Differences between the effects of thyroxine and tetraiodothyroacetic acid on TSH suppression and cardiac hypertrophy.

OBJECTIVE: We earlier reported marked qualitative differences between the effect of 3,5,3'-tri-iodothyroacetic acid (Triac) and tri-iodothyronine (T3) on cardiac hypertrophy at equivalent thyroid-stimulating hormone (TSH)-suppressive doses. We have now extended these studies to specific cardiac parameters. Due to its rapid metabolic clearance rate, Triac is not suitable for TSH suppression and therefore the slowly metabolized 3,5,3',5'-tetraiodothyroacetic acid (Tetrac), the precursor of Triac, was studied. METHODS: Hypothyroid rats were infused over 13 days with 1.5-40.5 nmol Tetrac/day per 100 g body weight (BW) or with 0.5-13.5 nmol thyroxine ((4)T4)/day per 100 g BW. RESULTS: The responses of serum TSH and of hepatic monodeiodinase type 1 were parallel for both hormones, their potency ratios could therefore be compared. Tetrac was revealed as being only half as active on hepatic monoiodinase type 1 despite a similar serum TSH levels. Tetrac can therefore be considered to have a preferential action on serum TSH suppression. The cardiac effects on Ca2+-ATPase (SERCA 2a) and monodeiodinase type 1 activity were qualitatively different and therefore one cannot give an overall quantitative estimate of these differences. The results showed clearly, however, that Tetrac is less efficient for all parameters studied, namely induction of cardiac hypertrophy, alpha-myosin heavy chain mRNA, monodeiodinase type 1 activity and mRNA levels of the sarcoplasmic SERCA 2a. CONCLUSION: We postulate therefore that, in the rat and possibly in man, Tetrac could represent a favorable alternative for suppression of serum TSH levels.

Expression of peroxisome proliferator-activated receptors in lean and obese Zucker rats.

OBJECTIVE: Examination of the pattern of expression of peroxisome proliferator-activated receptor (PPAR) isoforms alpha and gamma in a model of obesity. DESIGN: Examination of adipose tissue and primary adipocyte cultures from lean and obese Zucker rats at different ages (28 days and 12 weeks). METHODS: mRNA levels were measured by RNase protection assay. RESULTS: The highest levels of PPARalpha and gamma mRNA were present in brown adipose tissue (BAT), followed by liver and white adipose tissue (WAT) for the alpha and gamma subtypes, respectively, at both ages examined. PPARalpha was expressed 100-fold higher in BAT compared with WAT, and PPARgamma mRNA levels were 2-fold higher in the WAT of obese compared with lean rats. PPARalpha and gamma expression was minimal in m. soleus, although higher levels of PPARgamma were found in the diaphragm. In marked contrast to the findings in vivo, virtually no PPARalpha mRNA could be detected in BAT cultures differentiated in vitro. CONCLUSION: PPARalpha and gamma are most highly expressed in BAT in vivo. However, PPARalpha is undetectable in brown adipose cells in vitro, suggesting that the expression of this receptor is induced by some external stimuli. In addition, the expression of PPARgamma was increased in WAT from young obese animals, compatible with an early adaptive phenomenon. Finally, the presence of PPARgamma mRNA is detectable only in particular muscles, such as the diaphragm, suggesting the possibility of an influence of fiber type on its expression, although exercise did not influence the expression of PPARgamma in other skeletal muscles.

Regulation of the transcriptional activity of the peroxisome proliferator-activated receptor alpha by phosphorylation of a ligand-independent trans-activating domain.

The peroxisome proliferator-activated receptors (PPARs) are a subgroup of nuclear receptors activated by fatty acids and eicosanoids. In addition, they are subject to phosphorylation by insulin, resulting in the activation of PPARalpha, while inhibiting PPARgamma under certain conditions. However, it was hitherto unclear whether the stimulatory effect of insulin on PPARalpha was direct and by which mechanism it occurs. We now demonstrate that amino acids 1-92 of hPPARalpha contain an activation function (AF)-1-like domain, which is further activated by insulin through a pathway involving the mitogen-activated protein kinases p42 and p44. Further analysis of the amino-terminal region of PPARalpha revealed that the insulin-induced trans-activation occurs through the phosphorylation of two mitogen-activated protein kinase sites at positions 12 and 21, both of which are conserved across evolution. The characterization of a strong AF-1 region in PPARalpha, stimulating transcription one-fourth as strongly as the viral protein VP16, is compatible with the marked basal transcriptional activity of this isoform in transfection experiments. However, it is intriguing that the activity of this AF-1 region is modulated by the phosphorylation of two serine residues, both of which must be phosphorylated in order to activate transcription. This is in contrast to PPARgamma2, which was previously shown to be phosphorylated at a single site in a motif that is not homologous to the sites now described in PPARalpha. Although the molecular details involved in the phosphorylation-dependent enhancement of the transcriptional activity of PPARalpha remain to be elucidated, we demonstrate that the effect of insulin on the AF-1 region of PPARalpha can be mimicked by the addition of triiodothyronine receptor beta1, a strong binder of corepressor proteins. In addition, a triiodothyronine receptor beta1 mutant deficient in interacting with corepressors is unable to activate PPARalpha. These observations suggest that the AF-1 region of PPARalpha is partially silenced by corepressor proteins, which might interact in a phosphorylation-dependent manner.

Direct effects of leptin on brown and white adipose tissue.

Leptin is thought to exert its actions on energy homeostasis through the long form of the leptin receptor (OB-Rb), which is present in the hypothalamus and in certain peripheral organs, including adipose tissue. In this study, we examined whether leptin has direct effects on the function of brown and white adipose tissue (BAT and WAT, respectively) at the metabolic and molecular levels. The chronic peripheral intravenous administration of leptin in vivo for 4 d resulted in a 1.6-fold increase in the in vivo glucose utilization index of BAT, whereas no significant change was found after intracerebroventricular administration compared with pair-fed control rats, compatible with a direct effect of leptin on BAT. The effect of leptin on WAT fat pads from lean Zucker Fa/ fa rats was assessed ex vivo, where a 9- and 16-fold increase in the rate of lipolysis was observed after 2 h of exposure to 0.1 and 10 nM leptin, respectively. In contrast, no increase in lipolysis was observed in the fat pads from obese fa/fa rats, which harbor an inactivating mutation in the OB-Rb. At the level of gene expression, leptin treatment for 24 h increased malic enzyme and lipoprotein lipase RNA 1.8+/-0.17 and 1.9+/-0.14-fold, respectively, while aP2 mRNA levels were unaltered in primary cultures of brown adipocytes from lean Fa/fa rats. Importantly, however, no significant effect of leptin was observed on these genes in brown adipocytes from obese fa/fa animals. The presence of OB-Rb receptors in adipose tissue was substantiated by the detection of its transcripts by RT-PCR, and leptin treatment in vivo and in vitro activated the specific STATs implicated in the signaling pathway of the OB-Rb. Taken together, our data strongly suggest that leptin has direct effects on BAT and WAT, resulting in the activation of the Jak/STAT pathway and the increased expression of certain target genes, which may partially account for the observed increase in glucose utilization and lipolysis in leptin-treated adipose tissue.

The peroxisome proliferator-activated receptor alpha is a phosphoprotein: regulation by insulin.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily implicated in adipocyte differentiation. The observations that PPAR alpha is a regulator of hepatic lipid metabolism and that the insulin-sensitizing thiazolidinediones are ligands for PPAR gamma suggest that cross-talk might exist between insulin signaling and PPAR activity, possibly through insulin-induced PPAR phosphorylation. Immunoprecipitation of endogenous PPAR alpha from primary rat adipocytes prelabeled with [32P]-orthophosphate and pretreated for 2 h with vanadate and okadaic acid demonstrated for the first time that PPAR alpha is a phosphoprotein in vivo. Treatment with insulin induced a time-dependent increase in PPAR phosphorylation showing a 3-fold increase after 30 min. Insulin also increased the phosphorylation of human PPAR alpha expressed in CV-1 cells. These changes in phosphorylation were paralleled by enhanced transcriptional activity of PPAR alpha and gamma. Transfection studies in CV-1 cells and HepG2 cells revealed a nearly 2-fold increase of PPAR activity in the presence of insulin. In contrast, insulin had no effect on the transcriptional activity of transfected thyroid hormone receptor in CV-1 cells, suggesting a PPAR-specific effect. Thus, insulin stimulates PPAR alpha phosphorylation and enhances the transcriptional activity of PPAR, suggesting that the transcriptional activity of this nuclear hormone receptor might be modulated by insulin-mediated phosphorylation.

Intracellular calcium and phospholipid turnover are not involved in the inhibition of iodothyronine 5'-deiodinase type II activity by T4.

In glial cell cultures, iodothyronine 5'-deiodinase type II is stimulated by dibutyryl cAMP. Serum-free medium increases enzyme activity and prolongs the half-life of the enzyme. T4 and rT3 specifically inhibit this activity. We tested whether enzyme inactivation by T4 was mediated by changes in cytosolic free calcium concentration and/or phospholipid turnover. Intracellular calcium concentration was decreased either by chelation of extracellular calcium or by chelation of extracellular and intracellular calcium. Neither basal hypothyroid 5'-deiodinase activity nor its inactivation by T4 were modified in such experimental conditions, compared with control cells incubated in normal calcium-containing medium. T4 by itself had no effect on the cytosolic free calcium concentration for up to 20 min. Studies on phospholipid turnover included norepinephrine in parallel to T4 as positive stimulation control. While norepinephrine clearly accelerated phosphoinositide turnover, there was no effect of T4 on any phospholipid turnover. These results suggest that neither cytosolic free calcium nor phospholipid turnover is involved in T4-dependent modulation of 5'-deiodinase type II activity in astrocytes in culture.

Thyroxine-dependent modulation of actin polymerization in cultured astrocytes. A novel, extranuclear action of thyroid hormone.

Actin depolymerization specifically blocks the rapid thyroid hormone-dependent inactivation of type II iodothyronine 5'-deiodinase. Thyroid hormone appears to regulate enzyme inactivation by modulating actin-mediated internalization of this plasma membrane-bound protein. In this study, we examined the interrelationships between thyroxine-dependent enzyme inactivation and the organization of the actin cytoskeleton in cultured astrocytes. Steady-state enzyme levels were inversely related to actin content in dibutyryl cAMP-stimulated astrocytes, and increases in filamentous actin resulted in progressively shorter enzyme half-lives without affecting enzyme synthesis. In the absence of thyroxine, filamentous actin decreased by approximately 40% and soluble actin correspondingly increased; thyroxine normalized filamentous actin levels without changing total cell actin. Thyroxine treatment for only 10 min resulted in an approximately 50% loss of enzyme and increased filamentous actin 2-fold. Neither cycloheximide nor actinomycin D affected the thyroxine-induced actin polymerization. Astrocytes grown without thyroxine also showed a disorganized actin cytoskeleton, and 10 nM thyroxine or 10 nM reverse triiodothyronine normalized the actin cytoskeleton appearance within 20 min; 10 nM 3,3',5-triiodothyronine had no effect. These data show that thyroxine modulates the organization of the actin cytoskeleton in astrocytes and suggest that regulation of actin polymerization may contribute to thyroid hormone's influence on arborization, axonal transport, and cell-cell contact in the developing brain.

Regulation of type II iodothyronine 5'-deiodinase by thyroid hormone. Inhibition of actin polymerization blocks enzyme inactivation in cAMP-stimulated glial cells.

The cellular events mediating the rapid, thyroid hormone-dependent modulation of membrane-bound, type II iodothyronine 5'-deiodinase were studied in dibutyryl cyclic AMP(bt2cAMP)-treated brain astrocytes. Unstimulated cells had undetectable type II 5'-deiodinating activity. Treating the cells with bt2cAMP and hydrocortisone induced enzyme expression by increasing transcription of the enzyme or an essential enzyme related protein(s), with steady-state levels of type II 5'-deiodinase attained after 8 h of bt2cAMP treatment. Glial cells grown in the absence of thyroid hormone had 10-15-fold higher levels of 5'-deiodinating activity than cells grown in the presence of serum. The increased type II 5'-deiodinating activity observed in serum-free cultures was due to a prolonged enzyme half-life with no change in the rate of enzyme synthesis. Addition of thyroxine or 3,3',5'-triiodothyronine to the serum-free culture medium resulted in a concentration-dependent fall in steady-state enzyme levels, with EC50 values of approximately 0.4 nM. 3,5,3'-Triiodothyronine was at least 100-fold less effective. Chloroquine, NH4Cl, tunicamycin, colchicine, and monodansylcadavarine had no effect on the t1/2 of the enzyme, while both carbonyl cynanide m-chlorophenylhydrazone and cytochalasins completely blocked the inactivation of the type II 5'-deiodinase. These data indicate that in glial cells, an intact actin-cytoskeleton is required for thyroid hormone to modulate the energy-dependent regulation of the half-life of the short-lived, membrane-bound enzyme, type II 5'-deiodinase.

Peroxisomal oxidative capacity of brown adipose tissue depends on the thyroid status.

The induction of hypothyroidism in rats by methimazole affects interscapular brown adipose tissue (IBAT) mitochondrial and peroxisomal enzyme activities in opposite directions. Hypothyroidism, indeed, decreases both mitochondrial succinate dehydrogenase and beta-oxidation total activities by 35 and 45%, respectively and increases peroxisomal catalase and acyl coenzyme A (acyl CoA) oxidase total activities 3.2- and 1.6-fold, respectively. Administration of a thyroid hormone analogue (3'-isopropyl-3,5-diiodo-L-thyronine) prevents these enzymatic modifications. The effects of hypothyroidism on IBAT mitochondrial enzyme activities seem to be direct, i.e. due to the lack of thyroid hormones, while those on peroxisomal enzyme activities might be indirect, i.e. secondary to the increased thermogenic needs of the rat and mediated by adrenergic stimulation. It is noteworthy that the indirect effects of hypothyroidism on peroxisomes are not observed in liver where acyl CoA oxidase activity is in fact decreased by 40%. In hypothyroid rat IBAT, administration of the peroxisome proliferator nafenopin does not further stimulate the already increased peroxisomal enzyme activities and does not inhibit the already decreased mitochondrial enzyme activities.

Human recombinant interleukin-1 beta decreases plasma thyroid hormone and thyroid stimulating hormone levels in rats.

Thyroid function was investigated in rats treated sc with a single injection of human recombinant interleukin-1 beta (hrIL-1). In 5 h 12.5 micrograms hrIL-1 decreased total serum T4 levels by 30 +/- 2% (P less than 0.01) and serum T3 levels by 35 +/- 4% (P less than 0.001). However free T4 and T3 fractions increased markedly within the first 140 min by 162 +/- 20% (P less than 0.001) and by 55 +/- 4% (P less than 0.001) resulting in a 88 +/- 20% increase in the free T4 concentration (P less than 0.001) but no increase in the free T3 concentration. Serum TSH concentration fell in the 5 h after the hrIL-1 injection by 77 +/- 3% (P less than 0.001). A similar decrease was observed with 0.125 micrograms hrIL-1. Five hours of starvation did not change serum TSH levels, suggesting that the effect of hrIL-1 on TSH was not due to decreased food intake. In order to test whether the decrease in serum TSH was due to an intrapituitary increase in T3, hrIL-1 was injected in hypothyroid rats: the fall of serum TSH was not prevented and it fell in 5 h from 14.05 +/- 0.56 to 9.66 +/- 0.98 ng/ml (31%, P less than 0.01, n = 14). These results suggest that hrIL-1 acts independently of thyroid hormones. Peripheral metabolism of T4 was studied by implanting [125I]T4 secreting minipumps during 14 days. There was no difference in T4 plasma clearance rate between control and treated animals. The fall of serum T4 was therefore explained by decreased secretion and not by increased catabolism since ether link cleavage of T4 and changes in hepatic deiodinase could not be detected. We therefore suggest that hrIL-1 inhibits thyroid function mainly at the hypothalamic-hypophyseal level.