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.

Hyper and hypothyroidism change the expression and diurnal variation of thyroid hormone receptor isoforms in rat liver without major changes in their zonal distribution.

We investigated the effect of hypothyroidism or hyperthyroidism on mRNA and protein expression, diurnal variation and zonal distribution of thyroid hormone receptor (TR) isoforms TRalpha1, TRalpha2 and TRbeta1 in rat liver. Hypothyroidism results in increased isoform mRNA and protein expression whereas hyperthyroidism shows a decreased TRalpha1 and TRalpha2 mRNA and protein expression. During hyperthyroidism no change is seen in TRbeta1 mRNA, but TRbeta1 protein is upregulated in the light period and downregulated in the dark period. Diurnal changes (measured at 13:30 and 19:30 h) in the TR isoform proteins are abolished in hypothyroidism and hyperthyroidism, with the exception of a reversal in diurnal changes of TRbeta1 in hyperthyroidism. Zonal distribution of the isoforms is not affected by hypo- or hyperthyroidism. We therefore conclude that thyroid hormone influences both the levels and the diurnal expression of its receptor isoforms but not the zonal distribution.

Contribution of interleukin-12 to the pathogenesis of non-thyroidal illness.

Changes in both central and peripheral thyroid hormone (TH) metabolism occur during illness. These changes, known collectively as non-thyroidal illness, are apparently mediated by the proinflammatory cytokines IL-6, TNFalpha and IFNgamma. IL-12 is involved in regulation of IFNgamma and TNFalpha. The aim of this study was to evaluate the role of IL-12 in TH metabolism during illness. We studied TH metabolism both centrally (in the pituitary) and peripherally (in the liver) in IL-12 knock-out (IL-12 (-/-)) and wild type (WT) mice during illness induced by administration of bacterial endotoxin (LPS). LPS induced a similar decrease in serum T (3), T (4) and liver 5'-DI mRNA expression in IL-12 (-/-) and WT mice with the exception of a smaller reduction of serum T (4) in IL-12 (-/-) mice. In the pituitary, the LPS-induced decline in 5'-DI activity in WT mice was not observed in IL-12 (-/-) mice (p < 0.001), whereas the decrease in DII activity tended to be smaller in IL-12 (-/-) mice (p = 0.066). The lower decrease in pituitary activity of both DI and DII in IL-12 (-/-) mice is possibly related to the lower LPS-induced T (4) decrease. In conclusion, IL-12 is involved in the central regulation of the HPT axis during illness but not in the peripheral regulation.

Diurnal variation in rat liver thyroid hormone receptor (TR)-alpha messenger ribonucleic acid (mRNA) is dependent on the biological clock in the suprachiasmatic nucleus, whereas diurnal variation of TR beta 1 mRNA is modified by food intake.

Previous studies have shown a diurnal variation of certain isoforms of thyroid hormone receptors (TR) in rat liver. The genesis of these diurnal changes is still unknown. To clarify whether the biological clock, located in the hypothalamic suprachiasmatic nucleus (SCN), is involved, we made selective SCN lesions. Rats with an SCN lesion lost their circadian rhythm of plasma corticosterone and TSH when compared with intact animals. TR alpha 1 and TR alpha 2 mRNA expression of control rats was higher in the light period than in the dark period; changes that were abolished in the rats with SCN lesions. In contrast, liver TR beta 1 mRNA of intact rats showed a diurnal variation that failed to reach statistical significance. To evaluate whether these effects could be explained indirectly by the disappearance of rhythmic feeding behavior in rats with SCN lesions, we performed a second experiment in which otherwise intact animals were subjected to a regular feeding (RF) schedule, with one meal every 4 h. When compared with rats with free access to food, RF only affected TR beta 1 mRNA expression and had no effect on the diurnal changes in TR alpha 1 and TR alpha 2. We conclude that liver TR beta 1 expression is most clearly affected by food intake. Diurnal changes in liver TR alpha 1 and TR alpha 2 are controlled by the biological clock in the SCN but not via changes in the daily rhythm of food intake. The findings may have physiological relevance for diurnal variation of T(3)-dependent gene expression, which is supported by a diurnal variation in the expression of the 5'-deiodinase gene.

A survey of iodine intake and thyroid volume in Dutch schoolchildren: reference values in an iodine-sufficient area and the effect of puberty.

BACKGROUND: Iodine deficiency and endemic goiter have been reported in the past in The Netherlands, especially in the southeast. OBJECTIVE: To evaluate iodine intake and thyroid size in Dutch schoolchildren, contrasting those living in a formerly iodine-deficient region in the east (Doetinchem) with those living in an iodine-sufficient region in the west (Amsterdam area). DESIGN: Cross-sectional survey of 937 Dutch schoolchildren aged 6--18 years, of whom 390 lived in the eastern and 547 in the western part of the country. METHODS: Thyroid size was assessed by inspection and palpation as well as by ultrasound. Iodine intake was evaluated by questionnaires on dietary habits and by measurement of urinary iodine concentration. RESULTS: Eastern and western regions were similar with respect to median urinary iodine concentration (15.7 and 15.3 microg/dl, NS, Mann-Whitney U test), goiter prevalence by inspection and palpation (0.8 and 2.6%, P=0.08, chi-squared test), and thyroid volumes. The P97.5 values of thyroid volumes per age and body surface area group were all lower than the corresponding sex-specific normative WHO reference values. Iodized salt was not used by 45.7% of households. Daily bread consumption was five slices by boys and four slices by girls. Weekly milk consumption was 3 liters by boys and 2 liters by girls. Seafish was consumed once monthly. From these figures we calculated a mean daily iodine intake of 171 microg in boys and 143 microg in girls, in good agreement with the measured median urinary concentration of 16.7 microg/dl in boys and 14.5 microg/dl in girls. The sex difference in iodine excretion is fully accounted for by an extra daily consumption of one slice of bread (20 microg I) and one-seventh of a liter of milk (8.3 microg I) by boys. Thyroid volume increases with age, but a steep increase by 41% was observed in girls between 11 and 12 years, and by 55% in boys between 13 and 14 years, coinciding with peak height velocity. Girls have a larger thyroid volume at the ages of 12 and 13 years, but thyroid volume is larger in boys as of the age of 14 years. CONCLUSIONS: (1) Iodine deficiency disorders no longer exist in The Netherlands. (2) Bread consumption remains the main source of dietary iodine in The Netherlands; the contribution of iodized table salt and seafish is limited. (3) The earlier onset of puberty in girls renders their thyroid volume larger than in boys at the age of 12--13 years, but boys have a larger thyroid volume as of the age of 14 years.

The biological relevance of thyroid hormone receptors in immortalized human umbilical vein endothelial cells.

The gene expression of thyroid hormone receptors (TR) in ECRF24 immortalized human umbilical vein endothelial cells (HUVECs) was investigated at both the mRNA and the protein level. Endothelin-1 (ET-1) and von Willebrand factor (vWF) production were measured in response to triiodothyronine (T(3)) administration. A real-time PCR technique was used to quantify the presence of mRNAs encoding for the different isoforms of the TR. The binding of T(3) to nuclear TRs was studied in isolated endothelial cell nuclei by Scatchard analysis. Expression of TR at the protein level was investigated by immunocytochemistry and Western blotting using TR-isoform-specific polyclonal rabbit antisera. ET-1 and vWF were measured in cell supernatants with a two-site immunoenzymatic assay. Scatchard analysis yielded a maximum binding capacity of 55 fmol T(3)/mg DNA (+/-200 sites/cell) with a K(d) of 125 pmol/l. Messenger RNAs encoding for the TRalpha1 and the TRalpha2 and the TRbeta1 were observed. The approximate number of mRNA molecules per cell was at least 50 molecules per cell for TRalpha1, five for TRalpha2 and two for TRbeta1. Immunocytochemistry revealed (peri)nuclear staining for TRbeta1, TRalpha1 and TRalpha2. ET-1 and vWF secretion did not increase upon addition of T(3) (10(-10)-10(-6) M). Immortalized ECRF24 HUVECs express TR, but at low levels. The number of TRs per endothelial cell is probably too low to be functional and no change in ET-1 or vWF production was found after addition of T(3). Therefore we conclude that the genomic effects of T(3) are unlikely to occur in these immortalized HUVECs.

Immunoneutralization of interleukin-1, tumor necrosis factor, interleukin-6 or interferon does not prevent the LPS-induced sick euthyroid syndrome in mice.

The sick euthyroid syndrome is a state of altered thyroid hormone metabolism which occurs during illness. The pathogenesis is incompletely understood but recent studies indicate a role of cytokines. It is unknown if cytokines released during illness are directly responsible for the changes in thyroid hormone metabolism. Therefore we studied if previous immunoneutralization of cytokines can prevent endotoxin (lipopolysaccharide LPS), induced sick euthyroid syndrome. LPS administration resulted in systemic illness, an increase in serum tumor necrosis factor (TNF alpha) and interleukin (IL)-6 and a decrease in serum triiodothyronine (T3) and thyroxine (T4). Immunoneutralization of the effects of cytokines was accomplished by administration of monoclonal antibodies against mouse IL-1 type-1 receptor (IL-1R), TNF alpha, IL-6 or interferon (IFN gamma) prior to LPS. The LPS-induced release of cytokines was affected by previous immunoneutralization as compared with control experiments with normal immunoglobulin (IgG): anti-IL-1R did not affect serum TNF alpha but decreased serum IL-6, anti-TNF alpha decreased serum TNF alpha but not IL-6, anti-IL-6 did not affect serum TNF alpha but hugely increased IL-6 and anti-IFN gamma decreased both serum TNF alpha and IL-6. Specific immunoneutralization of IL-1, TNF alpha or IFN gamma did not prevent the LPS-induced decrease in serum T3, T4 and liver 5'-deiodinase mRNA. However, immunoneutralization of IL-6, although not preventing the fall in serum T3 and T4, did mitigate the LPS-induced decrease in liver 5'-deiodinase mRNA. In view of possible non-specific effects of the huge dose of immunoglobulins (1 mg), used only in the immunoneutralization of IL-6, we repeated the experiment with F(ab')2 fragments of anti-IL-6 antibodies. Compared with F(ab')2 fragments of control IgG, anti-IL-6 F(ab')2 did not affect the LPS-induced rise in serum TNF alpha or the decrease in serum T3 and T4 and liver 5'-deiodinase mRNA. Serum IL-6 levels induced by LPS were, however, cleared more rapidly from the circulation when anti-IL-6 F(ab')2 fragments rather than intact anti-IL-6 were administered. In conclusion, immunoneutralization of IL-1, TNF alpha or IFN gamma did not prevent the LPS-induced sick euthyroid syndrome in mice; immunoneutralization of IL-6, however, transiently inhibits the LPS-induced decrease of liver 5'-deiodinase mRNA.

Selective macrophage depletion in the liver does not prevent the development of the sick euthyroid syndrome in the mouse.

A decreased serum triiodothyronine (T3) level is one of the main characteristics of the sick euthyroid syndrome, caused mainly by a decreased 5'-deiodination of thyroxine (T4) in the liver. Cytokines have been implicated in the pathogenesis of the changes in thyroid hormone metabolism during illness. We therefore investigated the role of cytokines produced by the liver macrophages (Kupffer cells) in the development of the sick euthyroid syndrome, which was induced in mice by a single injection of bacterial endotoxin (lipopolysaccharide) or by 24-h starvation. Experiments were carried out with or without previous selective depletion of liver macrophages by intravenous administration of liposome-encapsulated dichloromethylene diphosphonate. Relative to saline-injected pair-fed controls, the administration of lipopolysaccharide caused a decrease of serum T3 and T4 and liver 5'-deiodinase mRNA. Selective depletion of liver macrophages, did not affect these changes. Starvation for 24h decreased serum T3 and T4, associated with a slight decrease of liver 5'-deiodinase mRNA. There were no differences between macrophage-depleted and non-depleted animals in this respect. In summary, selective depletion of liver macrophages did not affect the decrease in serum T3, T4 or liver 5'-deiodinase mRNA induced by lipopolysaccharide or 24-h starvation in mice. We conclude that cytokines produced by Kupffer cells are not involved in the pathogenesis of the sick euthyroid syndrome in this experimental model.

The role of cytokines in the lipopolysaccharide-induced sick euthyroid syndrome in mice.

To evaluate the role of cytokines in the sick euthyroid syndrome, we tried to establish an animal model of non-thyroidal illness in mice by the administration of a sub-lethal dose of bacterial endotoxin (lipopolysaccharide; LPS) which induces a variety of cytokines, including tumour necrosis factor (TNF alpha), interleukin-1 (IL-1 alpha), interleukin-6 (IL-6) and interferon-gamma (IFN gamma). When compared with pair-fed controls, a single dose of LPS resulted in (a) systemic illness, (b) induction of TNF alpha and IL-6 and (c) a decrease of liver 5'-deiodinase mRNA from 4 h onwards followed by a decrease of serum tri-iodothyronine (T3) and thyroxine (T4) at 8 h and of serum free T3 (fT3) and free T4 (fT4) at 24 h; serum TSH remained unchanged. We then studied whether a single dose or a combination of IL-1 alpha, TNF alpha, IL-6 or IFN gamma could induce the sick euthyroid syndrome in mice, again using pair-fed controls. None of the cytokines except IL-1 alpha caused systemic illness, and IL-1 alpha was the only cytokine that decreased liver 5'-deiodinase mRNA transiently. IL-1 alpha, TNF alpha or IL-6 did not decrease serum T3, T4 and TSH, but administration of IFN gamma decreased serum T4, T3 and fT3 in a dose-dependent manner without changes in serum TSH. Administration of all four cytokines together had no synergistic effects; observed changes were of a smaller magnitude than after LPS. The following conclusions were reached.(ABSTRACT TRUNCATED AT 250 WORDS)

Soluble cytokine receptors and the low 3,5,3'-triiodothyronine syndrome in patients with nonthyroidal disease.

Cytokines have been implicated in the pathogenesis of the low T3 syndrome during illness. This is supported by our recent observation of a strong negative relationship between serum T3 and serum interleukin-6 (IL-6) in nonthyroidal illness (NTI). In the last few years, soluble cytokine receptors and cytokine receptor antagonists have been discovered in human serum. These proteins have the potential to further regulate cytokine activity. Therefore, we now studied the association between serum T3 and serum levels of soluble tumor necrosis factor-alpha (sTNF alpha R p55 and sTNF alpha R p75), soluble interleukin-2 receptor (sIL-2R), and the interleukin-1 receptor antagonist (IL-1RA) in 100 consecutive hospital admissions with a wide variety of nonthyroidal diseases. Patients were divided into group A (T3, > or = 1.30 nmol/L; T4, > or = 75 nmol/L; n = 41), group B (T3, < 1.30 nmol/L; T4, > or = 75 nmol/L; n = 46), and group C (T3, < 1.30 nmol/L; T4, < 75 nmol/L; n = 13). Serum sTNF alpha R p55, sTNF alpha R p75, sIL-2R, and IL-1RA were lower in group A than in groups B and C [median values; sTNF alpha R p55, 1.25, 2.25, and 3.55 ng/mL (P < 0.001); sTNF alpha R p75, 2.02, 4.56, and 7.00 ng/mL (P < 0.001); sIL-2R, 184, 259, and 272 U/mL (P = 0.0004), respectively]. Serum IL-1RA levels were not different in the three groups (median values, 122, 193, and 258 pg/mL, respectively). Taking all patients together, a significant negative relation was found among serum T3 and sTNF alpha p55 (r = -0.59; P < 0.0001), sTNF alpha R p75 (r = -0.55; P < 0.0001), sIL-2R (r = -0.54; P < 0.0001), IL-1RA (r = -0.38; P = 0.001), and IL-6 (r = -0.56; P < 0.0001). A remarkable high correlation (r = -0.70; P < 0.0001) was found between serum T3 and a newly designed total score based on the summation of serum levels of IL-6 and the four soluble cytokine receptor proteins. IL-6 and the four cytokine receptor proteins were all significantly related to each other. Stepwise multiple regression indicated IL-6 and sTNF alpha R p75 as independent determinants of T3 [serum T3 = 2.09-0.32ln (sTNF alpha R p75) -0.15ln (IL-6); r = 0.70]. The variability in serum T3 was accounted for 35% by changes in ln (sTNF alpha R p75) and 14% by changes in ln (IL-6).(ABSTRACT TRUNCATED AT 400 WORDS)

Association between serum interleukin-6 and serum 3,5,3'-triiodothyronine in nonthyroidal illness.

Increased serum concentrations of FFA, bilirubin, and carboxyl-methyl-propyl-furanpropionic acid, accumulating in chronic renal failure in direct relationship with serum creatinine, have all been implicated in the pathogenesis of the low T3 syndrome during illness. Cytokines may also be involved in the sick euthyroid syndrome. In contrast to interleukin-1 (IL-1) and tumor necrosis factor-alpha, IL-6 is usually detectable in serum during illness and acts as a systemic hormone. We studied the association between serum T3 and IL-6 in consecutive hospital admissions with a wide variety of medical conditions. Patients were divided into group A (T3, > or = 1.30 nmol/L; T4, > or = 75 nmol/L; n = 41), group B (T3, < 1.30 nmol/L; T4, > or = 75 nmol/L; n = 46), and group C (T3, < 1.30 nmol/L; T4, < 75 nmol/L; n = 13). Serum IL-6 levels in groups C and B were higher than those in group A (median values 59, 39, and 9 U/mL, respectively; P < 0.01). Serum creatinine and bilirubin/albumin ratios were similar in the three groups, but the FFA/albumin ratio in group C was higher than in group A (P < 0.05). When all patients were analyzed together, serum T3 was negatively correlated to serum IL-6 (r = -0.56; P < 0.001), bilirubin/albumin ratio (r = -0.29; P = 0.004), and FFA/albumin ratio (r = -0.21; P = 0.03), but not with creatinine (r = -0.16; P = 0.11). Stepwise multiple regression resulted in the following equation: serum T3 = 2.13-0.18ln(IL-6)-0.15ln(creatinine)-0.094ln(bilirubin /albumin) (r = 0.61). The variability in serum T3 was accounted for 28% by ln(IL-6), 5% by ln(creatinine), and 4% by ln(bilirubin/albumin). FFA/albumin did not contribute in this respect. We conclude that the low T3 syndrome in nonthyroidial illness is associated with high serum IL-6 levels. However, even when IL-6 is assumed to play a causative role, the variation of serum T3 in NTI-patients remains largely unexplained.

Inhibition of nuclear T3 binding by fatty acids: dependence on chain length, unsaturated bonds, cis-trans configuration and esterification.

1. Fatty acids have the capacity for inhibition of nuclear T3 binding (INB). The present studies were undertaken to describe the INB-activity of fatty acids as a function of chain length, unsaturated bonds, cis-trans configuration, and esterification. 2. Isolated rat liver nuclei were incubated with [125I]T3 in the absence or presence of fatty acids in concentrations of 0.011, 0.033, 0.1 and 0.3 mM respectively. 3. INB-activity depended on the chain length, being greatest at 14 carbon atoms. 4. INB by unsaturated fatty acids was greater than that of saturated fatty acids, and increased with increasing number of double bonds. 5. Fatty acids in the cis configuration had greater INB-activity than those in trans configuration. 6. Esterification of fatty acids decreased INB-activity: monoglycerides still had some effect, but di- and triglycerides had no effect.