Treatment with amiodarone, a potent antiarrhythmic drug, is associated with a dose-dependent increase in plasma cholesterol resulting from a decreased number of liver low-density lipoprotein (LDL) receptors. Similar changes occur in hypothyroidism, and it has been suggested that amiodarone acts via induction of a local 'hypothyroid-like' state in extrathyroidal tissues. The present study was designed to evaluate whether exogenous tri-iodothyronine (T₃) could prevent the effects of amiodarone on LDL cholesterol.

Rats were treated for 14 days with water, amiodarone 10 mg/100 g body weight (BW), or amiodarone and 2·5, 5 or 10 μg T₃/100 g BW respectively. Relative to controls, amiodarone increased plasma LDL cholesterol by 31% and decreased liver LDL receptor mRNA by 56% and protein by 45%; liver T₃ content was reduced by 21%. Addition of T₃ to the treatment with amiodarone dose-dependently reversed all these changes, with a return to control values of plasma cholesterol and the number of liver LDL receptors, although LDL receptor mRNA remained slightly lower. Treatment of rats for 14 days with T₃ alone (5 μg/100 g BW) decreased plasma LDL cholesterol by 19% and increased liver LDL receptor mRNA by 41%.

In conclusion, T₃ prevents the amiodarone-induced changes in plasma LDL cholesterol and liver LDL receptor gene expression. These findings suggest that the inhibitory effect of amiodarone on LDL receptor gene expression is mediated by T₃-dependent pathways.

Journal of Endocrinology (1997) 152, 413–421

One of the main characteristics of nonthyroidal illness (NTI) is a decrease in serum triiodothyronine, partly caused by a decrease in liver deiodinase type 1 (D1) mRNA and activity. Proinflammatory cytokines have been associated with NTI in view of their capability to decrease D1 and thyroid hormone receptor (TR)β1 mRNA expression in hepatoma cells. Proinflammatory cytokine induction leads to activation of the inflammatory pathways nuclear factor (NF)κB and activator protein (AP)-1. The proinflammatory cytokine interleukin (IL)-1β decreases thyroid hormone receptor (TR)β1 mRNA in an NFκB-dependent way. The aim of this study was to unravel the effects of IL-1β on endogenous TRα gene expression in an animal model and in a liver cell line. The TRα gene product is alternatively spliced in TRα1 and TRα2, TRα2 is capable of inhibiting TRα1-induced gene transcription. We showed that both TRα1 and TRα2 mRNA decreased not only after lipopolysaccharide administration in liver of mice, but also after IL-1β stimulation of hepatoma cells (HepG2). Using the NFκB inhibitor sulfasalazine and the AP-1 inhibitor SP600125, it became clear that the IL-1β-induced decrease in TRα mRNA expression in HepG2 cells can only be abolished by simultaneous inhibition of NFκB and AP-1. The IL-1β-induced TRα1 and TRα2 mRNA decrease in HepG2 cells is the result of decreased TRα gene promoter activity, as evident from actinomycin D experiments. Cycloheximide experiments showed that the decreased promoter activity is independent of de novo protein synthesis and therefore most likely due to posttranslational modifications such as phosphorylation or subcellular relocalization.

One of the hallmarks of the sick euthyroid syndrome or non-thyroidal illness is a decrease of serum triiodothyronine, caused mainly by a decrease in liver deiodinase type 1 (D1) mRNA and activity. Proinflammatory cytokines like interleukin (IL)-1β are likely involved in this disease, but are also known to inhibit thyroid hormone receptor (TR)-β1 gene expression, which is of interest as the D1 promoter contains TREs. The aim of the present study was to evaluate whether the IL-1β-induced decrease of D1 and TRβ1 mRNA is mediated by the same cytokine signalling pathways in a human hepatoma cell line (HepG2). We observed a downregulation of both D1 and TRβ1 mRNA after 4 h of incubating the cells with IL-1β. Sulfasalazine was used to inhibit the nuclear factor-κB (NFκB) pathway and SP600125, a chemical inhibitor of the c-Jun N-terminal kinase, was used as an inhibitor of the activator protein-1 (AP-1) pathway. AP-1 inhibition did not affect the decrease of D1 and TRβ1 mRNA, but the TRβ1 mRNA decrease was completely abolished after inhibiting NFκB, while D1 mRNA was unaffected. Only simultaneous inhibition of both the NFκB and AP-1 pathways abolished the D1 mRNA decrease. We concluded that IL-1β stimulation of HepG2 cells results in a marked decrease of D1 and TRβ1 mRNA. The decrease of TRβ1 mRNA is exclusively mediated by the NFκB pathway, while the decrease of D1 mRNA requires inhibition of both the AP-1 and the NFκB pathways.

Abstract

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 (TNFα) 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), TNFα, IL-6 or interferon (IFNα) 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 TNFα but decreased serum IL-6, anti-TNFα decreased serum TNFα but not IL-6, anti-IL-6 did not affect serum TNFα but hugely increased IL-6 and anti-IFNγ decreased both serum TNFα and IL-6. Specific immunoneutralization of IL-1, TNFα or IFNγ 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 TNFα 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, TNFα or IFNγ 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.

Journal of Endocrinology (1997) 153, 115–122

During illness, changes in thyroid hormone metabolism occur, known as nonthyroidal illness and characterised by decreased serum triiodothyronine (T₃) and thyroxine (T₄) without an increase in TSH. A mouse model of chronic illness is local inflammation, induced by a turpentine injection in each hind limb. Although serum T₃ and T₄ are markedly decreased in this model, it is unknown whether turpentine administration affects the central part of the hypothalamus–pituitary–thyroid axis (HPT-axis). We therefore studied thyroid hormone metabolism in hypothalamus and pituitary of mice during chronic inflammation induced by turpentine injection. Using pair-fed controls, we could differentiate between the effects of chronic inflammation per se and the effects of restricted food intake as a result of illness. Chronic inflammation increased interleukin (IL)-1β mRNA expression in the hypothalamus more rapidly than in the pituitary. This hypothalamic cytokine response was associated with a rapid increase in local D2 mRNA expression. By contrast, no changes were present in pituitary D2 expression. TSHβ mRNA expression was altered compared with controls. Comparing chronic inflamed mice with pair-fed controls, both preproTSH releasing hormone (TRH) and D3 mRNA expression in the paraventricular nucleus were significantly lower 48 h after turpentine administration. The timecourse of TSHβ mRNA expression was completely different in inflamed mice compared with pair-fed mice. Turpentine administration resulted in significantly decreased TSHβ mRNA expression only after 24 h while later in time it was lower in pair-fed controls. In conclusion, central thyroid hormone metabolism is altered during chronic inflammation and this cannot solely be attributed to diminished food intake.

Abstract

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 (TNFα), interleukin-1 (IL-1α), interleukin-6 (IL-6) and interferon-γ (IFNγ). When compared with pair-fed controls, a single dose of LPS resulted in (a) systemic illness, (b) induction of TNFα 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-1α, TNFα, IL-6 or IFNγ could induce the sick euthyroid syndrome in mice, again using pair-fed controls. None of the cytokines except IL-1α caused systemic illness, and IL-1α was the only cytokine that decreased liver 5′-deiodinase mRNA transiently. IL-1α, TNFα or IL-6 did not decrease serum T₃, T₄ and TSH, but administration of IFNγ 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. (1) Administration of LPS in mice is a suitable experimental model for the acute induction of the sick euthyroid syndrome. (2) Acute administration of IL-1α, TNFα or IL-6 in mice does not induce changes in thyroid hormones but IFNγ results in a dose-dependent decrease of serum T₄, T₃ and fT₃ and IL-1α decreases liver 5′-deiodinase mRNA transiently. (3) Combined administration of IL-1α, TNFα, IL-6 and IFNγ had no synergistic effects; observed changes were of a smaller magnitude than after LPS. (4) The LPS-induced sick euthyroid syndrome is currently best explained by a direct thyroidal inhibition due to IFNγ and an extrathyroidal inhibition of liver 5′-deiodinase due to IL-1α, but other still unidentified factors seem to be involved as well.

Journal of Endocrinology (1995) 146, 475–483

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)β1. One might thus expect that lacking the TRβ gene would result in diminished downregulation of liver D1 expression and a smaller decrease in serum T₃ during illness. In this study, we used TRβ^−/− mice to evaluate the role of TRβ 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 TRβ. Furthermore, we observed basal differences in liver D1 mRNA and activity between TRβ^−/− and wild-type mice and TRβ^−/− 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 TRβ^−/− mice and increased in response to LPS in TRβ^−/− and WT mice. Other (TRβ 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.

Profound changes in thyroid hormone metabolism occur in the central part of the hypothalamus–pituitary–thyroid (HPT) axis during fasting. Hypothalamic changes are partly reversed by leptin administration, which decreases during fasting. It is unknown to what extent leptin affects the HPT axis at the level of the pituitary. We, therefore, studied fasting-induced alterations in pituitary thyroid hormone metabolism, as well as effects of leptin administration on these changes. Because refeeding rapidly increased serum leptin, the same parameters were studied after fasting followed by refeeding. Fasting for 24 h decreased serum T₃ and T₄ and pituitary TSHβ, type 2deiodinase (D2), and thyroid hormone receptor β2 (TRβ2) mRNA expression. The decrease in D2 and TRβ2 mRNA expression was prevented when 20 μg leptin was administered twice during fasting. By contrast, the decrease in TSHβ mRNA expression was unaffected. A single dose of leptin given after 24 h fasting did not affect decreased TSHβ, D2, and TRβ2 mRNA expression, while 4 h refeeding resulted in pituitary D2 and TRβ2 mRNA expression as observed in control mice. Serum leptin, T₃, and T₄ after refeeding were similar compared with leptin administration. We conclude that fasting decreases pituitary TSHβ, D2, and TRβ2 mRNA expression, which (with the exception of TSHβ) can be prevented by leptin administration during fasting. Following 24 h fasting, 4 h refeeding completely restores pituitary D2 and TRβ2 mRNA expression, while a single leptin dose is ineffective. This indicates that other postingestion signals may be necessary to modulate rapidly the fasting-induced decrease in pituitary D2 and TRβ2 mRNA expression.

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 α expression. Muscle interleukin-1β (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.

ABSTRACT

Stimulation of adrenal DNA synthesis by ACTH(1–39) and its fragments ACTH(1–24) (Synacthen) and ACTH(18–39) was investigated. Synthesis of DNA was measured as the increase in the percentage of cells in S-phase (Feulgen densitometry) in guinea-pig adrenal explants kept in organ culture and exposed to the peptides for 5 h at 37 °C. ACTH(1–39) and its C-terminal fragment ACTH(18–39) (corticotrophin-like intermediate lobe peptide) were found to be potent stimulators of in-vitro adrenal DNA synthesis. The dose–response kinetics were biphasic and optimal responsiveness was reached in both instances at 1 fmol/1–10 pmol/l (this biological effect of ACTH(18–39) has hitherto not been described). The N-terminal fragment ACTH(1–24) gave only minimal responses. Thyrotrophin and LH, tested as controls, did not induce adrenal DNA synthesis. Epidermal growth factor was a potent stimulator of adrenal DNA synthesis in vitro.

Our data suggest a trophic action of the C-terminal part (ACTH(18–39)) of the corticotrophic molecule. Clear trophic effects were also found for the N-terminal part of the pro-opiomelanocortin molecule N-POC(1–76) (optimum 0·1 nmol/l) and N-POC(51– 62) (optimum 0·1 pmol/l). The latter observations support earlier concepts that this part of the proopiomelanocortin molecule has a stimulatory effect on adrenal DNA synthesis.

J. Endocr. (1987) 115, 505–510