H C van Beeren

DESETHYLAMIODARONE IS A COMPETITIVE INHIBITOR OF THE BINDING OF THYROID HORMONE TO THE THYROID HORMONE ALPHA 1-RECEPTOR PROTEIN

Desethylamiodarone (DEA), the major metabolite of the potent antiarrythmic drug amiodarone, is a non-competitive inhibitor of the binding of thyroid hormone (T3) to the beta 1-thyroid hormone receptor (T3R). In the present study, we investigated whether DEA acts in a similar way with respect to the alpha 1-T3R. The chicken alpha 1-T3R, expressed in an E. coli system, was incubated in the presence or absence of DEA with [125I]T3 in buffer containing 0.05% Triton X-100, 0.05% BSA and 1% ethanol (v/v) in order to solubilise DEA. DEA, but not amiodarone, inhibited T3 binding in a dose-dependent manner; the IC50 value was 3.5 x 10(-5) M. Scatchard analyses in the presence of DEA demonstrated a dose-dependent decrease in Ka values, but no change in MBC. Lineweaver-Burk plots clearly indicated competitive inhibition by DEA. Pre-incubation of the alpha 1-receptor with DEA decreased maximal [125I]T3 binding, which was independent of the duration of pre-incubation. In conclusion, in contrast to the beta 1-T3R, where DEA acts as a non-competitive inhibitor, we now report as a new finding the competitive action of DEA to the alpha 1-T3R.

Skeletal muscle deiodinase type 2 regulation during illness in mice

We have previously shown that skeletal muscle deiodinase type 2 (D2) mRNA (listed as Dio2 in MGI Database) is upregulated in an animal model of acute illness. However, human studies on the expression of muscle D2 during illness report conflicting data. Therefore, we evaluated the expression of skeletal muscle D2 and D2-regulating factors in two mouse models of illness that differ in timing and severity of illness: 1) turpentine-induced inflammation, and 2) Streptococcus pneumoniae infection. During turpentine-induced inflammation, D2 mRNA and activity increased compared to pair-fed controls, most prominently at day 1 and 2, whereas after S. pneumoniae infection D2 mRNA decreased. We evaluated the association of D2 expression with serum thyroid hormones, (de-)ubiquitinating enzymes ubiquitin-specific peptidase 33 and WD repeat and SOCS box-containing 1 (Wsb1), cytokine expression and activation of inflammatory pathways and cAMP pathway. During chronic inflammation the increased muscle D2 expression is associated with the activation of the cAMP pathway. The normalization of D2 5 days after turpentine injection coincides with increased Wsb1 and tumor necrosis factor alpha expression. Muscle interleukin-1beta (Il1b) expression correlated with decreased D2 mRNA expression after S. pneumoniae infection. In conclusion, muscle D2 expression is differentially regulated during illness, probably related to differences in the inflammatory response and type of pathology. D2 mRNA and activity increases in skeletal muscle during the acute phase of chronic inflammation compared to pair-fed controls probably due to activation of the cAMP pathway. In contrast, muscle D2 mRNA decreases 48 h after a severe bacterial infection, which is associated with local Il1b mRNA expression and might also be due to diminished food-intake.

Lacking thyroid hormone receptor β gene does not influence alterations in peripheral thyroid hormone metabolism during acute illness

The downregulation of liver deiodinase type 1 (D1) is supposed to be one of the mechanisms behind the decrease in serum tri-iodothyronine (T3) observed during non-thyroidal illness (NTI). Liver D1 mRNA expression is positively regulated by T3, mainly via the thyroid hormone receptor (TR)beta1. One might thus expect that lacking the TRbeta gene would result in diminished downregulation of liver D1 expression and a smaller decrease in serum T3 during illness. In this study, we used TRbeta-/- mice to evaluate the role of TRbeta in lipopolysaccharide (LPS, a bacterial endotoxin)-induced changes in thyroid hormone metabolism. Our results show that the LPS-induced serum T3 and thyroxine and liver D1 decrease takes place despite the absence of TRbeta. Furthermore, we observed basal differences in liver D1 mRNA and activity between TRbeta-/- and wild-type mice and TRbeta-/- males and females, which did not result in differences in serum T3. Serum T3 decreased rapidly after LPS administration, followed by decreased liver D1, indicating that the contribution of liver D1 during NTI may be limited with respect to decreased serum T3 levels. Muscle D2 mRNA did not compensate for the low basal liver D1 observed in TRbeta-/- mice and increased in response to LPS in TRbeta-/- and WT mice. Other (TRbeta independent) mechanisms like decreased thyroidal secretion and decreased binding to thyroid hormone-binding proteins probably play a role in the early decrease in serum T3 observed in this study.

Desethylamiodarone interferes with the binding of co-activator GRIP-1 to the β1-thyroid hormone receptor

Ligand binding to the thyroid hormone nuclear receptor β1 (TRβ1) is inhibited by desethylamiodarone (DEA), the major metabolite of the widely used anti‐arrhythmic drug amiodarone. Gene expression of thyroid hormone (triiodothyronine, T3)‐regulated genes can therefore be affected by amiodarone due to less ligand binding to the receptor. Previous studies have indicated the possibility of still other explanations for the inhibitory effects of amiodarone on T3‐dependent gene expression, probably via interference with receptor/co‐activator and co‐repressor complex. The binding site of DEA is postulated to be on the outside surface of the receptor protein overlapping the regions where co‐activator and co‐repressor bind. Here we show the effect of a drug metabolite on the interaction of TRβ1 with the co‐activator GRIP‐1 (glucocorticoid receptor interacting protein‐1). The T3‐dependent binding of GRIP‐1 to the TRβ1 is disrupted by DEA. A DEA dose experiment showed that the drug metabolite acts like an antagonist under ‘normal’ conditions (at 10−7 M T3 and 5×10−6→10−3 M DEA), but as an agonist under extreme conditions (at 0 and 10−9 M T3 and >10−4 M DEA). To our knowledge, these results show for the first time that a metabolite of a drug which was not devised for this purpose can interfere with nuclear receptor/co‐activator interaction.

Effect of mutations in the β1‐thyroid hormone receptor on the inhibition of T3 binding by desethylamiodarone

Desethylamiodarone (DEA) acts as a competitive inhibitor of triiodothyronine (T3) binding to the α1‐thyroid hormone receptor (TRα1) but as a non‐competitive inhibitor with respect to TRβ1. To gain insight into the position of the binding site of desethylamiodarone on TRβ1 we investigated the naturally occurring mutants Y321C, R429Q, P453A, P453T and the artificial mutants L421R and E457A in the ligand binding domain of human TRβ1. The IC50 values (in μM) of DEA for P453A (50±11) and P453T (55±16) mutant TRβ1 are not different from that for the wild type TRβ1 (56±15), but the IC50 values of R429Q (32±7; P<0.001) and E457A (17±3; P<0.001) are significantly lower than of the wild type. Scatchard plots and Langmuir analyses indicate a non‐competitive nature of the inhibition by DEA of T3 binding to all four mutant TRβ1s tested. Mutants P453A and P453T do not influence overall electrostatic potential, and also do not influence the affinity for DEA compared to wild type. Mutant E457A causes a change from a negatively charged amino acid to a hydrophobic amino acid, enhancing the affinity for DEA. Mutant R429Q, located in helix 11, causes an electrostatic potential change from positive to uncharged, also resulting in greater affinity for DEA. We therefore postulate that amino acids R429 and E457 are at or close to the binding site for DEA, and that DEA does not bind in the T3 binding pocket itself, in line with the non‐competitive nature of the inhibition of T3 binding to TRβ1 by DEA.

Differential effects of fasting vs food restriction on liver thyroid hormone metabolism in male rats

A variety of illnesses that leads to profound changes in the hypothalamus-pituitary-thyroid (HPT) are axis collectively known as the nonthyroidal illness syndrome (NTIS). NTIS is characterized by decreased tri-iodothyronine (T3) and thyroxine (T4) and inappropriately low TSH serum concentrations, as well as altered hepatic thyroid hormone (TH) metabolism. Spontaneous caloric restriction often occurs during illness and may contribute to NTIS, but it is currently unknown to what extent. The role of diminished food intake is often studied using experimental fasting models, but partial food restriction might be a more physiologically relevant model. In this comparative study, we characterized hepatic TH metabolism in two models for caloric restriction: 36 h of complete fasting and 21 days of 50% food restriction. Both fasting and food restriction decreased serum T4 concentration, while after 36-h fasting serum T3 also decreased. Fasting decreased hepatic T3 but not T4 concentrations, while food restriction decreased both hepatic T3 and T4 concentrations. Fasting and food restriction both induced an upregulation of liver D3 expression and activity, D1 was not affected. A differential effect was seen in Mct10 mRNA expression, which was upregulated in the fasted rats but not in food-restricted rats. Other metabolic pathways of TH, such as sulfation and UDP-glucuronidation, were also differentially affected. The changes in hepatic TH concentrations were reflected by the expression of T3-responsive genes Fas and Spot14 only in the 36-h fasted rats. In conclusion, limited food intake induced marked changes in hepatic TH metabolism, which are likely to contribute to the changes observed during NTIS.

A model for chronic, intrahypothalamic thyroid hormone administration in rats.

In addition to the direct effects of thyroid hormone (TH) on peripheral organs, recent work showed metabolic effects of TH on the liver and brown adipose tissue via neural pathways originating in the hypothalamic paraventricular and ventromedial nucleus (PVN and VMH). So far, these experiments focused on short-term administration of TH. The aim of this study is to develop a technique for chronic and nucleus-specific intrahypothalamic administration of the biologically active TH tri-iodothyronine (T3). We used beeswax pellets loaded with an amount of T3 based on in vitro experiments showing stable T3 release (∼5 nmol l(-1)) for 32 days. Upon stereotactic bilateral implantation, T3 concentrations were increased 90-fold in the PVN region and 50-fold in the VMH region after placing T3-containing pellets in the rat PVN or VMH for 28 days respectively. Increased local T3 concentrations were reflected by selectively increased mRNA expression of the T3-responsive genes Dio3 and Hr in the PVN or in the VMH. After placement of T3-containing pellets in the PVN, Tshb mRNA was significantly decreased in the pituitary, without altered Trh mRNA in the PVN region. Plasma T3 and T4 concentrations decreased without altered plasma TSH. We observed no changes in pituitary Tshb mRNA, plasma TSH, or plasma TH in rats after placement of T3-containing pellets in the VMH. We developed a method to selectively and chronically deliver T3 to specific hypothalamic nuclei. This will enable future studies on the chronic effects of intrahypothalamic T3 on energy metabolism via the PVN or VMH.