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Therapeutic potential of metformin in obese/diabetic patients has been associated to its ability to combat insulin resistance. However, it remains largely unknown the signaling pathways involved and whether some cell types are particularly relevant for its beneficial effects. M1-activation of macrophages by bacterial lipopolysaccharide (LPS) promotes a paracrine activation of hypoxia-inducible factor-1α (HIF1α) in brown adipocytes which reduces insulin signaling and glucose uptake, as well as β-adrenergic sensitivity. Addition of metformin to M1-polarized macrophages blunted these signs of brown adipocyte dysfunction. At the molecular level, metformin inhibits an inflammatory program executed by HIF1α in macrophages by inducing its degradation through the inhibition of mitochondrial complex I activity, thereby reducing oxygen consumption in a reactive oxygen species (ROS)-independent manner. In obese mice, metformin reduced inflammatory features in brown adipose tissue (BAT) such as macrophage infiltration, proinflammatory signaling and gene expression, and restored the response to cold exposure. In conclusion, the impact of metformin on macrophages by suppressing a HIF1α-dependent proinflammatory program is likely responsible for a secondary beneficial effect on insulin-mediated glucose uptake and β-adrenergic responses in brown adipocytes., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) is the most common primary liver tumor. Hexachlorobenzene (HCB) is an endocrine disruptor and a liver tumor promoter. Deregulation of thyroid hormone (TH) homeostasis may play a significant role in early neoplastic transformation. The aim of this study was to evaluate the relation between TH metabolism and the regulation of cell growth in an in vivo and in vitro model. We examined the role of transforming growth factor-β1 (TGF-β1) on TH deiodinase expression and hepatocyte proliferation. An initiation (DEN)/promotion (HCB) tumor model from rat liver and HepG2 cells were used. We evaluated PCNA, p21, p27, SMAD2/3, TGF-β1, deiodinase 1 (D1), D3, protein expression levels; D1 and D3 mRNA expression; TH and TGF-β1, D1, D3, and GST-P protein levels in focal/non-focal areas. In vivo, HCB decreased triiodothyronine (T3) and D1 mRNA levels and increased thyroxine (T4) and D3 mRNA levels in liver from DEN+HCB vs. DEN group. HCB increased protein levels from D3, TGF-β1, and PCNA and decreased D1 in focal-areas. In vitro, HCB increased PCNA, pSMAD 2/3, and TGF-β1 protein levels and mRNA expression and decreased p21 and p27 protein levels. Exogenous T3 treatment prevent HCB induced molecular alterations related to hepatocyte proliferation whereas T4 did not have any effect. These effects were prevented by using a TGF-β1 receptor II inhibitor. Results suggest that alteration of TH homeostasis, through D1 function, play a key role in hepatocyte proliferation and that TGF-β1-SMAD pathway is involved in this process confirming their role in early neoplastic transformation in HCC.

Aims/hypothesis: Pleiotrophin, a developmentally regulated and highly conserved cytokine, exerts different functions including regulation of cell growth and survival. Here, we hypothesise that this cytokine can play a regulatory role in glucose and lipid homeostasis., Methods: To test this hypothesis, we performed a longitudinal study characterising the metabolic profile (circulating variables and tissue mRNA expression) of gene-targeted Ptn-deficient female mice and their corresponding wild-type counterparts at different ages from young adulthood (3 months) to older age (15 months). Metabolic cages were used to investigate the respiratory exchange ratio and energy expenditure, at both 24°C and 30°C. Undifferentiated immortalised mouse brown adipocytes (mBAs) were treated with 0.1 μg/ml pleiotrophin until day 6 of differentiation, and markers of mBA differentiation were analysed by quantitative real-time PCR (qPCR)., Results: Ptn deletion was associated with a reduction in total body fat (20.2% in Ptn +/+ vs 13.9% in Ptn -/- mice) and an enhanced lipolytic response to isoprenaline in isolated adipocytes from 15-month-old mice (189% in Ptn +/+ vs 273% in Ptn -/- mice). We found that Ptn -/- mice exhibited a significantly lower QUICKI value and an altered lipid profile; plasma triacylglycerols and NEFA did not increase with age, as happens in Ptn +/+ mice. Furthermore, the contribution of cold-induced thermogenesis to energy expenditure was greater in Ptn -/- than Ptn +/+ mice (42.6% and 33.6%, respectively). Body temperature and the activity and expression of deiodinase, T 3 and mitochondrial uncoupling protein-1 in the brown adipose tissue of Ptn -/- mice were higher than in wild-type controls. Finally, supplementing brown pre-adipocytes with pleiotrophin decreased the expression of the brown adipocyte markers Cidea (20% reduction), Prdm16 (21% reduction), and Pgc1-α (also known as Ppargc1a, 11% reduction)., Conclusions/interpretation: Our results reveal for the first time that pleiotrophin is a key player in preserving insulin sensitivity, driving the dynamics of adipose tissue lipid turnover and plasticity, and regulating energy metabolism and thermogenesis. These findings open therapeutic avenues for the treatment of metabolic disorders by targeting pleiotrophin in the crosstalk between white and brown adipose tissue.

Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-β1) and thyroid hormones (TH) regulation., Introduction and Aim: Hepatocellular carcinoma (HCC) represents 90% of liver tumors. Statins, may reduce the incidence of various tumors, including HCC. Antitumoral activities may be mediated by changes in transforming growth factor-beta (TGF-β1) and thyroid hormones (TH) regulation. Aim. The aim of our study is to establish the statins mechanism of action and the potential key molecules involved in an in vivo and in vitro HCC model., Materials and Methods: We used two models: in vivo (in rats) using diethylnitrosamine (DEN) and hexachlorobenzene (HCB) to develop HCC. We analyzed cell proliferation parameters (proliferating cel nuclear antigen, PCNA) and cholesterol metabolism (hydroxy-methylglutaryl-CoA reductase, HMGCoAR). In vitro (Hep-G2 cells) we evaluated the effects of different doses of Atorvastatin (AT) and Simvastatin (SM) on HCB induced proliferation and analyzed proliferative parameters, cholesterol metabolism, TGF-β1 mRNA, c-Src and TH levels., Results: In vivo, we observed that cell proliferation significantly increased as well as cholesterol serum levels in rats treated with HCB. In vitro, we observed the same results on PCNA as in vivo. The statins prevented the increase in HMG-CoAR mRNA levels induced by HCB, reaching levels similar to controls at maximum doses: AT (30 μM), and SM (20 μM). Increases in PCNA, TGF-β1, and pc-Src, and decreases in deiodinase I mRNA levels induced by HCB were not observed when cells were pre-treated with AT and SM at maximum doses., Conclusion: Statins can prevent the proliferative HCB effects on Hep-G2 cells. TGF-β1, c-Src and TH may be the statins molecular targets in hepatocarcinogenesis.

Thyroid hormone deficiency at early postnatal ages affects the cytoarchitecture and function of neocortical and telencephalic limbic areas, leading to impaired associative memory and in a wide spectrum of neurological and mental diseases. Neocortical areas project interhemispheric axons mostly through the corpus callosum and to a lesser extent through the anterior commissure (AC), while limbic areas mostly project through the AC and hippocampal commissures. Functional magnetic resonance data from children with late diagnosed congenital hypothyroidism and abnormal verbal memory processing, suggest altered ipsilateral and contralateral telencephalic connections. Gestational hypothyroidism affects AC development but the possible effect of transient and chronic postnatal hypothyroidism, as occurs in late diagnosed neonates with congenital hypothyroidism and in children growing up in iodine deficient areas, still remains unknown. We studied AC development using in vivo magnetic resonance imaging and electron microscopy in hypothyroid and control male rats. Four groups of methimazole (MMI) treated rats were studied. One group was MMI-treated from postnatal day (P) 0 to P21; some of these rats were also treated with L-thyroxine (T4) from P15 to P21, as a model for early transient hypothyroidism. Other rats were MMI-treated from P0 to P150 and from embryonic day (E) 10 to P170, as a chronic hypothyroidism group. The results were compared with age paired control rats. The normalized T2 signal using magnetic resonance image was higher in MMI-treated rats and correlated with the number and percentage of myelinated axons. Using electron microscopy, we observed decreased myelinated axon number and density in transient and chronic hypothyroid rats at P150, unmyelinated axon number increased slightly in chronic hypothyroid rats. In MMI-treated rats, the myelinated axon g-ratio and conduction velocity was similar to control rats, but with a decrease in conduction delays. These data show that early postnatal transient and chronic hypothyroidism alters AC maturation that may affect the transfer of information through the AC. The alterations cannot be recovered after delayed T4-treatment. Our data support the neurocognitive delay found in late T4-treated children with congenital hypothyroidism.

Background: The possibility that the intrinsic genomic activity of thyroxine (T4) is of physiological relevance has been frequently hypothesized. It might explain gene expression patterns in the brain found in type 2-deiodinase (Dio2)-deficient mice. These mice display normal expression of most thyroid hormone-dependent genes, despite decreased brain triiodothyronine (T3)., Methods: The relative effects of T4 and T3 on gene expression were analyzed in mouse neuro-2a (N2a) cells stably expressing the thyroid hormone receptor α1, and in primary mouse cerebrocortical cells enriched in astrocytes or in neurons. Cortical cells were derived from Dio2-deficient mice to prevent conversion of T4 to T3. T4 and T3 were measured in the media at the beginning and end of incubation, and T4 and T3 antibodies were used to block T4 and T3 action., Results: In all cell types, T4 had intrinsic genomic activity. In N2a cells, T4 activity was higher on negative regulation (1/5th of T3 activity) than on positive regulation (1/40th of T3 activity). T4 activity on positive regulation was dependent on the cell context, and was higher in primary cells than in N2a cells., Conclusion: T4 has intrinsic genomic activity. Positive regulation depends on the cell context, and primary cells appear much more sensitive than neuroblastoma cells. In all cells, negative regulation is more sensitive to T4 than positive regulation. These properties may explain the mostly normal gene expression in the brain of Dio2-deficient mice.

The discovery of active brown adipose tissue (BAT) in adult humans and the fact that it is reduced in obese and diabetic patients have put a spotlight on this tissue as a key player in obesity-induced metabolic disorders. BAT regulates energy expenditure through thermogenesis; therefore, harnessing its thermogenic fat-burning power is an attractive therapeutic approach. We aimed to enhance BAT thermogenesis by increasing its fatty acid oxidation (FAO) rate. Thus, we expressed carnitine palmitoyltransferase 1AM (CPT1AM), a permanently active mutant form of CPT1A (the rate-limiting enzyme in FAO), in a rat brown adipocyte (rBA) cell line through adenoviral infection. We found that CPT1AM-expressing rBA have increased FAO, lipolysis, UCP1 protein levels and mitochondrial activity. Additionally, enhanced FAO reduced the palmitate-induced increase in triglyceride content and the expression of obese and inflammatory markers. Thus, CPT1AM-expressing rBA had enhanced fat-burning capacity and improved lipid-induced derangements. This indicates that CPT1AM-mediated increase in brown adipocytes FAO may be a new approach to the treatment of obesity-induced disorders.

This review briefly summarizes: (1) the changes in maternal thyroid function that are imposed by the presence of the fetus and the high concentrations of human chorionic gonadotropin essential for the maintenance of the pregnancy, which result in high first trimester free thyroxine and triiodothyronine, requiring doubling of the iodine intake; (2) the changes in the fetal compartment up to midgestation, which result in increasing concentrations of triiodothyronine in the cerebral cortex generated locally from thyroxine by high activities of type 2 iodothyronine deiodinase; (3) the important role of the maternal contribution of thyroxine to the fetal circulation after onset of secretion of hormones by the fetal thyroid; and (4) the consequences of the interruption of the maternal supply of thyroid hormones that occur with prematurity. Efforts to devise appropriate strategies to avoid or shorten the postnatal hypothyroxinemia of infants born prematurely may well result in fewer and less severe neurodevelopmental deficits.

An inadequate supply of iodine during gestation results in damage to the foetal brain that is irreversible by mid-gestation unless timely interventions can correct the accompanying maternal hypothyroxinemia. Even mild to moderate maternal hypothyroxinemia may result in suboptimal neurodevelopment. This review mainly focuses on iodine and thyroid hormone economy up to mid-gestation, a period during which the mother is the only source for the developing brain of the foetus. The cerebral cortex of the foetus depends on maternal thyroxine (T4) for the production of the 3',3,5-tri-iodothyronine (T3) for nuclear receptor-binding and biological effectiveness. Maternal hypothyroxinemia early in pregnancy is potentially damaging for foetal brain development. Direct evidence has been obtained from experiments on animals: even a relatively mild and transient hypothyroxinemia during corticogenesis, which takes place mostly before mid-gestation in humans, affects the migration of radial neurons, which settle permanently in heterotopic locations within the cortex and hippocampus. Behavioural defects have also been detected. The conceptus imposes important early changes on maternal thyroid hormone economy that practically doubles the amount of T4 secreted something that requires a concordant increase in the availability of iodine, from 150 to 250-300 microg I day- 1. Women who are unable to increase their production of T4 early in pregnancy constitute a population at risk for having children with neurological disabilities. As a mild to moderate iodine deficiency is still the most widespread cause of maternal hypothyroxinemia, the birth of many children with learning disabilities may be prevented by advising women to take iodine supplements as soon as pregnancy starts, or earlier if possible, in order to ensure that their requirements for iodine are met.

Iodine deficiency remains the most frequent cause worldwide, after starvation, of preventable mental retardation in children. It causes maternal hypothyroxinemia, which affects pregnant women even in apparently iodine-sufficient areas, and often goes unnoticed because L-thyroxine (T4) levels remain within the normal range, and thyroid-stimulating hormone (TSH) is not increased. Even a mild hypothyroxinemia during pregnancy increases the risk of neurodevelopmental abnormalities, and experimental data clearly demonstrate that it damages the cortical cytoarchitecture of the fetal brain. The American Thyroid Association (ATA) recommends a supplement of 150 microg iodine/day during pregnancy and lactation, in addition to the use of iodized salt. We discuss the importance of iodine supplementation to ensure adequate T4 levels in all women who are considering conception and throughout pregnancy and lactation.

Treatment of male Wistar rats with hexachlorobenzene (HCB) (1000 mg/kg b.w.) for 3-30 days decreases circulating levels of thyroxine (T4) but does not affect triiodothyronine (T3). Time courses were determined for 5' deiodinase type I (5' D-I) activity in thyroid, liver, and kidney and 5' deiodinase type II (5' D-II) activity in brown adipose tissue (BAT) to test the possibility that increased deiodinase activity might contribute to the maintenance of the serum T3 level. Specific 5' D-I activity was increased in the thyroid at 21 days and thereafter. No significant changes were observed in the liver, however, total 5' D-I activity in this tissue was increased at 30 days of treatment as a consequence of liver weight enhancement. HCB decreased kidney 5' D-I activity after 15 days, and BAT 5' D-II activity after 21 days of treatment. Total body 5' D-I activity was significantly increased by 30 days of HCB-treatment. HCB increased the activity of hepatic T4 uridine diphosphoglucuronosyl transferase (UDPGT) in a time-dependent manner, without changes in T3 UDPGT. We propose that increased T4 to T3 conversion in the thyroid and in the greatly enlarged liver may account for the maintenance of serum T3 concentration in hypothyroxinemic HCB-treated rats.

Type II 5' deiodinase (D2) activity produces triiodothyronine (T3) from thyroxine (T4) and is induced by cold and norepinephrine (NE) in brown adipose tissue. T3 is required for and amplifies the adrenergic stimulation of D2 activity and mRNA in cultured brown adipocytes. D2 is upregulated by insulin and decrease in fasting. We now study the regulation by insulin of the adrenergically induced D2 activity and mRNA in primary cultures of rat brown adipocytes. Insulin alone does not increase D2 activity or mRNA. Insulin-depleted cells show a reduction in the adrenergically induced D2 activity, which is proportional to the length of insulin depletion and is restored after insulin addition. IGFs mimic this effect at higher doses. ERK 1/2 MAPK activity (p44/p42), stimulated by insulin, serum and NE, is an absolute requirement for the adrenergic stimulation of D2 activity and mRNA. PI3K is stimulated by insulin and serum, and NE increases the effect of insulin. The action of insulin on D2 is not due to changes in D2 half-life or in the proteasome-mediated degradation of D2, but it seems to modulate the transcriptional induction mediated by NE. D2 mRNA expression, induced by NE plus T3, is reduced when insulin is withdrawn at early differentiation stages. Insulin or IGF-I promotes increases in D2 mRNA. Insulin is required for the induction of D2 mRNA by T3. In conclusion, MAPK signaling is required for the adrenergic stimulation of D2 activity and mRNA, and insulin stimulates D2 activity via MAPK and PI3K and enhances the adrenergic pathways.

During the last few decades our understanding of the possible role of thyroid hormones during brain development has increased and contributed to resolve previously discordant hypotheses, although much remains to be clarified. Thyroid hormones of maternal origin are present in the fetal compartment, despite the very efficient uterine-placental 'barrier', necessary to avoid potentially toxic concentrations of free T4 and T3 from reaching fetal tissues before they are required for development. T3 remains low throughout pregnancy, whereas FT4 in fetal fluids increases rapidly to adult levels, and is determined by the maternal availability of T4. It is present in embryonic fluids 4 weeks after conception, with FT4 steadily increasing to biologically relevant values. T3, generated from T4 in the cerebral cortex, reaches adult values by mid-gestation and is partly bound to specific nuclear receptor isoforms. Iodothyronine deioidinases are important for the spatial and temporal regulation of T3 bioavailability, tailored to the differing and changing requirements of thyroid hormone-sensitive genes in different brain structures, but other regulatory mechanism(s) are likely to be involved. Maternal transfer constitutes a major fraction of fetal serum T4, even after onset of fetal thyroid secretion, and continues to have an important protective role in fetal neurodevelopment until birth. Prompt treatment of maternal hypothyroidism, identified by increased TSH, is being advocated to mitigate a negative effect on the woman and her child. However, even a moderate transient period of maternal hypothyroxinemia at the beginning of rat neurogenesis disrupts neuronal migration into cortical layers. These findings reinforce the epidemiological evidence that early maternal hypothyroxinemia-when neuronal migratory waves are starting-is potentially damaging for the child. Detection of an inappropiate first trimester FT4 surge that may not result in increased TSH, may be crucial for the prevention of learning disabilities in a significant number of unborn children.

Leptin regulates appetite, inhibits food intake, and seems to increase energy expenditure. We investigated the effect of triiodothyroacetic acid (Triac), a metabolite of T3, which seems to be more thermogenic than T3, on leptin secretion and mRNA expression. Rat primary cultures of white and brown adipocytes were treated with increasing concentrations of Triac and T3. The effect of different types of serum and insulin concentrations was also tested. Serum inhibited leptin secretion and mRNA expression. Leptin secretion was also clearly inhibited by Triac and T3 in a dose-dependent manner and with similar potency. In the presence of norepinephrine (NE), Triac and T3 had a similar inhibitory effect, but the inhibition was almost complete in white adipocytes. Parallel results were found at the mRNA level, where Triac and T3 had similar inhibitory potency, both alone and with NE. We also show that insulin induced dose- and time-dependent increases in leptin secretion, reaching maximum levels at 0.5 and 3 nM insulin for white and brown adipocytes, respectively. Leptin secretion was higher in white than in brown adipocytes. The increases in leptin secretion were preceded by increases in leptin mRNA. In conclusion, these data demonstrate for the first time that Triac, like T3 and serum, inhibits leptin secretion and expression in white and brown adipocytes, whereas insulin has the opposite effect.

Triiodothyroacetic acid (TRIAC) is a triiodothyronine (T3) metabolite with high affinity for T3 nuclear receptors. We compared the thermogenic action of TRIAC versus T3 in brown adipocytes, by studying target genes known to mediate thermogenic action: uncoupling protein 1 (UCP-1), a marker of brown adipocytes, and type II-5'deiodinase (D2), which provides the T3 required for thermogenesis. TRIAC is 10-50 times more potent than T3 at increasing the adrenergic induction of UCP-1 mRNA and D2 activities. TRIAC action on UCP-1 is exerted at the transcriptional level. In the presence of an adrenergic stimulus, TRIAC is also more potent than T3, inducing lipoprotein lipase mRNA and 5 deiodinase (D3) activity and mRNA. Maximal effects occur at very low concentrations (0.2 nM). The greater potency of TRIAC is not due to preferential cellular or nuclear uptake. Therefore, TRIAC is a potent thermogenic agent that might increase energy expenditure and regulate T3 production in brown adipocytes.

The effect of treatment with thyroxine (T(4)) on the hepatic deiodinase (5'D-I) activity and triiodothyronine (T(3)) content and on insulin-like growth factor-I (IGF-I) secretion and mRNA hepatic expression were studied in neonatal and adult diabetic (D) rats and compared with 4 thyroidectomized (Tx) groups: neonatal and adult Tx rats treated or not with T(4). Serum T(3) and T(4) decreased by 92% in both Tx populations and by 80% to 70% in D adults according to the severity of diabetes: -70 mg/kg body weight (BW) (D(70)) or 50 mg/kg BW (D(50)) of streptozotocin (STZ) injected, whereas only a 30% to 33% decrease was found in D neonates. A similar decrease of liver 5'D-I activity and T(3) concentrations was found in neonatal and adult Tx rats, whereas a significant reduction in those parameters was observed only in adult diabetics, either D(70) or D(50), but not in D neonates. Serum levels and liver mRNA expression of IGF-I determined by ribonuclease protection assay, plasma and pituitary growth hormone (GH), plasma insulin, and glycemia were also measured in both D populations. A decrease in circulating IGF-I, previously reported for Tx adult rats, was also found in both D populations. T(4) treatment recovered IGF-I and liver T(3) in both Tx groups and D neonates, but not in D adults. These results show an age-dependent adaptation of the liver thyroid economy in diabetes, as hepatic 5'D-I does not respond to diabetes in neonates and IGF-I is insensitive to T(4) treatment in adult diabetics and suggest a positive correlation between hepatic T(3) content and IGF-I expression in conditions of diabetes and Tx.

Brown fat differentiation in mice is fully achieved in fetuses at term and entails the acquisition of not only adipogenic but also thermogenic and oxidative mitochondrial capacities. The present study of the mice homozygous for a deletion in the gene for CCAAT/enhancer-binding protein alpha (C/EBPalpha-null mice) demonstrates that C/EBPalpha is essential for all of these processes. Developing brown fat from C/EBPalpha-null mice showed a lack of uncoupling protein-1 expression, impaired adipogenesis, and reduced size and number of mitochondria per cell when compared with wild-type mice. Furthermore, immature mitochondrial morphology was found in brown fat, but not in liver or heart, from C/EBPalpha-null mice. Concordantly, expression of both nuclear and mitochondrial genome-encoded genes for mitochondrial proteins was reduced in C/EBPalpha-null brown fat, although expression of mitochondrial rRNA and mitochondrial DNA content were unaltered. Expression of nuclear respiratory factor-2, thyroid hormone nuclear receptors, and peroxisome proliferator-activated receptor gamma coactivator-1, was delayed in C/EBPalpha-null brown fat. Iodothyronine 5'-deiodinase activity and thyroid hormone content were also reduced in brown fat from C/EBPalpha-null mice, indicating for the first time a crucial role for C/EBPalpha in controlling thyroid status in developing brown fat, which may contribute to impaired mitochondrial biogenesis and cell differentiation. When survival of C/EBPalpha-null mice was achieved by transgenically expressing C/EBPalpha only in the liver, a substantial recovery in brown fat differentiation was found by day 7 of postnatal age, which is associated with a compensatory overexpression of C/EBPdelta and C/EBPbeta.

Type II 5'-iodothyronine deiodinase (D2), produces triiodothyronine (T(3)) and is stimulated by cold exposure via norepinephrine (NE) release in brown adipose tissue. Cultured rat brown adipocytes require T(3) for the adrenergic stimulation of D2 activity. D2 mRNA expression in cultured brown adipocytes is undetectable with the use of basal conditions or NE without T(3). Full D2 expression is achieved using NE + T(3), especially after prolonged T(3) exposure. beta(3)-Adrenergic agonists mimic the NE action, whereas cAMP analogs do not. Prolonged exposure to T(3) alone increases D2 mRNA. High T(3) doses (500 nM) inhibit the adrenergic stimulation of D2 activity while increasing D2 mRNA. The effects obtained with NE + T(3) or T(3) alone are suppressed by actinomycin, but not by cycloheximide, which leads to accumulation of short D2 mRNA transcripts. Prolonged or short exposure to T(3) did not change D2 mRNA half-life, but T(3) seemed to elongate it. In conclusion, T(3) is an absolute requirement for the adrenergic stimulation of D2 mRNA in brown adipocytes. T(3) upregulates D2 mRNA, an effect that might involve stimulation of factors required for transcription or for stabilization of D2 mRNA.

To study the effect of the mitogens epidermal growth factor (EGF), acidic and basic fibroblast growth factors (aFGF and bFGF), and vasopressin on brown adipocyte differentiation, we analyzed the expression of uncoupling protein-1 (UCP-1) mRNA. Quiescent brown preadipocytes express high levels of UCP-1 mRNA in response to triiodothyronine (T3) and norepinephrine (NE). The addition of serum or the mitogenic condition aFGF + vasopressin + NE or EGF + vasopressin + NE decreases UCP-1 mRNA. A second addition of mitogens further decreases UCP-1 mRNA. Treatment with aFGF or bFGF alone increases UCP-1 mRNA, whereas the addition of EGF or vasopressin dramatically reduces UCP-1 mRNA levels. The continuous presence of T3 increases UCP-1 mRNA levels in cells treated with EGF, aFGF, or bFGF. The effect of T3 on the stimulation of DNA synthesis also was tested. T3 inhibits the mitogenic activity of aFGF and bFGF. In conclusion, mitogens like aFGF or bFGF allow brown adipocyte differentiation, whereas EGF and vasopressin inhibit the differentiation process. T3 behaves as an important hormone that regulates both brown adipocyte proliferation and differentiation.

Is the fetal thyroid already capable to increase its iodide uptake in response to iodine deficiency? To answer this question, we analyzed the expression of the Na(+)/I(-) symporter and several other genes in the thyroid of rat fetuses at 21 d of gestation from control mothers presenting a mild or more severe iodine deficiency. Female rats were placed on a low iodine diet, not supplemented, or supplemented with iodide or perchlorate for 3 months. The maternal and fetal thyroidal iodide uptake was measured 24 h after injection of 10 microCi Na (125)I into the dams. The absolute iodide uptake of the maternal thyroid was unchanged in a low iodine diet, not supplemented, compared with one supplemented with iodide. In contrast, the fetal thyroid absolute iodide uptake of a low iodine diet, not supplemented, and one supplemented with perchlorate was decreased by 70% and 95% compared with that supplemented with iodide. Na(+)/I(-) symporter mRNA was detected in the fetal thyroid of supplemented with iodide and increased about 2- and 4- fold in the thyroid of fetuses from a low iodine diet, not supplemented, and one supplemented with perchlorate, respectively. Na(+)/I(-) symporter expression was induced in the fetal side of the placenta in both a low iodine diet, not supplemented, and one supplemented with perchlorate; in contrast, Na(+)/I(-) symporter mRNA was never detected in the maternal side of the placenta. Fetal thyroid thyroglobulin and type I deiodinase mRNA contents were only significantly increased with a diet supplemented with perchlorate. Glucose transporter 4 mRNA was decreased in the fetal thyroid of both a low iodine diet, not supplemented, and one supplemented with perchlorate compared with one supplemented with iodide. In conclusion, although the up-regulation of Na(+)/I(-) symporter expression in fetal thyroid and placenta in the low iodine diet, not supplemented group did not lead to restoration of a normal absolute iodide uptake, our data show that all adaptive and/or defending mechanisms against iodine deficiency are already present in the fetus.

The influence of hypothalamic and pituitary type II 5'deiodinase (5'D-II) activities and T3 content on pituitary TSH content was investigated in streptozotocin (STZ)-induced diabetic rats (D). The results show, first, that hypothalamic and pituitary 5'D-II activities were lower in neonatal D rats versus control (C) rats, and the normal developmental pattern was altered. Secondly, when D and C rats were thyroidectomized (Tx) at 25 days of age (D+Tx, C+Tx), pituitary and hypothalamic 5'D-II activities increased ten days later in both populations vs. intact rats, but the percentage of increase was smaller in D+Tx than in C+Tx. The hypothalamic T3 to T4 ratios were also decreased in D+Tx animals (0.38) as compared to C+Tx rats (1.64). The hypothalamic T3 content was reduced by 30% in D as compared to C rats and by 80% in D+Tx as compared to C+Tx rats, showing a defect in hypothalamic T4 deiodination. Pituitary TSH content increased after Tx in D+Tx, but not in C+Tx. These results in diabetic rats indicate that the hypothalamic and pituitary 5'D-II activity and hypothalamic T3 content are affected by diabetes and play a role in the regulation of pituitary TSH content.

Several recent publications have drawn attention to the role of the thyroid hormone status of the mother on the future neuropsychological development of the child. The screening of pregnant women for clinical or subclinical hypothyroidism based on second trimester elevated maternal TSH values has been proposed. Here, we have summarized present epidemiological and experimental evidence strongly suggesting that conditions resulting in first trimester hypothyroxinemia (a low for gestational age circulating maternal free T4, whether or not TSH is increased) pose an increased risk for poor neuropsychological development of the fetus. This would be a consequence of decreased availability of maternal T4 to the developing brain, its only source of thyroid hormone during the first trimester; T4 is the required substrate for the ontogenically regulated generation of T3 in the amounts needed for optimal development in different brain structures, both temporally and spatially. Normal maternal T3 concentrations do not seem to prevent the potential damage of a low supply of T4, although they might prevent an increase in circulating TSH and detection of the hypothyroxinemia if only TSH is measured. Hypothyroxinemia seems to be much more frequent in pregnant women than either clinical or subclinical hypothyroidism and autoimmune thyroid disease, especially in regions where the iodine intake of the pregnant woman is inadequate to meet her increased needs for T4. It is proposed that the screening of pregnant women for thyroid disorders should include the determination of free T4 as soon as possible during the first trimester as a major test, because hypothyroxinemia has been related to poor developmental outcome, irrespective of the presence of high titers of thyroid autoantibodies or elevated serum TSH. The frequency with which this may occur is probably 150 times or more that of congenital hypothyroidism, for which successful screening programs have been instituted in many countries.

Uncoupling protein (UCP), the mitochondrial protein specific to brown adipose tissue, is activated transcriptionally in response to cold and adrenergic agents. We studied the role of triiodothyronine (T(3)) on the adrenergic stimulation of UCP mRNA expression by use of primary cultures of rat brown adipocytes. Basal UCP mRNA levels are undetectable. Norepinephrine (NE) increases UCP mRNA during differentiation, not during proliferation. In hypothyroid conditions, UCP mRNA response to NE is almost absent. The presence of T(3) (0.2-20 nM) greatly increases the adrenergic response (30-fold). The sensitivity of UCP mRNA responses to NE is potentiated approximately 100-fold by the presence of T(3). The effect is proportional to the dose and time of preexposure to T(3). The increases obtained with NE and T(3) are prevented by actinomycin and cycloheximide. T(3) greatly stabilizes UCP mRNA transcripts. The effects of thyroxine and retinoic acid are weaker than those of T(3). In conclusion, in cultured rat brown adipocytes, T(3) is required and both synergizes with NE to increase UCP mRNA and stabilizes its mRNA transcripts.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was studied in differentiating brown adipocytes. Northern blot analysis showed that GAPDH mRNA levels increased during differentiation of precursor cells into mature adipocytes, mainly in the initial stages of the differentiation process. Insulin, tri-iodothyronine (T(3)) and norepinephrine, the main regulators of brown adipose tissue function, upregulated GAPDH mRNA levels, whereas retinoic acid inhibited them. The effect of insulin was present on all culture days examined, was time- and dose-dependent, and was exerted through its own receptors, as demonstrated by comparing insulin and insulin-like growth factor (IGF)-I and -II potencies in this system. Using the transcriptional inhibitor, actinomycin D, we demonstrated that T(3), and to a lesser extent insulin, stabilized GAPDH mRNA. Experiments with cycloheximide indicated that both hormones require de novo protein synthesis to achieve their effects. Using cAMP analogs, we showed that the effect of norepinephrine is probably exerted through this second messenger. Co-operation was elucidated between norepinephrine- and insulin-mediated induction of GAPDH mRNA levels. In summary, we have demonstrated that GAPDH mRNA is subjected to multifactorial regulation in differentiating brown adipocytes that includes differentiation of precursor cells and the lipogenic/lipolytic regulators of the tissue.

We have measured 3,5,3'triiodothyronine (T3) in 12 tissues from thyroidectomized (Tx) rats infused with increasing doses of T3, and related them to their corresponding plasma levels. Young adult Wistar rats were surgically Tx. After 4 weeks, the animals were infused with placebo or T3 (0.25, 0.50, 0.75, 1.00 or 2.00 microg/100 g body weight/day). Placebo-infused intact rats served as euthyroid controls. Plasma and samples of cerebral cortex, cerebellum, brown adipose tissue (BAT), pituitary, liver, heart, lung, kidney, spleen, skeletal muscle, ovary and adrenal were obtained after 12-13 days of infusion. We determined plasma T3 and thyrotropin (TSH), and tissue T3 and thyroxine (T4), the latter being virtually undetectable. Results were compared with the relationships between tissue and plasma T3 in Tx rats on T4 infusions. Most tissues presented changes which paralleled those in plasma T3, irrespective of its source (infusion of T3, or generation from infused T4). However, at similar plasma T3 concentrations, cerebral cortex, cerebellum and BAT (containing type II 5' iodothyronine deiodinase (DII) activity), reached much lower T3 levels in the T3-infused Tx rats, than in Tx rats on T4, and required elevated plasma T3 levels for normal tissue T3. In these tissues, and in the pituitary, T3 concentrations were always lower than expected from plasma T3 levels. On the contrary, the lung and ovary of the T3-infused Tx rats contained more T3 than expected from plasma T3. Unexpectedly, both the ovary and adrenal attained higher tissue T3 concentrations in Tx rats on T3 than on T4 at comparable plasma T3 levels. In conclusion, the patterns of changes of the concentrations of T3 as a function of increasing plasma T3 are not only tissue-specific when T4 is provided, but also when circulating T3 is the only source of this iodothyronine. Further studies are needed to identify the mechanisms involved in the regulation of tissue T3 concentrations.

We have studied T4 and T3 concentrations, DNA and protein concentrations and 5' and 5 deiodinases in samples of brain tumors obtained at surgery from 49 patients, and, in most cases, also from surrounding normal tissue. T4 concentrations in normal cortical tissue (6.19+/-0.45 ng/g) were lower than in white matter, but the difference disappeared when referred to the DNA content (2.26+/-0.27 ng/mg DNA). No other differences were found between cortical and white matter, or among cortical lobes. T4 in normal tissue was higher than previously reported, mostly from autopsy samples, whereas T3 (0.99+/-0.07 ng/g) was similar. 5'D-I activity was negligible as compared to 5'D-II (8.11+/-1.09 fmol/h/mg protein). When expressed in relation to the different DNA contents of normal vs. tumoral tissue, 5'D-II activities were the same for both. 5D activity was highly variable in the tumoral tissue, with negligible activities in meningiomas and pituitary adenomas. When referred to the DNA content, T4 and 5'D-II were the same, but T3 concentrations were lower in the tumor (0.24+/-0.03 ng/mg DNA) as compared to normal (0.35+/-0.04 ng/mg DNA) tissue samples. Whether or not this decrease of T3 affects the expression of T3-sensitive processes remains to be studied., (Copyright 1998 Elsevier Science B.V.)

Iodothyronine deiodinases, types I, II, and III (D1, D2, and D3) activities were measured in tissues of fetal rats, at 18 and 21 days of gestation, at several levels of iodine deficiency (ID): mild ID diet (MID) and moderately severe ID, MID + 0.005% perchlorate (MID+P). D2 was present in fetal skin, increased between days 18 and 21, and also in MID and MID+P. In skin, D3 increased during ID at day 18, whereas there was a decrease at day 21. Skin T4 decreased in MID and MID+P, showing an inverse relationship with D2. Skin T3 decreased at day 18 in MID and MID+P but increased at day 21, probably because of the increased D2 and decreased D3, maintaining T3 concentrations. No effect of ID was observed on hepatic D1. D2 increased in brain and brown adipose tissue at day 21 in MID+P. No changes were found in maternal placental D2 and D3, but D2 and D3 increased in the fetal placenta at day 18 in MID+P. A higher level of D2 is present in fetal skin than in the brain. As the activity is increased, in even mild ID (and already at 18 days) it can be concluded that skin D2 is likely to be of considerable physiological importance, at least for fetal thyroid hormone economy, by contributing to the intracellular T3 content of the skin and, possibly, to the plasma T3.

The activity of the type III inner ring deiodinase (DIII), which converts T4 and T3 to inactive metabolites, is induced by serum and growth factors in primary cultures of rat brown adipocytes. The contribution of pretranslational mechanisms to this increase in DIII activity was examined in the present studies. DIII mRNA is undetectable in differentiated brown adipocytes when cultured in serum-free medium. However, exposure to epidermal growth factor (EGF), acidic or basic fibroblast growth factors (aFGF or bFGF) increase DIII transcript levels. Lesser inductions are found with platelet-derived growth factor, and insulin-like growth factor I has no effect. Maximal induction of DIII mRNA is obtained after 9 h of exposure to EGF, bFGF, or aFGF at a concentration of 10 ng/ml. The increase in DIII mRNA in response to aFGF, bFGF, and EGF requires gene transcription and protein synthesis, as the inductive effect on mRNA is completely blocked by actinomycin D or cycloheximide. The DIII mRNA half-life is 4 h when stimulated with bFGF and increases to 12 h when 10% serum, EGF, or aFGF is present. In conclusion, EGF, aFGF, and bFGF increase DIII mRNA expression in differentiated brown adipocytes. This effect appears to be exerted at the level of both enhanced transcription and mRNA stabilization.

Rat brown preadipocytes cultured in low serum conditions increase DNA synthesis and proliferate in response to serum and a variety of growth factors and hormones. Epidermal growth factor, platelet-derived growth factor, and acidic and basic fibroblast growth factors stimulate DNA synthesis in a dose-dependent manner and induce at least a 5-fold increase in [3H]thymidine incorporation after 40 h of exposure. The physiological activator of brown adipose tissue, norepinephrine, has a low mitogenic effect per se, but increases DNA synthesis stimulation exerted by serum, epidermal growth factor, basic fibroblast growth factor, and the neuropeptide vasopressin. The addition of vasopressin plus norepinephrine greatly potentiates the mitogenic effect of growth factors to levels comparable to the effect of 10% serum. Preadipocytes cultured in the presence of these mitogen combinations (growth factor, vasopressin, and norepinephrine) express a differentiation marker, the uncoupling protein. Thus, our results show 1) that a variety of growth factors and hormones induce DNA synthesis in a synergistic fashion in brown preadipocytes in primary culture; and 2) there is evidence for a role of norepinephrine in the regulation of brown adipocyte proliferation, potentiating the action of serum and mitogens, besides its role in uncoupling protein messenger RNA expression.

Thyroid hormone plays an essential role in mammalian brain maturation and function, in large part by regulating the expression of specific neuronal genes. In this tissue, the type 2 deiodinase (D2) appears to be essential for providing adequate levels of the active thyroid hormone 3,5,3'-triiodothyronine (T3) during the developmental period. We have studied the regional and cellular localization of D2 mRNA in the brain of 15-day-old neonatal rats. D2 is expressed in the cerebral cortex, olfactory bulb, hippocampus, caudate, thalamus, hypothalamus, and cerebellum and was absent from the white matter. At the cellular level, D2 is expressed predominantly, if not exclusively, in astrocytes and in the tanycytes lining the third ventricle and present in the median eminence. These results suggest a close metabolic coupling between subsets of glial cells and neurons, whereby thyroxine is taken up from the blood and/or cerebrospinal fluid by astrocytes and tanycytes, is deiodinated to T3, and then is released for utilization by neurons.

To provide new insights into the in vivo regulation of iodothyronine deiodinases in the different tissues of the rat, we have evaluated the effects on these enzymatic activities of T4 or T3 infusions into thyroidectomized rats. Thyroidectomized rats were infused with placebo, T4, or T3. Placebo-infused intact rats served as euthyroid controls. Plasma and samples of cerebral cortex, brown adipose tissue, pituitary, liver, and lung were obtained after 12-13 days of infusion. Plasma TSH, plasma and tissue T4 and T3, and iodothyronine deiodinase activities were determined. Type II 5'-deiodinase (DII) was increased in cortex, brown adipose tissue, and pituitary from animals infused with placebo. DII activity returned to normal only with T4 infusion, remaining elevated in the animals infused with T3 alone despite normal tissue T3 concentrations. Cortex type III 5-deiodinase was only increased when hyperthyroidism was induced by infusion of T3. Liver type I 5'-deiodinase (DI) paralleled the changes in plasma and tissue T3 regardless of whether T4 or T3 was infused. On the contrary, the increase in lung DI, proportional to the increases in plasma and tissue T3, was higher when T4 was infused. As a rule, the tissues with DII presented a tighter homeostasis in their T3 concentrations than the tissues with DI. In conclusion, the regulation of iodothyronine deiodinases depends on the hormone infused into the thyroidectomized animals and on the tissue in which the deiodinase is studied, demonstrating the existence of tissue-specific regulation of its thyroid hormone concentrations.

We have used the streptozotocin-induced diabetes mellitus pregnant rat as a model of maternal nonthyroidal illness. We measured the effects of different degrees of diabetes mellitus on maternal body weight, the outcome of pregnancy, circulating glucose, insulin, T4, T3, rT3, and TSH in mother and fetus, T4 and T3 in maternal and fetal tissues, and iodothyronine deiodinases in liver, lung, and brain. All of the changes in thyroid hormone status typical of nonthyroidal illnesses were observed in the mothers and were related to the degree of the metabolic imbalances. Most were controlled with a daily insulin dose of 0.5 U/100 g BW. Normalization of maternal placental T4, however, required higher insulin doses than in other maternal tissues. The number and body weight of the fetuses, their pituitary GH contents, and their thyroid hormone status were severely affected. The total extrathyroidal T4 and T3 pools decreased to one third of normal fetal values. T4 and T3 concentrations in the fetal brain were lower than normal, and the expected increase in type II 5'deiodinase activity was not observed. The low cerebral T3 only improved with adequate insulin treatment of the dams. It is concluded that maternal diabetes mellitus, and possibly other nonthyroidal illnesses that impair the availability of intracellular energy stores, may affect fetal brain T3 when thyroid hormones are essential for normal development.

Maintenance of normal maternal thyroxinemia prevents severe triiodothyronine (T3) deficiency of the fetus with primary thyroid failure (1). We have studied whether thyroxine (T4) would also protect the fetal brain when maternal hypothyroxinemia is caused by nonthyroidal illnesses. We have used the streptozotocin-induced diabetes mellitus pregnant rat as a model of maternal nonthyroidal illness. We measured the effects of diabetes mellitus, and of correction of the ensuing maternal hypothyroxinemia with T4 as compared to insulin, on maternal body weight, the outcome of pregnancy, glucose, insulin, T4, T3, reverse T3, and thyrotropin levels in the maternal and fetal circulation, as well as T4 and T3 concentrations in tissues, and iodothyronine deiodinases in liver, lung, and brain. The diabetic mothers showed changes in thyroid hormone status typical of nonthyroidal illnesses. Thyroid hormone status of the fetuses was severely affected: the total T4 and T3 pools decreased to one-third of normal values. T4 and T3 concentrations in the fetal brain were lower than normal and the expected increase in 5'-deiodinase activity was not observed. Although insulin treatment avoided or mitigated these changes, the low cerebral T3 did not improve with T4 treatment of the maternal hypothyroxinemia. Several findings indicated that treatment of the severely ill dams with T4 was actually harmful for the outcome of pregnancy. These negative effects were observed without the expected increase in the maternal or fetal T3 pools.

We studied the regulation of type II 5'-deiodinase (5'D-II) activity and uncoupling protein (UCP) messenger RNA (mRNA) by thyroid hormones in fetal brown adipose tissue (BAT). Fetuses were obtained from hypothyroid pregnant rats infused with increasing doses of T4 or T3. Infusion of T4 into hypothyroid pregnant rats increased T4 and T3 concentrations and inhibited 5'D-II activity in fetal BAT. In contrast, infusion of T3 increased BAT 5'D-II activities at low, normal, or high BAT T3 concentrations. The relationship between thyroid hormone concentrations in fetal BAT and plasma showed that BAT T3 concentrations are relatively stable, increasing less than 2-fold over a wide range of circulating T4 (3-fold) or T3 (8-fold) concentrations. Most T3 in fetal BAT are locally derived from T4 and not from plasma T3. UCP mRNA expression decreased to 30% of control values in hypothyroid fetuses. UCP mRNA levels were restored to normal in parallel with BAT T3 concentrations after the infusion of either T4 or T3. UCP mRNA levels correlate well with BAT T3 concentrations. Supraphysiological doses of T4 did not further increase either BAT T3 or UCP mRNA levels. T3 might regulate basal UCP mRNA expression during fetal life.

EMD 21388 displaces T4 from circulating transthyretin, is a potent in vitro inhibitor of outer-ring deiodination (5'D) of T4 and affects thyroid hormone secretion. To study its extrathyroidal effects on the thyroid hormone status of pregnant dams and their fetuses, we treated the dams with methimazole and infused them with T4 and with either 2.5 mg EMD 21388/rat per day [(EMD(+)], or placebo solution [EMD(-)]. EMD reduced total T4 and T3 in the maternal circulation, but free T4 increased and free T3 decreased. The total amount of T3 generated from T4 in the maternal compartment increased. Placental T3 also increased in EMD(+) animals, T4 remaining the same. EMD also reached the fetal circulation. The total fetal extrathyroidal T4 pool decreased to half that of EMD(-) fetuses, whereas T3 increased 1.8-fold, thus mitigating fetal T3 deficiency, especially in the lung. Thus, if the maternal supply of T4 is kept constant, EMD mitigates the T3 deficiency of many tissues of the hypothyroid fetus. Most of the effects of this dose of EMD could result from the displacement of T4 from circulating transthyretin. Liver 5'D-I activity did not decrease, but actually increased by 40% in dams and fetuses. The enhanced transfer of T4 into tissues would also increase the amount of substrate available to 5'D-I, leading to an increased amount of T3 in maternal and fetal pools. This had not been anticipated from the changes in circulating T4 and T3, whether maternal or fetal, total or free.

Nuclear T3 binding and T3 receptors (TR) expression were studied in brown adipocytes differentiated in culture from precursor cells. High affinity T3 receptors were found. During adipocyte differentiation maximal binding capacity (MBC) was doubled (up to 763 fmol T3/mg DNA), and an apparent decrease in receptor affinity was also observed (due to a decrease in the association rate constant). A very high disappearance rate of T3 was found in the cellular and nuclear fractions under binding assay conditions (serum-free medium). MBC increased 30% under hypothyroid conditions, but was not affected by physiological doses of T3 or retinoic acid. TR beta 1, TR alpha 1 and c-erbA-alpha 2 mRNAs were detected in brown adipocytes. During differentiation TR beta 1 decreased to 30%. Long exposure to T3 increased 2-fold TR beta 1 and decreased TR alpha 1 levels, when using insulin-depleted medium. Short exposure (4 h) to 10 nM insulin reduced both TR beta 1 and TR alpha mRNAs species.

Iodothyronine type II 5'-deiodinase (5'D-II) activities were studied in cultures of rat brown adipocytes. In the presence of serum, the adrenergically stimulated 5'D-II activities were very low. In the absence of serum, adenosine 3',5'-cyclic monophosphate (cAMP) analogues stimulated 5'D-II activity. Thyroxine (T4) inhibited these increases. Norepinephrine slightly increased 5'D-II activity in hypothyroid conditions, but 3,5,3'-triiodothyronine (T3) strongly potentiated the adrenergic stimulation of 5'D-II (20-fold). T3 amplification of the adrenergic stimulation was via beta-adrenergic receptors, specifically mimicked by beta3-agonists, but it was not observed using cAMP analogues. The stimulatory effect of T3 predominated over the inhibitory action of T4, increased with exposure to T3, and required de novo protein synthesis. The half-life of 5'D-II was 30 min, suggesting that stabilization of 5'D-II did not occur. The effect was only observed in differentiated adipocytes. Retinoic acid has similar although smaller effects than T3. In conclusion, the presence of T3 is required and strongly potentiates the noradrenergic stimulation of 5'D-II activity in rat brown adipocytes.

We have recently shown that it is not possible to restore euthyroidism completely in all tissues of thyroidectomized rats infused with T4 alone. The present study was undertaken to determine whether this is achieved when T3 is added to the continuous sc infusion of T4. Thyroidectomized rats were infused with placebo or T4 (0.80 and 0.90 microgram/100 g BW.day), alone or in combination with T3 (0.10, 0.15, or 0.20 microgram/100 g BW.day). Placebo-infused intact rats served as euthyroid controls. Plasma and 12 tissues were obtained after 12 days of infusion. Plasma TSH and plasma and tissue T4 and T3 were determined by RIA. Iodothyronine deiodinase activities were assayed using cerebral cortex, pituitary, brown adipose tissue, liver, and lung. Circulating and tissue T4 levels were normal in all the groups infused with thyroid hormones. On the contrary, T3 in plasma and most tissues and plasma TSH only reached normal levels when T3 was added to the T4 infusion. The combination of 0.9 microgram T4 and 0.15 microgram T3/100 g BW.day resulted in normal T4 and T3 concentrations in plasma and all tissues as well as normal circulating TSH and normal or near-normal 5'-deiodinase activities. Combined replacement therapy with T4 and T3 (in proportions similar to those secreted by the normal rat thyroid) completely restored euthyroidism in thyroidectomized rats at much lower doses of T4 than those needed to normalize T3 in most tissues when T4 alone was used. If pertinent to man, these results might well justify a change in the current therapy for hypothyroidism.

The kidney androgen-regulated protein (KAP) gene exhibits a cell-specific hormonal regulation of its expression in the epithelial cells of proximal tubules of mouse kidney, where T3 is required for constitutive expression in the straight segments and androgens for expression in the convoluted ones. By using different models of hypothyroidism, we demonstrate that maximal androgen-mediated induction of the gene depends on thyroid hormone as well. This constitutes a specific event, since vitamin D3 cannot mimic the effects of T3, albeit their remarkable functional relationship. It is also shown that while congenital hypothyroid hyt/hyt male mice, exposed to maternal T3 in the gestational period, exhibit diminished but existent androgen-dependent cortical responses, mice exposed to goitrogens during gestation and postnatally are unable to express the gene even at postnatal day ninety. Impairment of KAP cortical expression in hypothyroid animals does not correlate with lower levels of androgens or androgen receptor expression.

The levels of mRNA coding for the uncoupling protein (UCP) and for lipoprotein lipase (LPL) were monitored in the brown adipose tissue of newborn rat pups. At 5 h after birth, the mRNA levels of UCP and LPL were high in pups exposed singly to 28 degrees C and low in pups kept singly at thermoneutrality (36 degrees C); in pups staying with the dam, the UCP mRNA levels were intermediate. However, the LPL mRNA levels were lower in pups staying with the dam than in pups at 36 degrees C, implying that factors additional to environmental temperature influenced LPL gene expression. Injection of noradrenaline into pups at thermoneutrality (36 degrees C) led to increases in UCP and LPL gene expression, but noradrenaline injections had no further effect in cold-exposed pups. The adrenergic effects were mediated via beta-adrenergic receptors. The cold-induced increases in both UCP and LPL gene expression were abolished by the beta-adrenergic antagonist propranolol. Thus differences in adrenergic responsiveness could not explain the differential expression of the UCP and LPL genes observed in pups staying with the dam. The presence of a physiological suppressor was examined by feeding single pups at 28 degrees C with different foods: nothing, water, Intralipid, cow's milk, rat milk and rat colostrum. None of these agents led to suppression of UCP gene expression, but colostrum led to a selective suppression of LPL gene expression. It was concluded that the genes for UCP and LPL were responsive to adrenergic stimuli immediately after birth, and it is suggested that a component of rat colostrum can selectively suppress LPL gene expression.

We have studied whether, or not, tissue-specific regulatory mechanisms provide normal 3,5,3'-triiodothyronine (T3) concentrations simultaneously in all tissues of a hypothyroid animal receiving thyroxine (T4), an assumption implicit in the replacement therapy of hypothyroid patients with T4 alone. Thyroidectomized rats were infused with placebo or 1 of 10 T4 doses (0.2-8.0 micrograms per 100 grams of body weight per day). Placebo-infused intact rats served as controls. Plasma and 10 tissues were obtained after 12-13 d of infusion. Plasma thyrotropin and plasma and tissue T4 and T3 were determined by RIA. Iodothyronine-deiodinase activities were assayed using cerebral cortex, liver, and lung. No single dose of T4 was able to restore normal plasma thyrotropin, T4 and T3, as well as T4 and T3 in all tissues, or at least to restore T3 simultaneously in plasma and all tissues. Moreover, in most tissues, the dose of T4 needed to ensure normal T3 levels resulted in supraphysiological T4 concentrations. Notable exceptions were the cortex, brown adipose tissue, and cerebellum, which maintained T3 homeostasis over a wide range of plasma T4 and T3 levels. Deiodinase activities explained some, but not all, of the tissue-specific and dose related changes in tissue T3 concentrations. In conclusion, euthyroidism is not restored in plasma and all tissues of thyroidectomized rats on T4 alone. These results may well be pertinent to patients on T4 replacement therapy.

We found low T3 concentrations in rat brown adipocytes differentiated in vitro. This might be due to the high metabolic rate of T3, possibly caused by elevated type III iodothyronine 5-deiodinase activity (5DIII), induced by serum growth factors. We tested the ability of several growth factors to induce 5DIII activity. Epidermal growth factor and basic and acidic fibroblast growth factors produced a strong induction of 5DIII activity (25, 45-, and 50-fold, respectively). This process required gene transcription and de novo protein synthesis. The half-life of 5DIII was approximately 3 h. Heparin was required for full acidic fibroblast growth factor activity. Platelet-derived growth factor, vasopressin, and insulin-like growth factor-I induced lower 5DIII activities (3- to 6-fold). Vasopressin amplified basic fibroblast growth factor and epidermal growth factor inductions when used at submaximal doses. We found a Km of 22.5 nM using T3 as substrate. Although brown adipose tissue has undetectable 5DIII activities in vivo, the present data explain the low T3 concentrations found in cultured rat brown adipocytes and suggest that growth factors, by stimulating 5DIII, may lead to low T3 concentrations and indirectly inhibit the expression of some genes regulated by T3, e.g. the rat uncoupling protein.

The possible influence of hypothalamic and pituitary 5'-deiodinase II (5'-D-II) activity and 3,5,3'-triiodothyronine (T3) content on the modulation of thyroid-stimulating hormone (TSH) synthesis was studied. 1) Alterations in 5'-D-II activity and hypothalamic and pituitary T3 content produced by undernutrition were observed in fetal (21 days) and neonatal rats vs. controls. 2) After thyroidectomy, plasma TSH increased in both populations, undernourished and control, but pituitary TSH increased only in the former and not in the latter. The results obtained by giving small doses of thyroxine (T4; 0.5 micrograms/100 g body wt) to intact and thyroidectomized rats suggest a lower inhibitory effect by T4 on the pituitary in undernourished than in control rats. Although hypothalamic and pituitary 5'-D-II activity increased in both groups after thyroidectomy, the percentage increase was lower in undernourished vs. control rats, resulting in lower overall T3 content in tissues from undernourished animals. These studies on thyroid axis regulation show the in vivo regulation of TSH synthesis by hypothalamic and pituitary 5'-D-II activity and T3 content.

The kidney androgen-regulated protein (KAP) gene is expressed in epithelial cells of proximal convoluted tubules of mouse kidney. Although TSH proved to be necessary for the constitutive expression of the gene in the outer stripe of the outer medulla, androgens are responsible for expression in cortical segments of the proximal tubules. We have used the congenital thyroid hormone (TH)-deficient hyt/hyt mouse to demonstrate that TH, and not TSH, is responsible for the constitutive expression of the gene in the mouse kidney. Although the androgen-dependent cortical response is partially impaired in hypothyroid mice, the expression can be fully restored after the administration of TH or pharmacological doses of testosterone, suggesting some cooperativity between TH and androgens in promoting cortical KAP gene expression. Results in hyt/hyt mice after treatment with retinoic acid, alone or in combination with TH, demonstrated that this regulator does not have any effect on the regulation of the KAP gene in mouse kidney and that induction of the gene by T3 does not require heterodimerization of TR with retinoic acid-related receptors. By using immunocytochemical analysis and specific antibodies against alpha- and beta-TH receptors we have determined the presence of both types of receptors in all segments of the proximal tubules.

Concentrations of T4 and T3 were recently measured in rat fetal tissues, and the reported values were found to be more than 10-fold higher than those found by us. The differences have been explained by the assumption that previous analytical procedures, neither avoid deiodination during autopsy of the animals or during extraction and purification, because phloretin [(3'),4',4,6-(tetra)trihydroxyaurone], a potent inhibitor of 5'-iodothyronine deiodinase activity in vitro, had not been used to prevent such problems. We here show that perfusion with phloretin during autopsy does not affect 5'-iodothyronine activity or T4 and T3 concentrations in liver, kidney, or brain. Evidence is also provided that the addition of phloretin during the homogenization process is superfluous, as the use of 80% ethanol and 0.02 M NaOH for this step results in undetectable deiodinase activity. Data are presented showing that during the final sample drying, no losses or degradation of T4 and T3 occur, confirming the adequacy of the individual recovery corrections using radiolabeled iodothyronines as internal tracers. We also present quantitative information on the intralaboratory variability of the T4 and T3 concentrations found in tissues from normal fetuses and their mothers as well as in adult males and nonpregnant females. Results are comparable to those obtained by others using entirely different analytical procedures.

Primary cultures of brown adipocytes were used to investigate the regulation of malic enzyme (ME) gene expression by insulin and T3. No ME gene expression was detected in undifferentiated preadipocytes. The levels of ME mRNA increased slightly during cell differentiation. Physiological doses of insulin or T3 increased ME gene expression, which reached a maximum after 24 h, on whichever culture day they were added. The effects of insulin and T3 were at the transcriptional level, as measured by run-on assays. Both hormones also increased the stabilization of the transcripts and required ongoing protein synthesis to exert their effects. A comparison of the potencies of insulin and insulin-like growth factor-I and -II (IGF-I and -II) in this system indicated that induction by insulin is mediated via its own receptor. The effects of insulin and T3 were independent of the extracellular glucose concentration, but were additive to that of glucose. Moreover, insulin and T3 act additively to increase ME gene expression, suggesting that they interact either at the transcription level or that of mRNA stabilization.

We have studied the effects of maternal thyroid status on the effectiveness of the rat placenta near term as a barrier for the transfer of T4 and T3 to the fetus. Dams were given methimazole to minimize the fetal contribution to the T4 and T3 pools, so that the iodothyronines found in the conceptus are ultimately of maternal origin. The dams were infused with saline, or with T4 or T3 at doses ranging from 2.3-27.8 nmol T4 and from 0.77-20.7 nmol T3/100 g BW per day. A group of normal pregnant dams (C) was included. At 21 days of gestation T4, T3, and rT3 were measured by RIA in maternal and fetal plasma, and in maternal and fetal sides of the placenta. The total fetal extrathyroidal T4 and T3 pools were also determined. The dose-related changes in T4, T3, and rT3 levels in the placenta confirm the presence of both inner and outer ring iodothyronine deiodinase activities, and suggest increasing accumulation of the iodothyronines. Despite this, fetal extrathyroidal T4 and T3 increase progressively in T4-infused groups as a function of maternal circulating T4 levels. Fetal extrathyroidal T3 increases progressively in T3-infused groups as a function of maternal plasma T3. There was no evidence that the net maternal contribution of T4 or T3 would be proportionally less when the maternal pools became very high. It was concluded that the rat placenta is only a limited barrier for the transfer of T4 and T3 to the fetus.

Undernutrition was induced in rats submitted to food restriction from the fetal stage, and malnutrition was continued after birth until 70 days of life. Body weight was decreased to less than 50%. Plasma T4 and T3 and pituitary TSH content were determined between 8-70 days of life. In control rats, plasma T4 and T3 reached a maximum at 14 and 35 days of life, respectively, and TSH pituitary content at 45 days of life. In undernourished rats, after 8 days of life, plasma T4 and T3 and pituitary TSH content were decreased to about 50% or less, and the pattern of sequential changes observed in control rats was absent or modified. T4 and T3 concentrations were measured in heart, liver, and brain in the fetus (22 days old) and 8, 14, and 23 days after birth, as well as liver and brain 5'-deiodinases (5'D). Hepatic 5'D type I was always decreased in undernourished rats from 8-70 days after birth. Liver and heart T4 and T3 concentrations were decreased in 14-day-old undernourished rats as well as brain T3. Brain 5'D type II was decreased at 8 and 14 days, and total brain 5'D activities at 8 days. These changes occurred during the critical period for brain development (7th to 20th day) during which most processes of myelination take place and T3 brain normal levels are required.