Nongenomic signaling pathways triggered by thyroid hormones and their metabolite 3‐iodothyronamine on the cardiovascular system

Thyroid hormones play a wide range of important physiological activities in almost all organism. As changes in these hormones levels—observed in hypothyroidism and hyperthyroidism—promote serious derangements of the cardiovascular system, it is important to know their mechanisms of action. Although the classic genomic actions which are dependent on interaction with nuclear receptors to modulate cardiac myocytes genes expression, there is growing evidence about T₃ and T₄‐triggered nongenomic pathways, resulted from their binding to plasma membrane, cytoplasm, or mitocondrial receptors that leads to a rapidly regulation of cardiac functions. Interestingly both actions converge to amplify thyroid hormone effects on cardiovascular system. T₃ and T₄ nongenomic actions modify inotropic and chronotropic effects, cardiac action potential duration, cardiac growth, and myocyte shape by protein translation through protein kinases‐dependent signaling cascades, which include PKA, PKC, PI3K, and MAPK, and changes on ion channels and pumps activity. In respect to the decreased systemic vascular resistance seen in hyperthyroidism, T₃ appears to activate NOS or ATP‐sensitive K⁺ channels. In addition, a novel biologically active T₄‐derived metabolite has been described, 3‐iodothyronamine, T₁AM, which also acts through membrane receptors to mediate nongenomic cardiac effects. This metabolite influences the physiological manifestations of thyroid hormone actions by inducing opposite effects from those stimulated by T₃ and T₄, such as negative inotropic and chronotropic effects. Therefore, beyond genomic and nongenomic effects of thyroid hormones, it is crucial for there to be an equilibrium between T₃ or T₄ and T₁AM levels for maintaining cardiac homeostasis. J. Cell. Physiol. 226: 21–28, 2010. © 2010 Wiley‐Liss, Inc.     

Thyroid Hormone Upregulates Zinc-α2-glycoprotein Production in the Liver but Not in Adipose Tissue

Overproduction of zinc-α₂-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α₂-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α₂-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α₂-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α₂-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α₂-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α₂-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α₂-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α₂-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α₂-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α₂-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α₂-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α₂-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α₂-glycoprotein expression in adipose tissue could be the reason why zinc-α₂-glycoprotein is not related to weight loss in hyperthyroidism.     

Differential effects of thyroid hormone manipulation and β adrenoceptor agonist administration on uncoupling protein mRNA abundance in adipose tissue and thermoregulation in neonatal pigs

We have shown that there is significant disparity in the expression of uncoupling proteins (UCP) 2 and 3 between modern-commercial and ancient-Meishan porcine genotypes, commercial pigs also have higher plasma triiodothyronine (T₃) in on the first day of life. T₃ and the sympathetic nervous system are both known to regulate UCPs in rodents and humans; their role in regulating these proteins in the pig is unknown. This study examined whether thyroid hormone manipulation or administration of a selective β3 adrenoceptor agonist (ZD) influenced plasma hormones, colonic temperature and UCP expression in adipose tissue of two breeds of pig. To mimic the differences observed in thyroid hormone status, piglets from Meishan and commercial litters were randomly assigned to control (1 ml/kg water), T₃ (10 mg/kg) (Meishan only), methimazole (a commonly used antithyroid drug) (50 mg/kg) (commercial only) or ZD (10 mg/kg) oral administration for the first 4 days of postnatal life. Adipose tissue UCP2/3 mRNA abundance was measured on day 4 using PCR. T₃ administration raised plasma T₃ concentrations and increased colonic temperature on day 4. UCP3 mRNA abundance was higher in Meishan, than commercial piglets and was downregulated following T₃ administration. Irrespective of genotype, ZD increased UCP2 mRNA abundance. Expression of neither UCP2 nor 3 was related to colonic temperature, regardless of treatment. In conclusion, we have demonstrated a dissociation between thyroid hormones and the sympathetic nervous system in the regulation of UCPs in porcine adipose tissue. We have also suggested that expression of adipose tissue UCP2 and 3 are not related to body temperature in piglets.     

Differential effects of thyroid hormone manipulation and �� adrenoceptor agonist administration on uncoupling protein mRNA abundance in adipose tissue and thermoregulation in neonatal pigs

We have shown that there is significant disparity in the expression of uncoupling proteins (UCP) 2 and 3 between modern-commercial and ancient-Meishan porcine genotypes, commercial pigs also have higher plasma triiodothyronine (T₃) in on the first day of life. T₃ and the sympathetic nervous system are both known to regulate UCPs in rodents and humans; their role in regulating these proteins in the pig is unknown. This study examined whether thyroid hormone manipulation or administration of a selective β3 adrenoceptor agonist (ZD) influenced plasma hormones, colonic temperature and UCP expression in adipose tissue of two breeds of pig. To mimic the differences observed in thyroid hormone status, piglets from Meishan and commercial litters were randomly assigned to control (1 ml/kg water), T₃ (10 mg/kg) (Meishan only), methimazole (a commonly used antithyroid drug) (50 mg/kg) (commercial only) or ZD (10 mg/kg) oral administration for the first 4 days of postnatal life. Adipose tissue UCP2/3 mRNA abundance was measured on day 4 using PCR. T₃ administration raised plasma T₃ concentrations and increased colonic temperature on day 4. UCP3 mRNA abundance was higher in Meishan, than commercial piglets (p = 0.042) and was downregulated following T₃ administration (p = 0.014). Irrespective of genotype, ZD increased UCP2 mRNA abundance (Meishan p = 0.05, commercial p = 0.03). Expression of neither UCP2 nor 3 was related to colonic temperature, regardless of treatment. In conclusion, we have demonstrated a dissociation between thyroid hormones and the sympathetic nervous system in the regulation of UCPs in porcine adipose tissue. We have also suggested that expression of adipose tissue UCP2 and 3 are not related to body temperature in piglets.Key words: mitochondria, postnatal growth, metabolism     

Characterization of 3-Iodothyronamine In Vitro Dynamics by Mathematical Modeling

3-Iodothyronamine (T₁AM) is regarded as a hormone-like substance thanks to its endogenous nature, its interaction with specific receptors trace amine-associated receptor 1 and its biological effects. We characterized T₁AM transport and conversion in an in vitro culture of H9c2 murine cells, after a T₁AM bolus injection. Samples of cell medium culture and cell lysate were assayed by high-performance liquid chromatography coupled to tandem mass spectrometry. We performed comparative experiments by adding to T₁AM bolus amino oxidase inhibitors as iproniazid, pargyline (monoamine oxidase, MAO inhibitors), aminoguanidine, and semicarbazide (semicarbazide-sensitive amino oxidase, SSAO inhibitors). A mathematical model was developed, based on the assumption that T₁AM is transported with a mechanism that is typical of hormone transport (i.e., EGF or insulin). We noticed that surface receptors downregulation could play a major role in T₁AM dynamics. We also estimated that T₁AM catabolism is mainly affected by MAO inhibitors, which produce a dramatic decrease in the kinetic constants related to T₁AM degradation, while no significant changes were observed in experiments with SSAO inhibitors.     

Sex-specific phenotypes of hyperthyroidism and hypothyroidism in mice

Background

Thyroid dysfunction is more common in the female population, however, the impact of sex on disease characteristics has rarely been addressed. Using a murine model, we asked whether sex has an influence on phenotypes, thyroid hormone status, and thyroid hormone tissue response in hyper- and hypothyroidism.

Methods

Hypo- and hyperthyroidism were induced in 5-month-old female and male wildtype C57BL/6N mice, by LoI/MMI/ClO₄ ^? or T₄ i.p. treatment over 7 weeks, and control animals underwent sham treatment (N?=?8 animals/sex/treatment). Animals were investigated for impact of sex on body weight, food and water intake, body temperature, heart rate, behaviour (locomotor activity, motor coordination, and strength), liver function, serum thyroid hormone status, and cellular TH effects on gene expression in brown adipose tissue, heart, and liver.

Results

Male and female mice showed significant differences in behavioural, functional, metabolic, biochemical, and molecular traits of hyper- and hypothyroidism. Hyperthyroidism resulted in increased locomotor activity in female mice but decreased muscle strength and motor coordination preferably in male animals. Hypothyroidism led to increased water intake in male but not female mice and significantly higher serum cholesterol in male mice. Natural sex differences in body temperature, body weight gain, food and water intake were preserved under hyperthyroid conditions. In contrast, natural sex differences in heart rate disappeared with TH excess and deprivation. The variations of hyper- or hypothyroid traits of male and female mice were not explained by classical T₃/T₄ serum state. TH serum concentrations were significantly increased in female mice under hyperthyroidism, but no sex differences were found under eu- or hypothyroid conditions. Interestingly, analysis of expression of TH target genes and TH transporters revealed little sex dependency in heart, while sex differences in target genes were present in liver and brown adipose tissue in line with altered functional and metabolic traits of hyper- and hypothyroidism.

Conclusions

These data demonstrate that the phenotypes of hypo- and hyperthyroidism differ between male and female mice and indicate that sex is an important modifier of phenotypic manifestations.

     

Endocrine Regulation of Fetal Adipose Tissue Metabolism in the Pig: Interaction of Porcine Growth Hormone and Thyroxine

HAUSMAN, D.B., G.J. HAUSMAN, AND R.J. MARTIN. Endocrine regulation of fetal adipose tissue metabolism in the pig: interaction of porcine growth hormone and thyroxine. Obes Res. 1999;7:76–82. Objective : This study tested the hypothesis that combined treatment of thyroxine (T₄) and growth hormone (GH) could normalize cellular and metabolic aspects of adipose tissue development of hypophysectomized fetal pigs. Research Methods and Procedures : On day 70 of gestation, pig fetuses were hypophysectomized by microcauterization or remained intact. Hypophysectomized fetuses remained untreated or were treated from day 90 to day 105 of gestation with T₄, GH, or a combination of both hormones. Results : Body weights were unaffected by hypophysectomy or hormone treatment. De novo lipogenesis in subcutaneous adipose tissue was increased 10-fold by hypophysectomy, consistent with our previous results. This increase was abolished by GH treatment in the hypophysectomized fetuses. In contrast, T₄ treatment of the hypophysectomized fetuses resulted in a 12-fold further increase in adipose tissue lipogenesis, an effect that was negated by concomitant administration of GH. Lipolytic response to isoproterenol was decreased by hypophysectomy, unaffected by GH treatment, and restored to intact values by T₄ or by T₄+GH treatment in the hypophysectomized fetuses. Discussion : In contrast to T₄, GH does not influence serum insulin-like growth factor-I or adipose tissue lipolysis, but decreases lipogenesis in the fetal pig. However, replacing both T₄ and GH normalized hypophysectomized fetuses to a greater extent than either GH or T₄ alone. Thus, any influence of thyroid hormones on stimulating adipose tissue lipogenesis in the developing fetal pig may be normally counterregulated by pituitary-derived growth hormone.     

Gene Specific Actions of Thyroid Hormone Receptor Subtypes

There are two homologous thyroid hormone (TH) receptors (TRs α and β), which are members of the nuclear hormone receptor (NR) family. While TRs regulate different processes in vivo and other highly related NRs regulate distinct gene sets, initial studies of TR action revealed near complete overlaps in their actions at the level of individual genes. Here, we assessed the extent that TRα and TRβ differ in target gene regulation by comparing effects of equal levels of stably expressed exogenous TRs +/− T₃ in two cell backgrounds (HepG2 and HeLa). We find that hundreds of genes respond to T₃ or to unliganded TRs in both cell types, but were not able to detect verifiable examples of completely TR subtype-specific gene regulation. TR actions are, however, far from identical and we detect TR subtype-specific effects on global T₃ response kinetics in HepG2 cells and many examples of TR subtype specificity at the level of individual genes, including effects on magnitude of response to TR +/− T₃, TR regulation patterns and T₃ dose response. Cycloheximide (CHX) treatment confirms that at least some differential effects involve verifiable direct TR target genes. TR subtype/gene-specific effects emerge in the context of widespread variation in target gene response and we suggest that gene-selective effects on mechanism of TR action highlight differences in TR subtype function that emerge in the environment of specific genes. We propose that differential TR actions could influence physiologic and pharmacologic responses to THs and selective TR modulators (STRMs).     

Human Uncoupling Proteins and Obesity

SCHRAUWEN, PATRICK, KEN WALDER, AND ERIC RAVUSSIN. Human coupling proteins and obesity. Obes. Res. 1999;7:97–105. Uncoupling protein (UCP) 2 and UCP3 are newly discovered proteins that can uncouple ATP production from mitochondrial respiration, thereby dissipating energy as heat and affecting energy metabolism efficiency. In contrast to UCP1, which is only present in brown adipose tissue, UCP2 has a wide tissue distribution, whereas UCP3 is expressed predominantly in skeletal muscle. Some evidence of a role for UCPs in modulating metabolic rate was provided by linkage and association studies. Furthermore, UCP3 gene expression was found to correlate negatively with body mass index and positively with sleeping metabolic rate in Pima Indians. Treatment with thyroid hormone increases expression of the UCP2 and UCP3 genes. Other regulators of UCP2 and UCP3 gene expression are β₃-adrenergic agonists and glucocorticoids. Surprisingly, fasting has a stimulatory effect on UCP2 and UCP3 mRNA levels, possibly explained by the effects of free fatty acid on UCP2 and UCP3 gene expression.     

Effects of triiodothyronine on turnover rate and metabolizing enzymes for thyroxine in thyroidectomized rats

Aim

Previous studies in rats have indicated that surgical thyroidectomy represses turnover of serum thyroxine (T₄). However, the mechanism of this process has not been identified. To clarify the mechanism, we studied adaptive variation of metabolic enzymes involved in T₄ turnover.

Main methods

We compared serum T₄ turnover rates in thyroidectomized (Tx) rats with or without infusion of active thyroid hormone, triiodothyronine (T₃). Furthermore, the levels of mRNA expression and activity of the metabolizing enzymes, deiodinase type 1 (D1), type 2 (D2), uridine diphosphate-glucuronosyltransferase (UGT), and sulfotransferase were also compared in several tissues with or without T₃ infusion.

Key findings

After the T₃ infusion, the turnover rate of serum T₄ in Tx rats returned to normal. Although mRNA expression and activity of D1 decreased significantly in both liver and kidneys without T₃ infusion, D2 expression and activity increased markedly in the brain, brown adipose tissue, and skeletal muscle. Surprisingly, hepatic UGT mRNA expression and activity in Tx rats increased significantly in comparison with normal rats, and returned to normal after T₃ infusion.

Significance

This study suggests that repression of the disappearance of serum T₄ in rats after Tx is a homeostatic response to decreased serum T₃ concentrations. Additionally, T₄ glucuronide is a storage form of T₄, but may also have biological significance. These results suggest strongly that repression of deiodination of T₄ by D1 in the liver and kidneys plays a major role in thyroid hormone homeostasis in Tx rats, and that hepatic UGT also plays a key role in this mechanism.     

Analysis of thyroid hormone effects on myosin heavy chain gene expression in cardiac and soleus muscles using a novel dot-blot mRNA assay

Effects of thyroid hormone on myosin heavy chain (MHC) gene expression were compared in ventricle and soleus muscle of hypothyroid rats, since in this condition, both muscle types express predominately β-MHC mRNA. Changes in MHC mRNAs were analyzed using synthetic oligonucleotide probes complementary to the 3′ untranslated region of the MHC mRNAs. The results indicated that daily treatment with 3,5,3′-triiodo-L-thyronine (T₃) at a dose of 2 μg/100 g body weight increased α-MHC mRNA content in heart muscle by 600% and decreased β-MHC mRNA by 70% within 48 h. In soleus muscle, β-MHC mRNA levels were not affected by 9 wks of treatment, however, Fast IIa-MHC mRNA was increased by 150% at 7 wks and 300% after 9 wks of T₃ administration. Thus, regulation of MHC gene expression by thyroid hormone is both gene and tissue specific.     

The Influence of Human Growth Hormone (GH) and Thyroxine (T4) on the Differentiation of Adipose Tissue in the Fetus

Late term fetuses from genetically obese dams have slightly larger fat cells, greater adipose tissue lipoprotein lipase (LPL) activities, elevated levels of thyroid hormones, and depressed growth hormone (GH) levels when compared to fetuses from lean dams. We have investigated the influence of thyroid hormone and GH status per se on these and other adipose tissue traits by chronically treating hypophysectomized (hypox) fetuses (day 70) between day 90 and 105 of gestation with either thyroxine (T4) or human GH. Treatment with T4 decreased body weights (P<.05), increased serum T₄ levels (P<.05), and enhanced skin and hair development (P<.05). Quantitative analysis of sections of perirenal and subcutaneous adipose tissue indicated that T₄ increased LPL activity (P<.05), slightly increased fat cell size, and more than doubled (P<.05) lipid accretion. A hypox induced deficit in fat cell cluster number in the outer layer of subcutaneous tissue was normalized by T₄ (P<.05). Conversely, human GH (hGH) treatment had no influence on body weight, increased serum hGH levels, decreased fat cell size (P<.05) and LPL activity (P<.05) but had no influence on lipid accretion. Quantitative analysis of adipose tissue sections provided direct and indirect evidence of a “critical” or “sensitive” period between 90 and 105 days, since fetal hypox at day 70 severely impeded preadipocyte recruitmentheplication during this period. Furthermore, T₄ but not GH effectively normalized this hypox-induced deficiency in preadipocyte development. Therefore, T₄ may have a major role in preadipocyte recruitmentheplication during late fetal life.     

Inflammation Correlates With Markers of T‐Cell Subsets Including Regulatory T Cells in Adipose Tissue From Obese Patients

Accumulation of cytotoxic and T‐helper (T_h)1 cells together with a loss of regulatory T cells in gonadal adipose tissue was recently shown to contribute to obesity‐induced adipose tissue inflammation and insulin resistance in mice. Human data on T‐cell populations in obese adipose tissue and their potential functional relevance are very limited. We aimed to investigate abundance and proportion of T‐lymphocyte sub‐populations in human adipose tissue in obesity and potential correlations with anthropometric data, insulin resistance, and systemic and adipose tissue inflammation. Therefore, we analyzed expression of marker genes specific for pan‐T cells and T‐cell subsets in visceral and subcutaneous adipose tissue from highly obese patients (BMI >40 kg/m², n = 20) and lean to overweight control subjects matched for age and sex (BMI <30 kg/m²; n = 20). All T‐cell markers were significantly upregulated in obese adipose tissue and correlated with adipose tissue inflammation. Proportions of cytotoxic T cells and T_h1 cells were unchanged, whereas those of regulatory T cells and T_h2 were increased in visceral adipose tissue from obese compared to control subjects. Systemic and adipose tissue inflammation positively correlated with the visceral adipose abundance of cytotoxic T cells and T_h1 cells but also regulatory T cells within the obese group. Therefore, this study confirms a potential role of T cells in human obesity‐driven inflammation but does not support a loss of protective regulatory T cells to contribute to adipose tissue inflammation in obese patients as suggested by recent animal studies.