No abstract

Expression of the primary female sex behaviour, lordosis, in laboratory animals depends on oestrogen-induced expression of progesterone receptor (PgR) within a defined cell group in the ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMH). The minimal latency from oestradiol administration to lordosis is 18 hours. During that time, ligand-bound oestrogen receptors (ER), members of a nuclear receptor superfamily, recruit transcriptional coregulators, which induce covalent modifications of histone proteins thus leading to transcriptional activation or repression of target genes. The aim of this study was to investigate early molecular epigenetic events underlying oestrogen-regulated transcriptional activation of the Pgr gene in the VMH of female mice. Oestradiol (E2) administration induced rapid and transient global histone modifications in the VMH of ovariectomised female mice. Histone H3 N-terminus phosphorylation (H3S10phK14Ac), acetylation (H3Ac) and methylation (H3K4me3) exhibited distinct temporal patterns facilitative to the induction of transcription; and a transcriptional repressive (H3K9me3) modification showed a different temporal pattern. Collectively these should create a permissive environment for the transcriptional activity necessary for lordosis, within 3-6 hours after E2-treatment. In the VMH, changes in the H3Ac and H3K4me3 levels of histone H3 were also detected at the promoter region of Pgr gene within the same time window, but were delayed in the preoptic area. Moreover, examination of histone modifications associated with the promoter of another ER-target gene, oxytocin receptor (Oxtr), revealed gene-and brainregion specific effects of E2 treatment. In the VMH of female mice, E2-treatment resulted in the recruitment of ERα to the oestrogen-response-elements-containing putative enhancer site of Pgr gene ~200kb upstream of the transcription start site (TSS), but failed to increase ERα association with the more proximal promoter region. Finally, E2 administration led to significant changes in the mRNA expression of several ER coregulators in a brain-region dependent manner. Taken together, these data indicate that in the hypothalamus and preoptic area of female mice, early responses to E2-treatment involve highly specific changes in chromatin structure, dependent on cell group, gene, histone modification studied, promoter/enhancer site and time following E2.

n-3 polyunsaturated fatty acids (n-3 PUFA) from fish oil (FO) exert important lipid-lowering effects, an effect also ascribed to thyroid hormones (TH) and TH receptor b1 (TRb1)-specific agonists. n-3 PUFA effects are mediated by nuclear receptors, such as peroxisome proliferator-activated receptors (PPAR) and others. In this study, we investigated a role for TH signaling in n-3 PUFA effects. Euthyroid and hypothyroid adult rats (methimazole-treated for 5 weeks) received FO or soybean oil (control) by oral administration for 3 weeks. In euthyroid rats, FO treatment reduced serum triglycerides and cholesterol, diminished body fat, and increased protein content of the animals. In addition, FO-treated rats exhibited higher liver expression of TRb1 and mitochondrial a-glycerophosphate dehydrogenase (mGPD), at protein and mRNA levels, but no alteration of glutathione S-transferase or type 1 deiodinase. In hypothyroid condition, FO induced reduction in serum cholesterol and increase in body protein content, but lost the ability to reduce triglycerides and body fat, and to induce TRb1 and mGDP expression. FO did not change PPARa liver abundance regardless of thyroid state; however, hypothyroidism led to a marked increase in PPARa liver content but did not alter TRb1 or TRa expression. The data suggest that part of the effect of n-3 PUFA from FO on lipid metabolism is dependent on TH signaling in specific steps and together with the marked upregulation of PPARa in liver of hypothyroid rats suggest important in vivo consequences of the cross-talking between those fatty acids and TH pathways in liver metabolism.

Sirtuin 1 (SIRT1), a NAD C -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 TRb. 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.

Mice bearing the genomic mutation D337T on the thyroid hormone receptor b (TRb) gene present the classical signs of resistance to thyroid hormone (TH), with high serum TH and TSH. This mutant TR is unable to bind TH, remains constitutively bound to co-repressors, and has a dominant negative effect on normal TRs. In this study, we show that homozygous (TRbD337T) mice for this mutation have reduced body weight, length, and body fat content, despite augmented relative food intake and relative increase in serum leptin. TRbD337T mice exhibited normal glycemia and were more tolerant to an i.p. glucose load accompanied by reduced insulin secretion. Higher insulin sensitivity was observed after single insulin injection, when the TRbD337T mice developed a profound hypoglycemia. Impaired hepatic glucose production was confirmed by the reduction in glucose generation after pyruvate administration.In addition, hepatic glycogen content was lower in homozygous TRbD337T mice than in wild type. Collectively, the data suggest that TRbD337T mice have deficient hepatic glucose production, by reduced gluconeogenesis and lower glycogen deposits. Analysis of liver gluconeogenic gene expression showed a reduction in the mRNA of phosphoenolpyruvate carboxykinase, a rate-limiting enzyme, and of peroxisome proliferator-activated receptor-g coactivator 1a, a key transcriptional factor essential to gluconeogenesis. Reduction in both gene expressions is consistent with resistance to TH action via TRb, reproducing a hypothyroid phenotype. In conclusion, mice carrying the D337T-dominant negative mutation on the TRb are leaner, exhibit impaired hepatic glucose production, and are more sensitive to hypoglycemic effects of insulin.

Class GST (Gsta) represents an essential component of cellular antioxidant defense mechanisms in both the liver and the kidney. Estrogens and thyroid hormones (TH) play central roles in animal development, physiology, and behavior. Evidence of the overlapping functions of thyroid hormones and estrogens has been shown, although the molecular mechanisms are not always clear. We evaluated an interaction between TH and estradiol in regulating kidney Gsta expression and function. First, we observed that female mice expressed greater amounts of Gsta compared with males and showed an opposite pattern of expression in TR␤ knock-in mice. To further investigate these sex differences, hypothyroidism was induced by a 5-propyl-2-thiouracil diet, and hyperthyroidism was induced by daily T 3 injections. Hypothyroidism increased kidney Gsta expression in male mice but not in female mice, indicating that sex hormones could be influencing the regulation of Gsta by thyroid hormones. To analyze this hypothesis, ovariectomized females were subjected to hypo-and hyperthyroidism, which led to a male profile of Gsta expression. When hypo-or hyperthyroid ovariectomized mice were treated with 17␤-estradiol benzoate, we were able to confirm that estradiol was interfering with TH modulation; Gsta expression is increased by T 3 when estradiol is present and decreased by T 3 when estradiol is absent. Using proximal tubule cells, we also showed that estradiol and T 3 worked together to modulate Gsta expression in an overlapping fashion. In summary, 1) the sex difference in the basal expression of Gsta impacts the detoxification process, 2) kidney Gsta expression is regulated by TH in males and females but in opposite directions, and 3) T 3 and estradiol interact directly in renal proximal cells to regulate Gsta expression in females.triiodothyronine; proximal tubule; cross-talk; ovariectomy; estrogen GLUTATHIONE S-TRANSFERASES (GSTs) are a superfamily of ubiquitous dimeric detoxification isoenzymes that conjugate many substrates to reduced glutathione (GSH), including several xenobiotic and endogenous electrophiles (26). Mammalian cytosolic GSTs represent the largest family of such transferases and have been divided into seven different classes (␣, , , , , , and ) (2, 27, 28). The ␣-class of GST (GST␣) is found in several organs, such as the liver, kidney, lung, stomach, and gonads, some of which exhibit sexual dimorphism (35). In the liver, the important role played by GST in cellular detoxification and in many other known functions has already been well described (26). However, in other tissues, the role of GST is still not well characterized.It has been established that GST␣ is a biomarker of renal toxicity that aids in the detoxification of endogenous and exogenous compounds and in drug metabolism (2). Renal GST isoforms are differentially expressed along nephron segments; expression also depends on species. In the human kidney, GST␣ is found predominantly in the proximal convoluted tubule, and low levels of GST␣ have been detected in the...

As previously reported, the activity of liver glutathione S-transferases, an important family of enzymes for detoxification processes, is regulated by thyroid hormone levels. Here, we specifically studied glutathione S-transferase α (Gsta) gene expression in livers of mice. First, in wild-type (WT) mice, hypothyroidism was induced by 5 weeks of a diet containing 5-propyl-2-thiouracil plus water containing metimazole, whereas hyperthyroidism was induced by daily injections of 50 μg (100 g body weight)−1 of 3,3 , 5-triiodo-L-thyronine (L-T 3 ) for 15 days. Importantly, hypothyroidism induced liver Gsta mRNA (>500%) and protein levels (70%; P < 0.01), indicating an important role of baseline thyroid hormone levels to repress this gene; however, surprisingly, no differences were seen in hyperthyroid mice. To further investigate Gsta repression by T 3 , we used animals expressing a naturally occurring mutation of the gene for thyroid hormone receptor (TR)-β ( 337T), which prevents T 3 binding and causes a general resistance to thyroid hormone. At baseline, homozygous animals showed increased Gsta levels (mRNA 3.5 times, protein 1.3 times) similar to those found in hypothyroid animals. After a T 3 suppression test, we found a blunted response of liver Gsta after the lower doses of T 3 in homozygous animals, as expected. However, after the highest dose of T 3 , we observed a decrease in Gsta expression (80%), similar to normal animals, explained by a higher expression of TR-α1 (60%; P < 0.01) and a lower expression of Src1 (steroid coactivator receptor) in the mutant animals (50% decrease). In summary, a decrease in Gsta expression caused by T 3 was observed only in the hypothyroid state. In addition, an essential role of TR-β1 is to mediate Gsta suppression in response to T 3 and, in the absence of a functional TR-β, there is a compensatory action of TR-α1 that depends on low levels of Src1.

Cerebellum development is sensitive to thyroid hormone (TH) levels, as THs regulate neuronal migration, differentiation, and myelination. Most effects of THs are mediated by the thyroid hormone receptor (TR) isoforms TRβ1, TRβ2, and TRα1. Studies aimed at identifying TH target genes during cerebellum development have only achieved partial success, as some of these genes do not possess classical TH-responsive elements, and those that do are likely to be temporally and spatially regulated by THs. THs may also affect neurodevelopment by regulating transcription factors that control particular groups of genes. Furthermore, TH action can also be affected by TH transport, which is mediated mainly by monocarboxylate transporter family members. Studies involving transgenic animal models and genome-wide expression analyses have helped to address the unanswered questions regarding the role of TH in cerebellar development. Recently, a growing body of evidence has begun to clarify the molecular, cellular, and functional aspects of THs in the developing cerebellum. This review describes the current findings concerning the effects of THs on cerebellar development and maintenance as well as advances in the genetic animal models used in this field.