Identification of molecular mechanisms related to nonthyroidal illness syndrome in skeletal muscle and adipose tissue from patients with septic shock.

OBJECTIVE: Septic shock is one of various causes of nonthyroidal illness syndrome (NTIS). In humans, the molecular mechanisms involved in NTIS are mostly unknown. The aim of this study was to investigate, in patients with NTIS secondary to septic shock, changes in the expression of genes involved in the actions of thyroid hormones and in the activity of deiodinase enzymes, in two tissues important for protein and energy metabolism, skeletal muscle (SM) and subcutaneous adipose tissue (SAT). DESIGN: Hospitalized patients were divided into a control and a septic shock NTIS group. MEASUREMENT: Serum collection for biochemical measurements, and SM and SAT biopsies for mRNA expression analysis of thyroid hormone receptors (THRB1, THRA1), retinoid X receptors (RXRA, RXRB, RXRG), nuclear receptor corepressor (NCOR1), silencing mediator of retinoid and thyroid hormone receptor (SMRT), steroid receptor coactivator (SRC1), type 1 and 2 deiodinases (D1, D2), monocarboxylate transporter 8 (MCT8), SECIS binding protein 2 (SBP2) and uncoupling protein 3 (UCP3) as well as D1, D2 and D3 enzyme activity measurements. RESULTS: The NTIS group had lower serum TSH, and free T3 and higher rT3 than controls. D1 and D3 were detected in SAT, with no differences found between the two groups; SM had very low D2 activity and again no differences were found between groups; D3 activity in SM was higher in NTIS than controls. SM expression of THRB1, RXRG and D2 was lower and RXRA higher in NTIS than controls. SAT from NTIS patients had lower MCT8, THRB1, THRA1, RXRG and SMRT, and higher UCP3 expression than controls. CONCLUSIONS: In patients with septic shock NTIS tissue responses are orientated to decrease production and increase degradation (muscle) or decrease uptake (adipose tissue) of T3, as well as to decrease thyroid hormone actions.

Differential effects of maternal dexamethasone treatment on circulating thyroid hormone concentrations and tissue deiodinase activity in the pregnant ewe and fetus.

Clinically, treatment of pregnant women at risk of preterm delivery with synthetic glucocorticoids accelerates fetal maturation. This study investigated the effect of maternal dexamethasone treatment, in clinically relevant doses, on plasma thyroid hormone concentrations and tissue deiodinase activities (D1, D2, and D3) in ewes and their fetuses. From 125 d of gestation (term 145 +/- 2 d), pregnant ewes were injected twice im with either saline (2 ml of 0.9% NaCl, n = 11) or dexamethasone (2 x 12 mg in 2 ml of saline, n = 10) at 24-h intervals. Maternal dexamethasone treatment increased plasma T(3) and reverse T(3) (rT(3)), but not T(4), concentrations in the fetuses. In the dexamethasone-exposed fetuses, hepatic D1 activity was higher, and renal and placental D3 activities were lower, than in the saline-exposed fetuses. In the ewes, plasma concentrations of T(3) and T(4) were reduced, and rT(3) increased, by dexamethasone treatment without any change in tissue deiodinase activity. Therefore, maternal dexamethasone treatment has different effects on the thyroid hormone axis of the pregnant ewe and fetus. In the fetus, the dexamethasone-induced rise in circulating T(3) may be due to both increased hepatic production of T(3) from T(4), and reduced clearance of T(3) by the kidney and placenta. Changes in T(3) bioavailability may mediate some of the maturational effects of antenatal glucocorticoid treatment in the preterm fetus.

Developmental control of iodothyronine deiodinases by cortisol in the ovine fetus and placenta near term.

Preterm infants have low serum T4 and T3 levels, which may partly explain the immaturity of their tissues. Deiodinase enzymes are important in determining the bioavailability of thyroid hormones: deiodinases D1 and D2 convert T4 to T3, whereas deiodinase D3 inactivates T3 and produces rT3 from T4. In human and ovine fetuses, plasma T3 rises near term in association with the prepartum cortisol surge. This study investigated the developmental effects of cortisol and T3 on tissue deiodinases and plasma thyroid hormones in fetal sheep during late gestation. Plasma cortisol and T3 concentrations in utero were manipulated by exogenous hormone infusion and fetal adrenalectomy. Between 130 and 144 d of gestation (term 145+/-2 d), maturational increments in plasma cortisol and T3, and D1 (hepatic, renal, perirenal adipose tissue) and D3 (cerebral), and decrements in renal and placental D3 activities were abolished by fetal adrenalectomy. Between 125 and 130 d, iv cortisol infusion raised hepatic, renal, and perirenal adipose tissue D1 and reduced renal and placental D3 activities. Infusion with T3 alone increased hepatic D1 and decreased renal D3 activities. Therefore, in the sheep fetus, the prepartum cortisol surge induces tissue-specific changes in deiodinase activity that, by promoting production and suppressing clearance of T3, may be responsible for the rise in plasma T3 concentration near term. Some of the maturational effects of cortisol on deiodinase activity may be mediated by T3.

Tissue-Specific Suppression of Thyroid Hormone Signaling in Various Mouse Models of Aging.

DNA damage contributes to the process of aging, as underscored by premature aging syndromes caused by defective DNA repair. Thyroid state changes during aging, but underlying mechanisms remain elusive. Since thyroid hormone (TH) is a key regulator of metabolism, changes in TH signaling have widespread effects. Here, we reveal a significant common transcriptomic signature in livers from hypothyroid mice, DNA repair-deficient mice with severe (Csbm/m/Xpa-/-) or intermediate (Ercc1-/Δ-7) progeria and naturally aged mice. A strong induction of TH-inactivating deiodinase D3 and decrease of TH-activating D1 activities are observed in Csbm/m/Xpa-/- livers. Similar findings are noticed in Ercc1-/Δ-7, in naturally aged animals and in wild-type mice exposed to a chronic subtoxic dose of DNA-damaging agents. In contrast, TH signaling in muscle, heart and brain appears unaltered. These data show a strong suppression of TH signaling in specific peripheral organs in premature and normal aging, probably lowering metabolism, while other tissues appear to preserve metabolism. D3-mediated TH inactivation is unexpected, given its expression mainly in fetal tissues. Our studies highlight the importance of DNA damage as the underlying mechanism of changes in thyroid state. Tissue-specific regulation of deiodinase activities, ensuring diminished TH signaling, may contribute importantly to the protective metabolic response in aging.

Daily variations in type II iodothyronine deiodinase activity in the rat brain as controlled by the biological clock.

Type II deiodinase (D2) plays a key role in regulating thyroid hormone-dependent processes in, among others, the central nervous system (CNS) by accelerating the intracellular conversion of T4 into active T3. Just like the well-known daily rhythm of the hormones of the hypothalamo-pituitary-thyroid axis, D2 activity also appears to show daily variations. However, the mechanisms involved in generating these daily variations, especially in the CNS, are not known. Therefore, we decided to investigate the role the master biological clock, located in the hypothalamus, plays with respect to D2 activity in the rat CNS as well as the role of one of its main hormonal outputs, i.e. plasma corticosterone. D2 activity showed a significant daily rhythm in the pineal and pituitary gland as well as hypothalamic and cortical brain tissue, albeit with a different timing of its acrophase in the different tissues. Ablation of the biological clock abolished the daily variations of D2 activity in all four tissues studied. The main effect of the knockout of the suprachiasmatic nuclei (SCN) was a reduction of nocturnal peak levels in D2 activity. Moreover, contrary to previous observations in SCN-intact animals, in SCN-lesioned animals, the decreased levels of D2 activity are accompanied by decreased plasma levels of the thyroid hormones, suggesting that the SCN separately stimulates D2 activity as well as the hypothalamo-pituitary-thyroid axis.

Serum 3,3',5'-triiodothyronine (rT3) and 3,5,3'-triiodothyronine/rT3 are prognostic markers in critically ill patients and are associated with postmortem tissue deiodinase activities.

INTRODUCTION AND METHODS: Critical illness is associated with reduced TSH and thyroid hormone secretion, and with changes in peripheral thyroid hormone metabolism, resulting in low serum T3 and high rT3. In 451 critically ill patients who received intensive care for more than 5 d, serum thyroid parameters were determined on d 1, 5, 15, and last day (LD). All patients had been randomized for intensive or conventional insulin treatment. Seventy-one patients died, and postmortem liver and skeletal muscle biopsies were obtained from 50 of them for analysis of deiodinase (D1-3) activities. RESULTS: Insulin treatment did not affect thyroid parameters. On d 1, rT3 was higher and T3/rT3 was lower in nonsurvivors as compared with survivors (P = 0.001). Odds ratio for survival of the highest vs. the lowest quartile was 0.3 for rT3 and 2.9 for T3/rT3. TSH, T4, and T3 were lower in nonsurvivors from d 5 until LD (P < 0.001). TSH, T4, T3, and T3/rT3 increased over time in survivors, but decreased or remained unaltered in nonsurvivors. Liver D1 activity was positively correlated with LD serum T3/rT3 (R = 0.83, P < 0.001) and negatively correlated with rT3 (R = -0.69, P < 0.001). Both liver and skeletal muscle D3 activity were positively correlated with LD serum rT3 (R = 0.32, P = 0.02 and R = 0.31, P = 0.03). CONCLUSION: In critically ill patients who required more than 5 d of intensive care, rT3 and T3/rT3 were already prognostic for survival on d 1. On d 5, T4, T3, but also TSH levels are higher in patients who will survive. Serum rT3 and T3/rT3 were correlated with postmortem tissue deiodinase activities.

Tissue thyroid hormone levels in critical illness.

CONTEXT: Pronounced alterations in serum thyroid hormone levels occur during critical illness. T3 decreases and rT3 increases, the magnitudes of which are related to the severity of disease. It is unclear whether these changes are associated with decreased tissue T3 concentrations and, thus, reduced thyroid hormone bioactivity. PATIENTS AND STUDY QUESTIONS: We therefore investigated, in 79 patients who died after intensive care and who did or did not receive thyroid hormone treatment, whether total serum thyroid hormone levels correspond to tissue levels in liver and muscle. Furthermore, we investigated the relationship between tissue thyroid hormone levels, deiodinase activities, and monocarboxylate transporter 8 expression. RESULTS: Tissue iodothyronine levels were positively correlated with serum levels, indicating that the decrease in serum T3 during illness is associated with decreased levels of tissue T3. Higher serum T3 levels in patients who received thyroid hormone treatment were accompanied by higher levels of liver and muscle T3, with evidence for tissue-specific regulation. Tissue rT3 and the T3/rT3 ratio were correlated with tissue deiodinase activities. Monocarboxylate transporter 8 expression was not related to the ratio of the serum over tissue concentration of the different iodothyronines. CONCLUSION: Our results suggest that, in addition to changes in the hypothalamus-pituitary-thyroid axis, tissue-specific mechanisms are involved in the reduced supply of bioactive thyroid hormone in critical illness.

Increased thyroxine sulfate levels in critically ill patients as a result of a decreased hepatic type I deiodinase activity.

INTRODUCTION: Marked changes in peripheral thyroid hormone metabolism occur in critical illness, resulting in low serum T3 and high rT3 levels. In this study, we investigated whether T4S levels are increased in patients who died after intensive care and whether T4S levels are correlated with liver type I deiodinase (D1) or sulfotransferase (SULT) activity. METHODS: A total of 64 blood samples and 65 liver biopsies were obtained within minutes after death from 79 intensive care patients, randomized for intensive or conventional insulin treatment. Serum T4S and the activities of hepatic D1 and 3,3'-diiodothyronine (T2)-SULT and estrogen-SULT were determined. RESULTS: No differences in T4S or hepatic SULT activities were found between patients treated with intensive or with conventional insulin therapy. T4S levels were significantly elevated compared with healthy references. Furthermore, hepatic D1, but not SULT activity, showed a strong correlation with serum T4S (R = -0.53; P < 0.001) and T4S/T4 ratio (R = -0.62; P < 0.001). Cause of death was significantly correlated with hepatic T2- and estrogen-SULT activities (P < 0.01), with SULT activities being highest in the patients who died of severe brain damage and lowest in the patients who died of a cardiovascular collapse. A longer period of intensive care was associated with higher levels of T4S (P = 0.005), and high levels of bilirubin were associated with low T2-SULT (P = 0.04) activities and high levels of T4S (P < 0.001). CONCLUSION: Serum T4S levels were clearly elevated compared with healthy references, and the decreased deiodination by liver D1 during critical illness appears to play a role in this increase in serum T4S levels.

Characteristics and thyroid state-dependent regulation of iodothyronine deiodinases in pigs.

Three iodothyronine deiodinases (D1, D2, and D3) regulate local and systemic availability of thyroid hormone. D1 and D2 activate the prohormone T4 to the thyromimetic T3, and D3 inactivates T4 and T3 to rT3 and 3,3'-diiodothyronine, respectively. The expression of the three deiodinases is tightly regulated with regard to developmental stage and cell type to provide fine tuning of T3 supply to target cells. Most studies regarding distribution and regulation of deiodinases have been carried out in rodents. However, in different respects, rodents do not seem to be the optimal experimental model for human thyroid hormone physiology. For instance, D2 expression has been observed in human thyroid and skeletal muscle but not in these tissues in rodents. In this study, we have explored the pig as an alternative model. Porcine D1, D2, and D3 were cloned by RT-PCR, and their catalytic properties were shown to be virtually identical to those reported for human and rodent deiodinases. The tissue distribution of deiodinases was studied in normal pigs and in pigs made hypothyroid by methimazole treatment or in pigs made hyperthyroid by T4 treatment. D1 activity in liver and kidney was increased in T4-treated pigs. D2 activities in cerebrum and pituitary were decreased after T4 treatment and strongly increased after methimazole treatment. Remarkably, D2 activity in thyroid and skeletal muscle was induced in hypothyroid pigs. Significant expression of D3 was observed in cerebrum and was positively regulated by thyroid state. In conclusion, the pig appears to be a valuable model for human thyroid hormone physiology. The expression of D2 activity in thyroid and skeletal muscle is of particular interest for studies on the importance of this enzyme in (hypothyroid) humans.

Induction of thyroid hormone-degrading deiodinase in cardiac hypertrophy and failure.

The similarities between the changes in cardiac gene expression in pathological ventricular hypertrophy and hypothyroidism suggest a role of impaired cardiac thyroid hormone (TH) action in the development of contractile dysfunction during chronic cardiac pressure overload. Here we studied the possible involvement of altered cardiac TH metabolism using a rat model of right-ventricular (RV) hypertrophy induced by pressure-overload. Pathological RV hypertrophy was indicated by decreased mRNA levels of sarcoplasmic reticulum(SR) Ca2-ATPase type 2a (SERCA2a) and myosin heavy chain a (MHCalpha), and increased levels of MHCbeta mRNA. Enzyme activity of type HI deiodinase (D3), which converts T4 and T3 to the inactive compounds rT3 and 3,3'-T2, respectively, was identified in ventricular tissue. This activity was stimulated up to five fold in hypertrophic RV, but remained unaltered in the non-hypertrophic left ventricle (LV). A low level of type Ideiodinase activity was also detected, which decreased significantly in both RV and LV. Stimulation of RV D3 activity was significantly higher in those animals in which hypertrophy progressed to heart failure, compared to animals that developed compensatory hypertrophy. The induction of a cardiac TR-degrading deiodinase maybe expected to result in reduced cellular levels of T3 and thereby contribute to a local hypothyroid state in the hypertrophic and, particularly, in the failing ventricle.

Characterization of iodothyronine sulfatase activities in human and rat liver and placenta.

In conditions associated with high serum iodothyronine sulfate concentrations, e.g. during fetal development, desulfation of these conjugates may be important in the regulation of thyroid hormone homeostasis. However, little is known about which sulfatases are involved in this process. Therefore, we investigated the hydrolysis of iodothyronine sulfates by homogenates of V79 cells expressing the human arylsulfatases A (ARSA), B (ARSB), or C (ARSC; steroid sulfatase), as well as tissue fractions of human and rat liver and placenta. We found that only the microsomal fraction from liver and placenta hydrolyzed iodothyronine sulfates. Among the recombinant enzymes only the endoplasmic reticulum-associated ARSC showed activity toward iodothyronine sulfates; the soluble lysosomal ARSA and ARSB were inactive. Recombinant ARSC as well as human placenta microsomes hydrolyzed iodothyronine sulfates with a substrate preference for 3,3'-diiodothyronine sulfate (3,3'-T(2)S) approximately T(3) sulfate (T(3)S) >> rT(3)S approximately T(4)S, whereas human and rat liver microsomes showed a preference for 3,3'-T(2)S > T(3)S >> rT(3)S approximately T(4)S. ARSC and the tissue microsomal sulfatases were all characterized by high apparent K(m) values (>50 microM) for 3,3'-T(2)S and T(3)S. Iodothyronine sulfatase activity determined using 3,3'-T(2)S as a substrate was much higher in human liver microsomes than in human placenta microsomes, although ARSC is expressed at higher levels in human placenta than in human liver. The ratio of estrone sulfate to T(2)S hydrolysis in human liver microsomes (0.2) differed largely from that in ARSC homogenate (80) and human placenta microsomes (150). These results suggest that ARSC accounts for the relatively low iodothyronine sulfatase activity of human placenta, and that additional arylsulfatase(s) contributes to the high iodothyronine sulfatase activity in human liver. Further research is needed to identify these iodothyronine sulfatases, and to study the physiological importance of the reversible sulfation of iodothyronines in thyroid hormone metabolism.

Molecular basis for the substrate selectivity of cat type I iodothyronine deiodinase.

The type I iodothyronine deiodinase (D1) catalyzes the activation of T4 to T3 as well as the degradation of T3 (rT3) and sulfated iodothyronines. A comparison of the catalytic activities of D1 in liver microsomal preparations from several species revealed a remarkable difference between cat D1 on one hand and rat/human D1 on the other hand. The Michaelis constant (Km) of cat D1 for rT3 (11 microm) is 30-fold higher than that of rat and human D1 (0.2-0.5 microm). Deiodination of rT3 by cat D1 is facilitated by sulfation [maximal velocity (Vmax)/Km rT3 = 3 and Vmax/Km rT3S = 81]. To understand the molecular basis for the difference in substrate interaction the cat D1 cDNA was cloned, and the deduced amino acid sequence was compared with rat/human D1 protein. In the region between amino acid residues 40 and 70 of cat D1, various differences with rat/human D1 are concentrated. By site-directed mutagenesis of cat D1 it was found that a combination of mutations was necessary to improve the deiodination of rT3 by cat D1 enzyme. For efficient rT3 deiodination, a Phe at position 65 and the insertion of the Thr-Gly-Met-Thr-Arg48-52 sequence as well as the amino acids Gly and Glu at position 45-46 are essential. Either of these changes alone resulted in only a limited improvement of rT3 deiodination. At the same time the combination of the described mutations did not affect the already quite efficient outer ring deiodination of rT3S nor the inner ring deiodination of T3S, whereas each of the described changes alone did affect rT3S deiodination. Our findings suggest great flexibility of the active site in D1 that adapts to its various substrates. The active site of wild-type cat D1 is less flexible than the active site of rat/human D1 and favors sulfated iodothyronines.

Reduced activation and increased inactivation of thyroid hormone in tissues of critically ill patients.

Critical illness is often associated with reduced TSH and thyroid hormone secretion as well as marked changes in peripheral thyroid hormone metabolism, resulting in low serum T(3) and high rT(3) levels. To study the mechanism(s) of the latter changes, we determined serum thyroid hormone levels and the expression of the type 1, 2, and 3 iodothyronine deiodinases (D1, D2, and D3) in liver and skeletal muscle from deceased intensive care patients. To study mechanisms underlying these changes, 65 blood samples, 65 liver, and 66 skeletal muscle biopsies were obtained within minutes after death from 80 intensive care unit patients randomized for intensive or conventional insulin treatment. Serum thyroid parameters and the expression of tissue D1-D3 were determined. Serum TSH, T(4), T(3), and the T(3)/rT(3) ratio were lower, whereas serum rT(3) was higher than in normal subjects (P < 0.0001). Liver D1 activity was down-regulated and D3 activity was induced in liver and skeletal muscle. Serum T(3)/rT(3) ratio correlated positively with liver D1 activity (P < 0.001) and negatively with liver D3 activity (ns). These parameters were independent of the type of insulin treatment. Liver D1 and serum T(3)/rT(3) were highest in patients who died from severe brain damage, intermediate in those who died from sepsis or excessive inflammation, and lowest in patients who died from cardiovascular collapse (P < 0.01). Liver D3 showed an opposite relationship. Acute renal failure requiring dialysis and need of inotropes were associated with low liver D1 activity (P < 0.01 and P = 0.06) and high liver D3 (P < 0.01) and skeletal muscle D3 (P < 0.05) activity. Liver D1 activity was negatively correlated with plasma urea (P = 0.002), creatinine (P = 0.06), and bilirubin (P < 0.0001). D1 and D3 mRNA levels corresponded with enzyme activities (both P < 0.001), suggesting regulation of the expression of both deiodinases at the pretranslational level. This is the first study relating tissue deiodinase activities with serum thyroid hormone levels and clinical parameters in a large group of critically ill patients. Liver D1 is down-regulated and D3 (which is not present in liver and skeletal muscle of healthy individuals) is induced, particularly in disease states associated with poor tissue perfusion. These observed changes, in correlation with a low T(3)/rT(3) ratio, may represent tissue-specific ways to reduce thyroid hormone bioactivity during cellular hypoxia and contribute to the low T(3) syndrome of severe illness.

Left-ventricular remodeling after myocardial infarction is associated with a cardiomyocyte-specific hypothyroid condition.

Similarities in cardiac gene expression in hypothyroidism and left ventricular (LV) pathological remodeling after myocardial infarction (MI) suggest a role for impaired cardiac thyroid hormone (TH) signaling in the development of heart failure. Increased ventricular activity of the TH-degrading enzyme type 3 deiodinase (D3) is recognized as a potential cause. In the present study, we investigated the cardiac expression and activity of D3 over an 8-wk period after MI in C57Bl/6J mice. Pathological remodeling of the noninfarcted part of the LV was evident from cardiomyocyte hypertrophy, interstitial fibrosis, and impairment of contractility. These changes were maximal and stable from the first week onward, as was the degree of LV dilation. A strong induction of D3 activity was found, which was similarly stable for the period examined. Plasma T(4) levels were transiently decreased at 1 wk after MI, but T(3) levels remained normal. The high D3 activity was associated with increased D3 mRNA expression at 1 but not at 4 and 8 wk after MI. Immunohistochemistry localized D3 protein to cardiomyocytes. In vivo measurement of TH-dependent transcription activity in cardiomyocytes using a luciferase reporter assay indicated a 48% decrease in post-MI mice relative to sham-operated animals, and this was associated with a 50% decrease in LV tissue T(3) concentration. In conclusion, pathological ventricular remodeling after MI in the mouse leads to high and stable induction of D3 activity in cardiomyocytes and a local hypothyroid condition.

Characterization of rat iodothyronine sulfotransferases.

Sulfation appears to be an important pathway for the reversible inactivation of thyroid hormone during fetal development. The rat is an often used animal model to study the regulation of fetal thyroid hormone status. The present study was done to determine which sulfotransferases (SULTs) are important for iodothyronine sulfation in the rat, using radioactive T4, T3, rT3, and 3,3'-T2 as substrates, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as cofactor, and rat liver, kidney and brain cytosol, and recombinant rat SULT1A1, -1B1, -1C1, -1E1, -2A1, -2A2, and -2A3 as enzymes. Recombinant rat SULT1A1, -1E1, -2A1, -2A2, and -2A3 failed to catalyze iodothyronine sulfation. For all tissue SULTs and for rSULT1B1 and rSULT1C1, 3,3'-T2 was by far the preferred substrate. Apparent Km values for 3,3'-T2 amounted to 1.9 microM in male liver, 4.4 microM in female liver, 0.76 microM in male kidney, 0.23 microM in male brain, 7.7 microM for SULT1B1, and 0.62 microM for SULT1C1, whereas apparent Km values for PAPS showed less variation (2.0-6.9 microM). Sulfation of 3,3'-T2 was inhibited dose dependently by other iodothyronines, with similar structure-activity relationships for most enzymes except for the SULT activity in rat brain. The apparent Km values of 3,3'-T2 in liver cytosol were between those determined for SULT1B1 and -1C1, supporting the importance of these enzymes for the sulfation of iodothyronines in rat liver, with a greater contribution of SULT1C1 in male than in female rat liver. The results further suggest that rSULT1C1 also contributes to iodothyronine sulfation in rat kidney, whereas other, yet-unidentified forms appear more important for the sulfation of thyroid hormone in rat brain.