderate hyperthyroid mice or rats (Karakoc et al. 2004, Hernandez et al. 2007, Calvino et al. 2012). In our experiment, hypothyroid mice lost body weight possibly by its lower food intake, which was not due to a unpalatable PTU diet, as other hypothyroid mice models induced by methimazole and potassium perchlorate also achieved body weight loss (Hernandez et al. 2007). On the contrary, in our hyperthyroid model, T3 stimulus on food intake might have exceeded the stimulus on energy expenditure and by this, mice gained weight, which was also observed by others (Kong et al. 2004, Hernandez et al. 2007). As SIRT1 expression may vary according to nutrient availability (Cohen et al. 2004, Kanfi et al. 2008), we questioned whether decreased SIRT1 content observed in hyperthyroidism was a direct effect of TH, or whether it was secondary to increased food ingestion and body weight gain induced by the hormone excess. Hyperthyroid mice submitted to food restriction, receiving an amount of chow equalized to the mean consumption of euthyroid group, exhibited attenuation of the decrease in SIRT1 expression induced by TH, suggesting a role for the higher ingestion and body weight in the SIRT1 repression observed in hyperthyroid mice. However, the same percentage of food restriction and body weight lost in euthyroid mice did not change SIRT1 protein levels, which suggests that thyroid and nutritional status, in conjunction, regulate SIRT1 protein levels in the liver and BAT.
Calorie restriction-based studies have been reported to increase SIRT1 protein levels in several tissues (Cohen et al. 2004); even though in the liver, the opposite regulation has also been proposed (Chen et al. 2008). However, the experimental paradigms vary in duration and intensity of food restriction leading to particular adaptation mechanisms (Wang et al. 2006). In our study, 2 weeks of 40% food restriction of euthyroid mice did not result in significant changes in liver or BAT SIRT1. Others, employing the same percentage of food restriction, but for a longer period of time, found either increases or decreases in liver SIRT1 protein levels (Cohen et al. 2004, Chen et al. 2008), and, as far as we know, there is no report on SIRT1 variations on BAT during food restriction. Additionally, serum THs are also expected to reduce in response to food restriction protocols (Araujo et al. 2009), although there is disagreement among reports that have shown decreased or unaltered TH levels (Araujo et al. 2009, Luvizotto et al. 2010), which seems to be related not only to duration but to intensity of food deprivation. In our study, serum T3 was decreased, but T4 reduction did not reach statistical significance. Therefore, the short-term duration of food restriction (2 weeks) may explain the unaltered levels of liver and BAT SIRT1 and the borderline suppression of thyroid function.
Our data suggest that TRβ seems to participate in the mechanism by which SIRT1 is downregulated by TH as mice bearing the Δ337T TRβ, unable to bind T3, exhibited increased liver SIRT1 protein content, even in the presence of high serum TH levels, and characterizing a phenotype similar to TH deficiency. Hence, unbound TRβ may interfere in cellular SIRT1 protein abundance, but the mechanisms involved remain to be elucidated. As Sirt1 mRNA levels were unchanged, we wondered about the involvement of non-transcriptional mechanisms. It has already been demonstrated that proteasomes participate in hypothalamic regulation of SIRT1 protein levels exerted by nutrient availability (Sasaki et al. 2010). Beyond that, other reports described the importance of unbound TRβ in stabilizing cytoplasmic proteins and, by contrast, when TRβ interacts with T3, proteasomal degradation was no longer avoided (Furuya et al. 2007, Guigon et al. 2008). Therefore, it is possible that unbounded TRβ may reduce SIRT1 protein degradation rates; however, more experiments are necessary to give support to this hypothesis. Another possibility is that the increased T3 levels in mutant TRβ mice could generate an augmented 3,5-diiodothyronine (T2) pool, which has been demonstrated to be able to increase SIRT1 activity (de Lange et al. 2011).
Other pieces of evidence suggest the interplay between the actions of SIRT1 and TH. Transgenic mice with moderate overexpression of SIRT1 (SirBACO) and hypothyroid mice have a phenotype of energy conservation, exhibiting lower energy expenditure, lower body temperature, and lower food intake (Banks et al. 2008), although, to the best of our knowledge, the TH levels were not measured in those animals. On the other hand, SRT1720, a SIRT1-specific activator that attenuated the deleterious effects of high-fat diet intake, mostly by triggering lipid oxidation in metabolic active tissues, as skeletal muscle, liver, and BAT (Feige et al. 2008), and, specifically in BAT, SRT1720 were also able to increase TRα and TRβ mRNA expression (Feige et al. 2008). In addition, both T2 and TRC150094, a pharmacological functional analog of T2, reduced deleterious metabolic effects of high-fat diet in rats, and the mechanism may be related to their ability to increase SIRT1 activity in the liver (Cioffi et al. 2010, de Lange et al. 2011). Interestingly, pituitary SIRT1 is preferentially co-localized with TSH cells and SIRT1 overexpression and knockdown in pituitary cells resulted in increased and decreased TSH secretion respectively (Akieda-Asai et al. 2010). These data, in conjunction with ours in this study, strongly suggest that TH signaling pathways and SIRT1 seem to have physiologically important relationships in the control of energy homeostasis.
In conclusion, our study points to TH as an important negative regulator of SIRT1 protein expression, and moreover, the study reveals a new role for TH suppression during fasting, which is to allow the upregulation of SIRT1, with important consequences to the integrated responses in adaptation to fasting.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This research was supported by Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Conselho Nacional de Pesquisa e Desenvolvimento (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES).
References
Akamine R, Yamamoto T, Watanabe M, Yamazaki N, Kataoka M, Ishikawa M, Ooie T, Baba Y & Shinohara Y 2007 Usefulness of the 5′ region of the cDNA encoding acidic ribosomal phosphoprotein P0 conserved among rats, mice, and humans as a standard probe for gene expression analysis in different tissues and animal species. Journal of Biochemical and Biophysical Methods 70 481–486. (doi:10.1016/j.jbbm.2006.11.008)
Akieda-Asai S, Zaima N, Ikegami K, Kahyo T, Yao I, Hatanaka T, Iemura S, Sugiyama R, Yokozeki T & Eishi Y et al. 2010 SIRT1 regulates thyroid-stimulating hormone release by enhancing PIP5Kgamma activity through deacetylation of specific lysine residues in mammals. PLoS ONE 5 e11755. (doi:10.1371/journal.pone.0011755)
Allard JS, Heilbronn LK, Smith C, Hunt ND, Ingram DK, Ravussin E & de Cabo R 2008 In vitro cellular adaptations of indicators of longevity in response to treatment with serum collected from humans on calorie restricted diets. PLoS ONE 3 e3211. (doi:10.1371/journal.pone.0003211)
Araujo RL, Andrade BM, da Silva ML, Ferreira AC & Carvalho DP 2009 Tissue-specific deiodinase regulation during food restriction and low replacement dose of leptin in rats. American Journal of Physiology. Endocrinology and Metabolism 296 E1157–E1163. (doi:10.1152/ajpendo.90869.2008)
Attia RR, Connnaughton S, Boone LR, Wang F, Elam MB, Ness GC, Cook GA & Park EA 2010 Regulation of pyruvate dehydrogenase kinase 4 (PDK4) by thyroid hormone: role of the peroxisome proliferator-activated receptor gamma coactivator (PGC-1α). Journal of Biological Chemistry 285 2375–2385. (doi:10.1074/jbc.M109.039081)
Banks AS, Kon N, Knight C, Matsumoto M, Gutierrez-Juarez R, Rossetti L, Gu W & Accili D 2008 SirT1 gain of function increases energy efficiency and prevents diabetes in mice. Cell Metabolism 8 333–341. (doi:10.1016/j.cmet.2008.08.014)
Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G & Lewis K et al. 2006 Resveratrol improves health and survival of mice on a high-calorie diet. Nature 444 337–342. (doi:10.1038/nature05354)
Boelen A, Wiersinga WM & Fliers E 2008 Fasting-induced changes in the hypothalamus–pituitary–thyroid axis. Thyroid 18 123–129. (doi:10.1089/thy.2007.0253)
Boily G, Seifert EL, Bevilacqua L, He XH, Sabourin G, Estey C, Moffat C, Crawford S, Saliba S & Jardine K et al. 2008 SirT1 regulates energy metabolism and response to caloric restriction in mice. PLoS ONE 3 e1759. (doi:10.1371/journal.pone.0001759)
Burnett C, Valentini S, Cabreiro F, Goss M, Somogyvari M, Piper MD, Hoddinott M, Sutphin GL, Leko V & McElwee JJ et al. 2011 Absence of effects of Sir2 overexpression on lifespan in C. elegans and Drosophila. Nature 477 482–485. (doi:10.1038/nature10296)
Cakir I, Perello M, Lansari O, Messier NJ, Vaslet CA & Nillni EA 2009 Hypothalamic Sirt1 regulates food intake in a rodent model system. PLoS ONE 4 e8322. (doi:10.1371/journal.pone.0008322)
Calvino C, Souza LL, Costa ESRH, Almeida NA, Trevenzoli IH & Pazos-Moura CC 2012 Hypothyroidism reduces ObRb-STAT3 leptin signalling in the hypothalamus and pituitary of rats associated with resistance to leptin acute anorectic action. Journal of Endocrinology 215 129–135. (doi:10.1530/JOE-11-0476)
Chen D, Bruno J, Easlon E, Lin SJ, Cheng HL, Alt FW & Guarente L 2008 Tissue-specific regulation of SIRT1 by calorie restriction. Genes and Development 22 1753–1757. (doi:10.1101/gad.1650608)
Cioffi F, Zambad SP, Chhipa L, Senese R, Busiello RA, Tuli D, Munshi S, Moreno M, Lombardi A & Gupta RC et al. 2010 TRC150094, a novel functional analog of iodothyronines, reduces adiposity by increasing energy expenditure and fatty acid oxidation in rats receiving a high-fat diet. FASEB Journal 24 3451–3461. (doi:10.1096/fj.10-157115)
Cohen HY, Miller C, Bitterman KJ, Wall NR, Hekking B, Kessler B, Howitz KT, Gorospe M, de Cabo R & Sinclair DA 2004 Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase. Science 305 390–392. (doi:10.1126/science.1099196)
Colman RJ, Anderson RM, Johnson SC, Kastman EK, Kosmatka KJ, Beasley TM, Allison DB, Cruzen C, Simmons HA & Kemnitz JW et al. 2009 Caloric restriction delays disease onset and mortality in rhesus monkeys. Science 325 201–204. (doi:10.1126/science.1173635)
Deng XQ, Chen LL & Li NX 2007 The expression of SIRT1 in nonalcoholic fatty liver disease induced by high-fat diet in rats. Liver International 27 708–715. (doi:10.1111/j.1478-3231.2007.01497.x)
Diano S, Naftolin F, Goglia F & Horvath TL 1998 Fasting-induced increase in type II iodothyronine deiodinase activity and messenger ribonucleic acid levels is not reversed by thyroxine in the rat hypothalamus. Endocrinology 139 2879–2884. (doi:10.1210/en.139.6.2879)
Faustino LC, Pires RM, Lima AC, Cordeiro A, Souza LL & Ortiga-Carvalho TM 2011 Liver glutathione S-transferase expression is decreased by 3,5,3-triiodothyronine in hypothyroid but not in euthyroid mice. Experimental Physiology 96 790–800. (doi:10.1113/expphysiol.2011.058172)
Faustino LC, Almeida NA, Pereira GF, Ramos RG, Soares RM, Morales MM, Pazos-Moura CC & Ortiga-Carvalho TM 2012 Thyroid hormone and estradiol have overlapping effects on kidney glutathione S-transferase-α gene expression. American Journal of Physiology. Endocrinology and Metabolism 303 E787–E797. (doi:10.1152/ajpendo.00223.2012)
Feige JN, Lagouge M, Canto C, Strehle A, Houten SM, Milne JC, Lambert PD, Mataki C, Elliott PJ & Auwerx J 2008 Specific SIRT1 activation mimics low energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation. Cell Metabolism 8 347–358. (doi:10.1016/j.cmet.2008.08.017)
Furuya F, Ying H, Zhao L & Cheng SY 2007 Novel functions of thyroid hormone receptor mutants: beyond nucleus-initiated transcription. Steroids 72 171–179. (doi:10.1016/j.steroids.2006.11.005)
Gao Z, Zhang J, Kheterpal I, Kennedy N, Davis RJ & Ye J 2011 Sirtuin 1 (SIRT1) protein degradation in response to persistent c-Jun N-terminal kinase 1 (JNK1) activation contributes to hepatic steatosis in obesity. Journal of Biological Chemistry 286 22227–22234. (doi:10.1074/jbc.M111.228874)
Guigon CJ, Zhao L, Lu C, Willingham MC & Cheng SY 2008 Regulation of β-catenin by a novel nongenomic action of thyroid hormone β receptor. Molecular and Cellular Biology 28 4598–4608. (doi:10.1128/MCB.02192-07)
Hallows WC, Lee S & Denu JM 2006 Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases. PNAS 103 10230–10235. (doi:10.1073/pnas.0604392103)
Hashimoto K, Curty FH, Borges PP, Lee CE, Abel ED, Elmquist JK, Cohen RN & Wondisford FE 2001 An unliganded thyroid hormone receptor causes severe neurological dysfunction. PNAS 98 3998–4003. (doi:10.1073/pnas.051454698)
Hashimoto K, Cohen RN, Yamada M, Markan KR, Monden T, Satoh T, Mori M & Wondisford FE 2006 Cross-talk between thyroid hormone receptor and liver X receptor regulatory pathways is revealed in a thyroid hormone resistance mouse model. Journal of Biological Chemistry 281 295–302. (doi:10.1074/jbc.M507877200)
Hernandez A, Martinez ME, Liao XH, Van Sande J, Refetoff S, Galton VA & St Germain DL 2007 Type 3 deiodinase deficiency results in functional abnormalities at multiple levels of the thyroid axis. Endocrinology 148 5680–5687. (doi:10.1210/en.2007-0652)
Kanfi Y, Peshti V, Gozlan YM, Rathaus M, Gil R & Cohen HY 2008 Regulation of SIRT1 protein levels by nutrient availability. FEBS Letters 582 2417–2423. (doi:10.1016/j.febslet.2008.06.005)
Karakoc A, Ayvaz G, Taneri F, Toruner F, Yilmaz M, Cakir N & Arslan M 2004 The effects of hypothyroidism in rats on serum leptin concentrations and leptin mRNA levels in adipose tissue and relationship with body fat composition. Endocrine Research 30 247–255. (doi:10.1081/ERC-120039569)
Kim B 2008 Thyroid hormone as a determinant of energy expenditure and the basal metabolic rate. Thyroid 18 141–144. (doi:10.1089/thy.2007.0266)
Klieverik LP, Coomans CP, Endert E, Sauerwein HP, Havekes LM, Voshol PJ, Rensen PC, Romijn JA, Kalsbeek A & Fliers E 2009 Thyroid hormone effects on whole-body energy homeostasis and tissue-specific fatty acid uptake in vivo. Endocrinology 150 5639–5648. (doi:10.1210/en.2009-0297)
Kong WM, Martin NM, Smith KL, Gardiner JV, Connoley IP, Stephens DA, Dhillo WS, Ghatei MA, Small CJ & Bloom SR 2004 Triiodothyronine stimulates food intake via the hypothalamic ventromedial nucleus independent of changes in energy expenditure. Endocrinology 145 5252–5258. (doi:10.1210/en.2004-0545)
de Lange P, Cioffi F, Senese R, Moreno M, Lombardi A, Silvestri E, De Matteis R, Lionetti L, Mollica MP & Goglia F et al. 2011 Nonthyrotoxic prevention of diet-induced insulin resistance by 3,5-diiodo-l-thyronine in rats. Diabetes 60 2730–2739. (doi:10.2337/db11-0207)
Lazar MA 2003 Thyroid hormone action: a binding contract. Journal of Clinical Investigation 112 497–499.
Li X, Zhang S, Blander G, Tse JG, Krieger M & Guarente L 2007 SIRT1 deacetylates and positively regulates the nuclear receptor LXR. Molecular Cell 28 91–106. (doi:10.1016/j.molcel.2007.07.032)
Lin SJ, Defossez PA & Guarente L 2000 Requirement of NAD and SIR2 for life-span extension by calorie restriction in Saccharomyces cerevisiae. Science 289 2126–2128. (doi:10.1126/science.289.5487.2126)
Livak KJ & Schmittgen TD 2001 Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25 402–408. (doi:10.1006/meth.2001.1262)
Luvizotto RA, Conde SJ, Sibio MT, Nascimento AF, Lima-Leopoldo AP, Leopoldo AS, Padovani CR, Cicogna AC & Nogueira CR 2010 Administration of physiologic levels of triiodothyronine increases leptin expression in calorie-restricted obese rats, but does not influence weight loss. Metabolism 59 1–6. (doi:10.1016/j.metabol.2009.06.017)
Machado DS, Sabet A, Santiago LA, Sidhaye AR, Chiamolera MI, Ortiga-Carvalho TM & Wondisford FE 2009 A thyroid hormone receptor mutation that dissociates thyroid hormone regulation of gene expression in vivo. PNAS 106 9441–9446. (doi:10.1073/pnas.0903227106)
Mattagajasingh I, Kim CS, Naqvi A, Yamamori T, Hoffman TA, Jung SB, DeRicco J, Kasuno K & Irani K 2007 SIRT1 promotes endothelium-dependent vascular relaxation by activating endothelial nitric oxide synthase. PNAS 104 14855–14860. (doi:10.1073/pnas.0704329104)
Nemoto S, Fergusson MM & Finkel T 2005 SIRT1 functionally interacts with the metabolic regulator and transcriptional coactivator PGC-1α. Journal of Biological Chemistry 280 16456–16460. (doi:10.1074/jbc.M501485200)
Ness GC, Pendleton LC, Li YC & Chiang JY 1990 Effect of thyroid hormone on hepatic cholesterol 7 α hydroxylase, LDL receptor, HMG-CoA reductase, farnesyl pyrophosphate synthetase and apolipoprotein A-I mRNA levels in hypophysectomized rats. Biochemical and Biophysical Research Communications 172 1150–1156. (doi:10.1016/0006-291X(90)91568-D)
Oliveira KJ, Ortiga-Carvalho TM, Cabanelas A, Veiga MA, Aoki K, Ohki-Hamazaki H, Wada K, Wada E & Pazos-Moura CC 2006 Disruption of neuromedin B receptor gene results in dysregulation of the pituitary–thyroid axis. Journal of Molecular Endocrinology 36 73–80. (doi:10.1677/jme.1.01892)
Oppenheimer JH, Schwartz HL, Lane JT & Thompson MP 1991 Functional relationship of thyroid hormone-induced lipogenesis, lipolysis, and thermogenesis in the rat. Journal of Clinical Investigation 87 125–132. (doi:10.1172/JCI114961)
Paula GS, Souza LL, Cabanelas A, Bloise FF, Mello-Coelho V, Wada E, Ortiga-Carvalho TM, Oliveira KJ & Pazos-Moura CC 2010 Female mice target deleted for the neuromedin B receptor have partial resistance to diet-induced obesity. Journal of Physiology 588 1635–1645. (doi:10.1113/jphysiol.2009.185322)
Pfluger PT, Herranz D, Velasco-Miguel S, Serrano M & Tschop MH 2008 Sirt1 protects against high-fat diet-induced metabolic damage. PNAS 105 9793–9798. (doi:10.1073/pnas.0802917105)
Picard F, Kurtev M, Chung N, Topark-Ngarm A, Senawong T, Machado De Oliveira R, Leid M, McBurney MW & Guarente L 2004 Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature 429 771–776. (doi:10.1038/nature02583)
Purushotham A, Schug TT, Xu Q, Surapureddi S, Guo X & Li X 2009 Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation. Cell Metabolism 9 327–338. (doi:10.1016/j.cmet.2009.02.006)
Redman LM, Huffman KM, Landerman LR, Pieper CF, Bain JR, Muehlbauer MJ, Stevens RD, Wenner BR, Kraus VB & Newgard CB et al. 2011 Effect of caloric restriction with and without exercise on metabolic intermediates in nonobese men and women. Journal of Clinical Endocrinology and Metabolism 96 E312–E321. (doi:10.1210/jc.2010-1971)
Rodgers JT & Puigserver P 2007 Fasting-dependent glucose and lipid metabolic response through hepatic sirtuin 1. PNAS 104 12861–12866. (doi:10.1073/pnas.0702509104)
Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM & Puigserver P 2005 Nutrient control of glucose homeostasis through a complex of PGC-1α and SIRT1. Nature 434 113–118. (doi:10.1038/nature03354)
Rogina B & Helfand SL 2004 Sir2 mediates longevity in the fly through a pathway related to calorie restriction. PNAS 101 15998–16003. (doi:10.1073/pnas.0404184101)
Santiago LA, Santiago DA, Faustino LC, Cordeiro A, Lisboa PC, Wondisford FE, Pazos-Moura CC & Ortiga-Carvalho TM 2011 The Delta337T mutation on the TRβ causes alterations in growth, adiposity, and hepatic glucose homeostasis in mice. Journal of Endocrinology 211 39–46. (doi:10.1530/JOE-11-0194)
Sasaki T, Kim HJ, Kobayashi M, Kitamura YI, Yokota-Hashimoto H, Shiuchi T, Minokoshi Y & Kitamura T 2010 Induction of hypothalamic Sirt1 leads to cessation of feeding via agouti-related peptide. Endocrinology 151 2556–2566. (doi:10.1210/en.2009-1319)
Setsuie R, Suzuki M, Kabuta T, Fujita H, Miura S, Ichihara N, Yamada D, Wang YL, Ezaki O & Suzuki Y et al. 2009 Ubiquitin C-terminal hydrolase-L3-knockout mice are resistant to diet-induced obesity and show increased activation of AMP-activated protein kinase in skeletal muscle. FASEB Journal 23 4148–4157. (doi:10.1096/fj.09-132217)
Sun C, Zhang F, Ge X, Yan T, Chen X, Shi X & Zhai Q 2007 SIRT1 improves insulin sensitivity under insulin-resistant conditions by repressing PTP1B. Cell Metabolism 6 307–319. (doi:10.1016/j.cmet.2007.08.014)
Tanny JC & Moazed D 2001 Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: evidence for acetyl transfer from substrate to an NAD breakdown product. PNAS 98 415–420. (doi:10.1073/pnas.98.2.415)
Vaquero A, Scher M, Lee D, Erdjument-Bromage H, Tempst P & Reinberg D 2004 Human SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin. Molecular Cell 16 93–105. (doi:10.1016/j.molcel.2004.08.031)
Vaziri H, Dessain SK, Ng Eaton E, Imai SI, Frye RA, Pandita TK, Guarente L & Weinberg RA 2001 hSIR2(SIRT1) functions as an NAD-dependent p53 deacetylase. Cell 107 149–159. (doi:10.1016/S0092-8674(01)00527-X)
Wang T, Hung CC & Randall DJ 2006 The comparative physiology of food deprivation: from feast to famine. Annual Review of Physiology 68 223–251. (doi:10.1146/annurev.physiol.68.040104.105739)
Weindruch R & Walford RL 1982 Dietary restriction in mice beginning at 1 year of age: effect on life-span and spontaneous cancer incidence. Science 215 1415–1418. (doi:10.1126/science.7063854)
Yen PM 2001 Physiological and molecular basis of thyroid hormone action. Physiological Reviews 81 1097–1142.
Zschoernig B & Mahlknecht U 2008 SIRTUIN 1: regulating the regulator. Biochemical and Biophysical Research Communications 376 251–255. (doi:10.1016/j.bbrc.2008.08.137)