Thyroid hormones (TH) are of central importance for thermogenesis, energy homeostasis and metabolism. Here, we will discuss these aspects by focussing on the physiological aspects of TH-dependent regulation in response to cold exposure and fasting, which will be compared to alterations in primary hyperthyroidism and hypothyroidism. In particular, we will summarise current knowledge on regional thyroid hormone status in the central nervous system (CNS) and in peripheral cells. In contrast to hyperthyroidism and hypothyroidism, where parallel changes are observed, local alterations in the CNS differ to peripheral compartments when induced by cold exposure or fasting. Cold exposure is associated with low hypothalamic TH concentrations but increased TH levels in the periphery. Fasting results in a reversed TH pattern. Primary hypothyroidism and hyperthyroidism disrupt these fine-tuned adaptive mechanisms and both, the hypothalamus and the periphery, will have the same TH status. These important mechanisms need to be considered when discussing thyroid hormone replacement and other therapeutical interventions to modulate TH status.
K Alexander Iwen, Rebecca Oelkrug and Georg Brabant
Edwin J W Geven, Folkert Verkaar, Gert Flik and Peter H M Klaren
The effect of experimental hyperthyroidism, realized by T4 injection, on central mediators of the hypothalamo–pituitary–interrenal axis (HPI-axis) in common carp (Cyprinus carpio L.) was studied. Our results show that hyperthyroidism evokes a marked 3.2-fold reduction in basal plasma cortisol levels. Corticotropin-releasing hormone-binding protein (CRH-BP) mRNA levels in the hypothalamus, measured by real-time quantitative PCR, were significantly elevated by 40%, but CRH, urotensin-I, prepro-TRH, prohormone convertase-1 (PC1), and POMC mRNA levels were unchanged. In the pituitary pars distalis, PC1, CRH receptor-1, and POMC mRNA levels were unaffected, as was ACTH content. Plasma α-MSH concentrations were significantly elevated by 30% in hyperthyroid fish, and this was reflected in PC1 and POMC mRNA levels in pituitary pars intermedia that were increased 1.5- and 2.4-fold respectively. The α-MSH content of the pars intermedia was unchanged. Hyperthyroidism has profound effects on the basal levels of a central mediator, i.e., CRH-BP, of HPI-axis function in unstressed carp in vivo, and we conclude that HPI- and hypothalamo–pituitary–thyroid-axis functions are strongly interrelated. We suggest that the changes in plasma cortisol, thyroid hormone, and α-MSH levels reflect their concerted actions on energy metabolism.
M R Thomas, J P Miell, A M Taylor, R J M Ross, J R Arnao, D E Jewitt and A M McGregor
Thyroid hormones are essential for the normal growth and development of many tissues. In the rat, hypothyroidism is associated with growth impairment, and hyperthyroidism with the development of a hypercatabolic state and skeletal muscle wasting but, paradoxically, cardiac hypertrophy. The mechanism by which thyroid hormone produces cardiac hypertrophy and myosin isoenzyme changes remains unclear. The role of IGF-I, an anabolic hormone with both paracrine and endocrine actions, in producing cardiac hypertrophy was investigated during this study in hyperthyroid, hypothyroid and control rats. A treated hypothyroid group was also included in order to assess the effect of acute normalization of thyroid function.
Body weight was significantly lower in the hyperthyroid (mean±s.e.m.; 535·5±24·9 g, P<0·05), hypothyroid (245·3±9·8 g, P<0·001) and treated hypothyroid (265·3±9·8 g, P<0·001) animals when compared with controls (618·5±28·6 g). Heart weight/body weight ratios were, however, significantly increased in the hyperthyroid (2·74 ± 0·11×10−3, P<0·01) and treated hypothyroid (2·87±0·07 ×10−3, P<0·001) animals when compared with controls (2·26±0·03 × 10−3). Serum IGF-I concentrations were similar in the control and hyperthyroid rats (0·91±0·07 vs 0·78±0·04 U/ml, P=0·26), but bioactivity was reduced by 70% in hyperthyroid serum, suggesting a circulating inhibitor of IGF. Serum IGF-I levels (0·12±0·03 U/ml, P<0·001) and bioactivity (0·12±0·04 U/ml, P<0·001) were significantly lower in the hypothyroid group. Liver IGF-I mRNA levels were not statistically different in the control and hyperthyroid animals, but were significantly reduced in the hypothyroid animals (P<0·05 vs control). Heart IGF-I mRNA levels were similar in the control and hypothyroid rats, but were significantly increased in the hyperthyroid and treated hypothyroid animals (increased by 32% in hyperthyroidism, P<0·05; increased by 57% in treated hypothyroidism, P<0·01). Cardiac IGF-I was significantly elevated in hyperthyroidism (0·16±0·01 U/mg heart tissue, P<0·01), was low in hypothyroidism (0·08±0·01 U/mg, P<0·01) and was normalized in the treated hypothyroid group (0·11 ± 0·01 U/mg vs control, 0·13±0·01 U/mg).
Low body mass during both hypothyroidism and hyperthyroidism is therefore associated with reduced systemic IGF bioactivity. In hypothyroidism there is a primary defect in the endocrine function of IGF-I, while in hyperthyroidism serum IGF bioactivity is reduced in the presence of normal endocrine production of this anabolic hormone. In contrast, the paracrine actions of IGF-I are increased in the heart during hyperthyroidism, and this hormone appears to play a part in the development of hyperthyroid cardiac hypertrophy.
A. J. Pickles, N. Peers, W. R. Robertson and A. Lambert
The microheterogeneity of pituitary thyroid-stimulating hormone (TSH) is dependent on variations in the hormone's carbohydrate moieties. In this study, changes in the pattern of heterogeneity have been assessed by chromatofocusing, which separates the isospecies on the basis of their isoelectric points (pI). Rats (n = 6 per group) were either untreated or rendered hypo- or hyperthyroid by including in the drinking water either propylthiouracil (0·05% for 8 weeks) or thyroxine (T4; 4 mg/l for 6 weeks) before they were killed at 16 weeks. On autopsy, serum TSH and total T4 were (means±s.e.m.): 2±0·3 μg TSH/l and 64±5 nmol T4/l (control); <1 μg TSH/1 and 133±6 nmol T4/l (hyperthyroid); 58±6 μg TSH/1 and 32±6 nmol T4/l (hypothyroid). The pituitaries were individually homogenized and the TSH isoforms separated by chromatofocusing over a pH range of 7–4. Fractions were assayed for TSH by radioimmunoassay. TSH from the control group was distributed into seven major peaks with pI values of (means ±s.e.m., n=6) 6·9±0·1, 6·6±0·1, 6·2±0·1, 5·8±0·1, 5·5±0·1, 5·2±0·1 and 4·8±0·1; 7±3% of the TSH had a pI of <40. Six peaks of TSH were conserved in the hypothyroid group (with pI values of 6·8±0·1, 6·5±0·1, 6·2±0·1, 5·8±0·1, 5·4±0·1 and 5·2±0·1), and 11±4% of the hormone had a pI of <40. In contrast to the other two groups, only one major peak (with a pI of 5·8±0·1) was detected in the pituitaries from the hyperthyroid group; 13 ± 5% of the TSH had a pI of <40. In the pH range of 5·5– 60, the per cent distribution of TSH was 58±15 (hyperthyroid) compared with 17 ± 3 (hypothyroid) and 22±3 (euthyroid). Above pH 6, only 25±13% of the TSH (hyperthyroid) was present compared with 46±5% (hypothyroid) and 45±5% (euthyroid). Below pH 5·5, the per cent distribution of TSH was 19±5 (hyperthyroid), 37±5 (hypothyroid) and 35±3 (euthyroid). In conclusion, both hyper- and hypothyroidism are associated with changes in the composition of pituitary TSH. This change was most marked in the hyperthyroid group, where there was a selective loss of several isoforms of TSH.
C Massart, J Gibassier, M L Raoul, F Le Gall, G Lancien, N Genetet, A Denais, F Darcel and C Lucas
We have studied the action of peripheral blood lymphocytes (PBLs) and intrathyroidal lymphocytes (ITLs) on the biochemical and hormonal metabolism of autologous thyrocytes cultured in follicles in a collagen gel. The production of tumour necrosis factor α (TNF-α) in culture was also measured. Thyroid tissues and lymphocytes were obtained from ten patients with Graves' disease and from five control subjects. Lymphocyte-induced cytotoxicity was evaluated in autologous thyrocytes cultured in a collagen gel by several tests: neutral red uptake, lactate dehydrogenase activity and glutathione level. Hormonal metabolism was assessed by evaluating tri-iodothyronine (T3) and total cAMP production under TSH stimulation. TNF-α levels were measured in supernatants after 5 days of coculture. PBLs altered biochemical metabolism, T3 synthesis and cAMP production in autologous thyroid follicles. These inhibitions were greater than those obtained with ITLs. No difference was seen between cells obtained from patients with Graves' disease and those from normal subjects. TNF-α levels secreted by PBLs were higher than those secreted by ITLs. The concentrations of this cytokine decreased in coculture. Significant correlations were observed between the decrease in biochemical and hormonal parameters and TNF-α levels. Exogenous TNF-α and high doses of interferon γ inhibited follicle metabolism, especially hormone secretion.
In conclusion, thyrocytes cultured in follicles provide a more sensitive model than monolayer cultures for analysis of lymphocyte-induced interactions. Lymphocytes gradually inhibit the biochemical and hormonal metabolism of autologous thyroid follicles depending on the isolation method. These alterations may be particularly attributed to TNF-α secreted by lymphocytes. The cytokine-induced inhibition of thyroid hormonal function apparently involves the adenylate cyclase system.
Julika Lietzow, Janine Golchert, Georg Homuth, Uwe Völker, Wenke Jonas and Josef Köhrle
The endogenous thyroid hormone (TH) metabolite 3,5-diiodo-l-thyronine (3,5-T2) acts as a metabolically active substance affecting whole-body energy metabolism and hepatic lipid handling in a desirable manner. Considering possible adverse effects regarding thyromimetic action of 3,5-T2 treatment in rodents, the current literature remains largely controversial. To obtain further insights into molecular mechanisms and to identify novel target genes of 3,5-T2 in liver, we performed a microarray-based liver tissue transcriptome analysis of male lean and diet-induced obese euthyroid mice treated for 4 weeks with a dose of 2.5 µg/g bw 3,5-T2. Our results revealed that 3,5-T2 modulates the expression of genes encoding Phase I and Phase II enzymes as well as Phase III transporters, which play central roles in metabolism and detoxification of xenobiotics. Additionally, 3,5-T2 changes the expression of TH responsive genes, suggesting a thyromimetic action of 3,5-T2 in mouse liver. Interestingly, 3,5-T2 in obese but not in lean mice influences the expression of genes relevant for cholesterol and steroid biosynthesis, suggesting a novel role of 3,5-T2 in steroid metabolism of obese mice. We concluded that treatment with 3,5-T2 in lean and diet-induced obese male mice alters the expression of genes encoding hepatic xenobiotic-metabolizing enzymes that play a substantial role in catabolism and inactivation of xenobiotics and TH and are also involved in hepatic steroid and lipid metabolism. The administration of this high dose of 3,5-T2 might exert adverse hepatic effects. Accordingly, the conceivable use of 3,5-T2 as pharmacological hypolipidemic agent should be considered with caution.
Chen-Tian Shen, Wei-Jun Wei, Zhong-Ling Qiu, Hong-Jun Song, Xin-Yun Zhang, Zhen-Kui Sun and Quan-Yong Luo
More aggressive thyroid cancer cells show a higher activity of glycometabolism. Targeting cancer cell metabolism has emerged as a novel approach to prevent or treat malignant tumors. Glucose metabolism regulation effect of metformin in papillary thyroid cancer was investigated in the current study. Human papillary thyroid carcinoma (PTC) cell lines BCPAP and KTC1 were used. Cell viability was detected by CCK8 assay. Glucose uptake and relative gene expression were measured in metformin (0–10 mM for 48 h)-treated cells by 18F-FDG uptake assay and western blotting analysis, respectively. MicroPET/CT imaging was performed to detect 18F-FDG uptake in vivo. After treatment with metformin at 0, 2.5, 5 and 10 mM for 48 h, the ratio of p-AMPK to total AMPK showed significant rising in a dose-dependent manner in both BCPAP and KTC1, whereas p-AKT and p-mTOR expression level were downregulated. 18F-FDG uptake reduced after metformin treatment in a dose-dependent manner, corresponding to the reduced expression level of HK2 and GLUT1 in vitro. Xenograft model of PTC using BCPAP cells was achieved successfully. MicroPET/CT imaging showed that in vivo 18F-FDG uptake decreased after treatment with metformin. Immunohistochemistry staining further confirmed the reduction of HK2 and GLUT1 expression in the tumor tissue of metformin-treated PTC xenograft model. In conclusion, metformin could reduce glucose metabolism of PTC in vitro and in vivo. Metformin, by targeting glycometabolism of cancer cells, could be a promising adjuvant therapy alternative in the treatment modality of advanced thyroid carcinoma.
A S R Araujo, P Schenkel, A T Enzveiler, T R G Fernandes, W A Partata, S Llesuy, M F M Ribeiro, N Khaper, P K Singal and A Belló-Klein
This study was conducted to test whether oxidative stress activates the intracellular protein kinase B (AKT1) signaling pathway, which culminates with cardiac hypertrophy in experimental hyperthyroidism. Male Wistar rats were divided into four groups: control, vitamin E, thyroxine (T4), and T4+vitamin E. Hyperthyroidism was induced by T4 administration (12 mg/l in drinking water for 28 days). Vitamin E treatment was given during the same period via s.c. injections (20 mg/kg per day). Morphometric and hemodynamic parameters were evaluated at the end of the 4-week treatment period. Protein oxidation, redox state (reduced glutathione, GSH/glutathione dissulfide, GSSG), vitamin C, total radical-trapping antioxidant potential (TRAP), hydrogen peroxide (H2O2), and nitric oxide metabolites (NOX) were measured in heart homogenates. The p-AKT1/AKT1 ratio, p-glycogen-synthase kinase (GSK)3B/GSK3B ratio, FOS, and JUN myocardial protein expression were also quantified by western blot after 4 weeks. Increases in biochemical parameters, such as protein oxidation (41%), H2O2 (62%), and NOX (218%), and increase in the left ventricular end-diastolic pressure were observed in the T4 group. T4 treatment also caused a decrease in GSH/GSSG ratio (83%), vitamin C (34%), and TRAP (55%). These alterations were attenuated by vitamin E administration to the hyperthyroid rats. Expression of p-AKT1/AKT1, p-GSK3B/GSK3B, FOS, and JUN were elevated in the T4 group (by 69, 37, 130, and 33% respectively), whereas vitamin E administration promoted a significant reduction in their expression. These results indicate that oxidative stress plays an important role in cardiac hypertrophy, and suggest redox activation of AKT1 and JUN/FOS signaling pathways with H2O2 acting as a possible intracellular mediator in this adaptive response to experimental hyperthyroidism.
C Massart, J Gibassier, N Genetet, M L Raoul, M Baron, F Le Gall and C Lucas
We studied the lymphocyte-induced alterations in hormonal metabolism and the production of tumour necrosis factor α (TNF-α) during coculture of thyrocytes and autologous lymphocytes from 20 patients with Graves' disease and from five normal subjects. Thyroglobulin (Tg) mRNA was assessed by slot-blot analysis under TSH stimulation. Tg, tri-iodothyronine (T3) and cAMP secretion in the presence of TSH were measured by RIA after 3 or 5 days of coculture. TNF-α levels produced after 5 days incubation were also assayed in lymphocyte culture and coculture media.
Lymphocytes isolated from peripheral blood (PBLs) altered the production of Tg, T3 and cAMP in autologous thyrocytes. Intrathyroidal lymphocytes (ITLs) decreased Tg and cAMP secretion but had no effect on T3 secretion. The reductions in Tg and cAMP levels obtained with mechanically isolated ITLs (M-ITLs) were generally higher than those obtained with ITLs isolated by dispase (D-ITLs). No difference was seen between Graves' disease and normal cocultures. PBLs secreted large concentrations of TNF-α, larger than those obtained with M-ITLs whereas D-ITLs produced low amounts of this cytokine. In coculture, TNF-α levels were lower than those observed in lymphocyte culture. Significant correlations were obtained between TNF-α levels and the decrease in Tg, T3 and cAMP concentrations. The percentage of T lymphocytes was higher in PBLs and D-ITLs than in M-ITLs. B lymphocytes levels were higher in ITLs, especially M-ITLs, than in PBLs. TNF-α production by B lymphocytes was maximal in M-ITLs.
In conclusion, lymphocytes induced a decrease in hormonal thyroid metabolism when cocultured with autologous thyrocytes. These perturbations may be attributed, at least partly, to TNF-α secreted by lymphocytes. TNF-α interacts via the adenylate cyclase pathway of TSH signal transduction.
X Shen, QL Li, GA Brent and TC Friedman
Most pro-neuropeptides are processed by the prohormone convertases, PC1 and PC2. We previously reported that changes in thyroid status altered anterior pituitary PC1 mRNA and this regulation was due to triiodothyronine (T(3))-dependent interaction of thyroid hormone receptor (TR) with negative thyroid hormone response elements (nTREs) contained in a large region of the human PC1 promoter. In this study, we demonstrated that hypothyroidism stimulated, while hyperthyroidism suppressed, PC1 mRNA levels in rat hypothalamus and cerebral cortex, but not in hippocampus. In situ hybridization was used to confirm real-time PCR changes and localize the regulation within the hypothalamus and cortex. Using a human PC1 (hPC1) promoter construct (with and without deletions in two regions that each contain a negative TRE) transiently transfected into GH3 cells, we found that T(3) negatively regulated hPC1 promoter activity, and this regulation required both of these two regions. Electrophoretic mobility shift assays (EMSAs) using purified thyroid hormone receptor alpha1 (TRalpha1) and retinoid X receptor beta (RXRbeta) proteins demonstrated that RXR and TRalpha both bound the PC1 promoter. Addition of TRalpha1/RXRbeta to the wild-type PC1 probe demonstrated binding as both homodimers and a heterodimer. EMSAs with oligonucleotides containing deletion mutations of the putative nTREs demonstrated that the proximal nTRE binds more strongly to TR and RXR than the distal nTRE, but that both regions exhibit specific binding. We conclude that there are multiple novel TRE-like sequences in the hPC1 promoter and that these regions act in a unique manner to facilitate the negative effect of thyroid hormone on PC1.