ABSTRACT
Triiodothyronine (T3) is important for thermogenesis in brown adipose tissue (BAT). Type II deiodinase (DIO2) produces T3 required for intracellular needs in BAT. Brown adipocytes in culture require T3 for the adrenergic stimulation of DIO2. Glucocorticoids induce adipocyte differentiation (lipogenesis). We investigated the regulation of DIO2 activity, Dio2 mRNA and Dio2 promoter activity by glucocorticoids in primary cultures of rat brown adipocytes using dexamethasone (DEX) and hydrocortisone (HC). DEX and HC regulated the adrenergic stimulation of DIO2 activity in a dose- and time-dependent manner, inhibiting DIO2 activity at short treatment times and large doses (1-10 µM) and stimulating DIO2 at low HC doses (1-100 nM) and longer times (DEX). Insulin depletion reduced DIO2 activity but the response to glucocorticoids remained unchanged. DEX and HC inhibited basal DIO2 activity. DEX had no effect on DIO2 half-life, whereas HC stabilized DIO2 activity. DEX and HC inhibited the adrenergic stimulation of Dio2 mRNA expression (100-10000 nM, 14-96 h), but stabilized Dio2 mRNA, particularly DEX. DEX increased basal Dio2 mRNA levels, possibly through stabilization of Dio2 mRNA. An 807 bp construct of the murine Dio2 proximal promoter showed maximal reporter activity, with the cAMP response element (CRE) essential for transcriptional activity. DEX caused inhibition in most constructs containing the CRE element whereas HC stimulated reporter activity in the 807 bp construct. Glucocorticoids inhibited the adrenergic stimulation of Dio2 at the transcriptional level in brown adipocytes, although DIO2 activity increased with HC, possibly due to stabilization of Dio2 activity and mRNA. The CRE and cEBP elements of the Dio2 promoter seem involved in the regulation by glucocorticoids.
Subject(s)
Adipocytes, Brown/metabolism , Gene Expression Regulation, Enzymologic/drug effects , Glucocorticoids/pharmacology , Iodide Peroxidase/genetics , Promoter Regions, Genetic , Adipocytes, Brown/drug effects , Animals , Cells, Cultured , Dexamethasone/pharmacology , Half-Life , Hydrocortisone/pharmacology , Iodide Peroxidase/metabolism , Mice , Phenotype , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats, Sprague-Dawley , Uncoupling Protein 1/metabolismABSTRACT
TSH receptor (TSHR) is present in the thyroid and other tissues, as adipose tissue. In brown adipose tissue (BAT) TSH increases UCP1 expression and lipolysis. We have studied the regulation of Tshr mRNA expression and the effect of TSH on Ucp1 and Dio2 mRNA, on D2 activity and O2 consumption in rat brown adipocytes and the TSH signaling pathways. Tshr increased during brown adipocyte differentiation, was up-regulated by insulin and low TSH concentrations and down-regulated by high TSH concentrations, T3 and/or NE. TSH increased basal Ucp1 mRNA in a dose-dependent way acting synergistically with T3, while had no effect when NE was present. High TSH concentrations increased basal Dio2 mRNA (12-fold) and were synergistic with T3 (100-fold), but decreased Dio2 mRNA in T3+NE-treated cells. TSH increased D2 activities in T3-treated cells and inhibition of ERK pathway decreased the TSH effect by 55%. In T3+NE treated-cells TSH decreased D2 activity by 50%, in a dose-dependent manner. TSH activated Akt and Erk phosphorylation, while inhibition of PKA promoted Akt phosphorylation. TSH inhibited leptin mRNA. TSH increased O2 consumption by 20% and T3 enhanced its effect. Tshr is expressed in brown adipocytes and is regulated by insulin, TSH, T3 and NE. TSH increases basal and T3-stimulated Ucp1 and Dio2 expression and D2 activity only when T3 is present, but decreases Dio2 mRNA and D2 activity stimulated by NE+T3. TSH increases O2 consumption, confirming the role of TSH in the maintenance of thermogenesis.
Subject(s)
Adipocytes, Brown/cytology , Receptors, Thyrotropin/genetics , Receptors, Thyrotropin/metabolism , Thermogenesis/drug effects , Thyrotropin/pharmacology , Adipocytes, Brown/drug effects , Animals , Cell Differentiation , Cells, Cultured , Dose-Response Relationship, Drug , Insulin/pharmacology , Iodide Peroxidase/genetics , Iodide Peroxidase/metabolism , Ion Channels/genetics , Leptin/genetics , MAP Kinase Signaling System/drug effects , Mitochondrial Proteins/genetics , Oxygen Consumption/drug effects , Rats , Rats, Sprague-Dawley , Uncoupling Protein 1 , Iodothyronine Deiodinase Type IIABSTRACT
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.
Subject(s)
Insulin/metabolism , Iodide Peroxidase/metabolism , Mitogen-Activated Protein Kinases/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Adipocytes/metabolism , Adipose Tissue, Brown/metabolism , Animals , Iodide Peroxidase/genetics , RNA, Messenger/metabolism , Rats , Receptors, Somatomedin/metabolismABSTRACT
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.