help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-1443
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yu, J.
Right arrow Articles by Koenig, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, J.
Right arrow Articles by Koenig, R. J.
Endocrinology Vol. 147, No. 7 3580-3585
Copyright © 2006 by The Endocrine Society

Induction of Type 1 Iodothyronine Deiodinase to Prevent the Nonthyroidal Illness Syndrome in Mice

Jingcheng Yu and Ronald J. Koenig

Division of Metabolism, Endocrinology, and Diabetes, University of Michigan Medical Center, Ann Arbor, Michigan 48109

Address all correspondence and requests for reprints to: Dr. Ronald J. Koenig, University of Michigan, 5560 MSRB-2, 1150 West Medical Center Drive, Ann Arbor, Michigan 48109-0678. E-mail: rkoenig{at}umich.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Essentially all serious illness is associated with a decrease in circulating T3, a condition known as the nonthyroidal illness syndrome. Substantial evidence suggests that a contributing factor to this syndrome is a cytokine-induced decrease in hepatic type 1 iodothyronine deiodinase (D1), an enzyme that converts T4 to T3. The type 1 deiodinase is induced at the transcriptional level by T3, but illness-associated cytokines block this induction, resulting in decreased T3 and hence a further decline in D1 expression. We demonstrated that IL-1 blocks the ability of T3 to induce D1 in rat hepatocyte primary cultures and that forced expression of steroid receptor co- activator 1 (SRC-1) prevents this cytokine effect. This led us to test whether forced hepatic expression of SRC-1 can prevent the nonthyroidal illness syndrome in vivo. Pretreatment of endotoxin-treated mice with an adenovirus that expresses SRC-1, compared with a control adenovirus, prevented the endotoxin-induced decreases in hepatic D1 and plasma T3. The data suggest that a cytokine-induced defect in T3 receptor coactivators is an important component of this animal model of nonthyroidal illness and that the syndrome can be overcome by forced expression of the coactivator.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE NONTHYROIDAL ILLNESS syndrome (NTIS), also known as the sick euthyroid syndrome, is a generalized response to essentially any illness that is characterized by a decreased level of circulating T3 in the absence of any intrinsic defect in the hypothalamic pituitary thyroid axis (reviewed in Refs.1 and 2). The TSH level is inappropriately normal or low, and the T4 is usually normal, although it also can be low in the most severe illnesses. These abnormalities resolve upon recovery from the illness, and in fact the TSH can be elevated transiently as the T3 rises toward normal. It is commonly argued that the NTIS evolved to conserve energy in times of illness or stress, because tissue hypothyroidism should decrease oxygen consumption. However, even if this syndrome was adaptive in evolution, it is uncertain whether it is still adaptive in the modern era of intensive care unit medicine. The standard of care is not to treat the NTIS, but the data that guide this practice are sparse. There currently is no way to identify which, if any, patients might benefit from treatment, nor what that treatment should be. A better understanding of the pathophysiology of the NTIS could help resolve these uncertainties.

The pathophysiology of the NTIS is complex and involves multiple organs that participate in thyroid axis homeostasis. The T3 is low despite a normal T4 largely because of decreased T4 deiodination in peripheral organs (3, 4). A central mechanism, probably in the hypothalamus (5, 6), prevents the TSH from rising in response to the low T3. Multiple factors probably underlie these abnormalities. Considerable attention has been focused on the role of cytokines, given that the NTIS occurs in response to virtually any illness and cytokines are elevated as a generalized response to illness (7, 8).

Previously, we have examined the role of type 1 deiodinase (D1), the hepatocyte enzyme that converts T4 to T3, in the NTIS (9). This enzyme is unusual in that the D1 gene (DIO1) is induced at the transcriptional level by the enzyme’s end product, T3 (10). Using primary cultures of rat hepatocytes, we found that IL-1 and -6 impair the T3 induction of D1 mRNA and enzyme activity, thus converting D1 expression from a euthyroid to a hypothyroid level, which in turn serves to further decrease T3 production and D1 expression (9). The inhibitory effect on T3 induction also was manifest when a human DIO1 promoter-luciferase construct was transiently transfected into the primary hepatocyte cultures. The IL-1 blockade of DIO1-luciferase expression was overcome by forced expression of steroid receptor coactivator 1 (SRC-1, nuclear receptor coactivator 1), suggesting that cytokines were disturbing the normal function of this or related thyroid hormone receptor coactivators. However, because the transfection efficiency of hepatocytes is low, it was not possible to test whether SRC-1 also overcame the inhibitory cytokine effect on the endogenous Dio1 gene.

We have now examined this question by infecting primary rat hepatocyte cultures with an adenoviral SRC-1 expression vector (or a LacZ control adenovirus) and then exposing the cells to IL-1. These studies demonstrate that forced expression of SRC-1 also overcomes the inhibitory effect of IL-1 on expression of the endogenous rat Dio1 gene. This led us to test whether a similar treatment might prevent the NTIS in vivo. Mice received either SRC-1 or LacZ adenovirus by tail vein injection and subsequently were given an ip injection of lipopolysaccharide (LPS) to induce the NTIS. The LPS-induced decreases in hepatic D1 and plasma T3 were prevented by the SRC-1 adenovirus, suggesting that disruption of the normal function of hepatocyte SRC-1 is an important component in the pathophysiology of the NTIS.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Recombinant adenovirus vectors
Recombinant adenoviruses were produced using the AdEasy Adenoviral Vector System vectors pShuttle-CMV and pAdEasy-1 according to the vendor’s protocol (Stratagene, La Jolla, CA). The recombinant adenoviruses were grown and purified by the University of Michigan Vector Core. The number of viral particles was determined by measuring the absorbance at 260 nm where one OD unit corresponds to 1.1 x 1012 viral particles.

Hepatocyte cultures
Hepatocytes were isolated from male Harlan Sprague Dawley rats (Harlan, Indianapolis, IN) weighing 280–350 g, as described previously (9). Hepatocytes in serum-free Williams E media were plated at 1.25 x 105 cells/cm2 into Falcon Primaria six-well clusters and maintained at 37 C, 5% CO2. After 24 h, hepatocytes were infected with either a LacZ- or SRC-1-expressing adenovirus (1680 particles per cell). This dose was chosen based upon preliminary experiments demonstrating uniform X-gal staining of hepatocytes 48 or 72 h post infection with the LacZ adenovirus (data not shown). Two days later, the media were replenished, and 50 nM T3 was added or not. In addition, at the same time, recombinant rat IL-1ß (PharMingen, San Diego, CA) was added at concentrations of 0, 1, 3, or 10 ng/ml. RNA was harvested after an additional 24 h for measurement of endogenous D1 mRNA expression.

Analysis of D1 expression in hepatocyte cultures
The influence of T3 and IL-1ß on the expression of D1 was assessed by Northern blot. Total RNA was prepared using Trizol (Invitrogen, Carlsbad, CA). Twenty micrograms of RNA were electrophoresed per lane of a 1% agarose, 2.2 M formaldehyde gel and transferred to a nylon membrane. Membranes were hybridized with 32P-labeled cDNA probes derived from rat D1 and rat glyceraldehyde-3-phosphate dehydrogenase (Gapdh) as a neutral control. Probes were labeled using a Strip-EZ random priming kit (Ambion, Austin, TX) to facilitate stripping and reprobing. Blots were analyzed using a Bio-Rad Phosphorimager. For each experiment, the normalized value of D1/Gapdh expression in the absence of T3 or cytokines was assigned a value of 1, and all other experimental conditions were expressed relative to that assigned value. Three independent experiments were performed.

In vivo experiments
Male C57BL/6J mice (8–10 wk old, 18–22 g; The Jackson Laboratory, Bar Harbor, ME) were kept in 12-h light, 12-h dark cycles in a temperature-controlled room (22 C) with food and water available ad libitum. A week before the experiment, the mice were housed in groups based upon treatment. Beginning the day of adenovirus injection, the mice were fed a 45 kcal% fat diet (catalog no. D12451; Research Diets, Inc., New Brunswick, NJ) to assure adequate caloric intake despite illness. Mice were injected with 0.3 ml SRC-1 or LacZ adenovirus (3 x 1011 particles) via the tail vein. As above, this dose was chosen based upon preliminary experiments demonstrating uniform X-gal staining of hepatocytes 48 or 72 h after infection with the LacZ adenovirus (data not shown). Two days after the adenovirus injection, 25 µg/kg LPS (Sigma Chemical Co., St. Louis, MO) diluted in 0.5 ml of 0.15 M NaCl, or diluent alone, was given ip. After another 2 d, the mice were anesthetized by ip injection of 0.3 ml per 30 g body weight of a mixture of 2.5 mg/ml ketamine and 1.5 mg/ml xylazine. Blood was taken from the abdominal aortas to measure plasma T3 and T4, and the livers were quickly excised, frozen in liquid nitrogen, and then stored at –80 C until further processing to measure the expression of D1, type 3 deiodinase (D3), Gapdh, and SRC-1. Each of the four treatment groups included six mice.

A separate series of experiments was undertaken to test whether the control (LacZ) adenovirus itself induces the NTIS. The experimental paradigm was similar to that described above, except that there were two groups of six mice. One group received 0.3 ml LacZ adenovirus and the other received 0.3 ml of 0.15 M NaCl via tail vein injection. Two days later, all mice received 0.5 ml of 0.15 M NaCl ip.

Animal use (rat and mouse) was approved by the University of Michigan Committee on the Use and Care of Animals.

Real-time PCR
Total RNA was extracted from mouse livers using Trizol reagent (Invitrogen), visualized by electrophoresis to ensure integrity, and used to synthesize cDNA using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen) with random hexamer primers. Real-time PCR was performed on an Applied Biosystems 7500 Real Time PCR System using SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA). Samples were assayed in triplicate. Standard curves using the cDNA equivalents of 1, 10, and 100 ng input RNA were analyzed in each assay plate and used as calibrators to determine the relative expression of each gene for each sample, which was calculated by the Applied Biosystems software based upon threshold (Ct) values. Typically, the equivalent cDNA of 10 ng RNA was used for the real-time PCR of each sample. All values were normalized using Gapdh as an internal control (a Gapdh standard curve also was run in each assay plate). Phosphoglycerate kinase 1 was used as an additional neutral control mRNA, again with its own standard curve run in each assay plate. The primers were as follows: 5'-TGGCACTGGAATCAATCCTCAG-3' and 5'-GGGCTTAGAGATGGAGCAAAGTTG-3' amplify human SRC-1 cDNA; 5'-CAACCCCAATGACCAAACCTG-3' and 5'-ATCCATCCTGTCTGTCTC GCAC-3' amplify mouse SRC-1 cDNA; 5'-CATCTGGGATTTCATTCAAGGC-3' and 5'-TGGAGGCAAAGTCATCTACGAGTC-3' amplify mouse D1 cDNA; 5'-TGACAT CAAGAAGGTGGTGAAGC-3' and 5'-CCCTGTTGCTGTAGCCGTATTC-3' amplify mouse Gapdh cDNA; and 5'-TGACTTTGGACAAGCTGGACGTGA-3' and 5'-CAGCAGCCTTGATCCTTTGGTTGT-3' amplify mouse phosphoglycerate kinase 1. Real-time PCR primers for mouse D3 have been described previously (11).

D1 enzyme activity
The enzyme activity of liver homogenates was measured as the release of 125I from [125I]rT3 as described (12). In brief, liver samples were homogenized individually on ice in 0.25 M sucrose in 100 mM potassium phosphate buffer (pH 6.9) containing 10 mM dithiothreitol and frozen immediately at –80 C. Protein concentrations were determined by Bio-Rad Protein Assay reagent (Bio-Rad, Hercules, CA). D1 activity was assayed by incubating diluted aliquots of the homogenates for 45 min at 37 C in 100 mM potassium phosphate buffer with 300 nM nonradiolabeled rT3 (Sigma), 10 mM dithiothreitol, and 100,000 cpm freshly purified [125I]rT3 (~1000 µCi/µg; PerkinElmer Life Sciences, Boston, MA). [125I]Iodide was separated by trichloroacetic acid precipitation after the addition of horse serum and was quantified in a {gamma}-counter. Results were calculated as picomoles iodide per microgram protein per hour.

Total T3, free T3, and total T4 measurements
Plasma total T3, free T3, and total T4 values were determined using RIA kits (MP Biomedicals, Solon, OH). When assaying free T3 from the experiment testing the effect of forced SRC-1 expression on the NTIS, insufficient plasma was available for individual measurements from three of six mice in the LacZ adenovirus/saline group, three of six mice in the LacZ adenovirus/LPS group, and two of six mice in the SRC-1 adenovirus/saline group. In each of those cases, equal amounts of plasma from the mice were pooled and assayed as single samples. These values were used along with the individual free T3 values from the remaining mice to calculate the average free T3 for each group. Therefore, it was not possible to calculate SE for these free T3 measurements.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Prevention of the IL-1-induced decrease in D1 expression by forced expression of SRC-1 in cultured rat hepatocytes
Primary cultures of rat hepatocytes, maintained in the absence of cytokines or T3, were infected with either a LacZ- or SRC-1-expressing adenovirus. Two days later, the culture media were changed to include or not include T3 and IL-1. RNA was harvested after an additional 24 h for measurement of endogenous D1 mRNA by Northern blot. In the control (LacZ) adenovirus-treated cells, T3 induced D1 mRNA approximately 6-fold (Fig. 1Go, A and B, left). IL-1 inhibited this T3 induction in a dose-related manner, reducing it to 2-fold at 10 ng/ml IL-1, without having a significant effect on D1 expression in the absence of T3. Forced expression of SRC-1 prevented the IL-1 effect; even in the presence of 10 ng/ml IL-1, T3 still induced D1 mRNA approximately 6-fold (Fig. 1Go, A and B, right). It is interesting to note that SRC-1 had a negligible effect on D1 expression in the absence of IL-1. This suggests that endogenous SRC-1 is not rate limiting for T3 induction of D1 in the absence of cytokines but is rate limiting in the presence of IL-1. The ability of SRC-1 to block the inhibitory effect of IL-1 on expression of the endogenous rat Dio1 gene is similar to what we observed previously in transfections with a human DIO1 promoter-luciferase vector (9) and suggests that disruption of thyroid hormone receptor coactivator function is an integral part of the NTIS. These findings led us to test whether adenovirally expressed SRC-1 would influence the NTIS in vivo.


Figure 1
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 1. IL-1 inhibits the T3 induction of endogenous D1 in primary cultures of rat hepatocytes, and forced expression of SRC-1 overcomes this inhibition. A, Rat hepatocytes, cultured in the absence of T3, were infected with either a LacZ- or SRC-1-expressing adenovirus. Two days later, the media were replaced with new media with or without 50 nM T3 and with 0, 1, 3, or 10 ng/ml IL-1ß. RNA was harvested 24 h later for measurement of D1 and Gapdh mRNAs by Northern blot, quantified by phosphorimager analysis. For each experiment, D1/Gapdh expression was calculated relative to a normalized value of 1 in the absence of T3 or cytokines. Results are mean ± SE of three experiments. B, The data from A are recalculated to show T3 inductions (mean ± SE for three experiments).

 
Forced expression of SRC-1 in an in vivo model of the NTIS
LPS-treated mice were used as a model of the NTIS to test whether forced expression of SRC-1 can prevent the syndrome in vivo. Mice received by tail vein injection either a LacZ- or SRC-1-expressing adenovirus. Two days later, the mice received LPS (25 µg/kg) or saline ip. After another 2 d, blood was taken for thyroid hormone measurements, the mice were killed, and the livers were removed for measurements of D1 and SRC-1.

The hypothesis being tested is that forced expression of SRC-1 would prevent the LPS-induced decline in D1, and this in turn would prevent the decline in plasma T3. Therefore, the expression of SRC-1 and D1 was assessed in the livers of these mice. By real-time RT-PCR, human SRC-1 was, as expected, undetectable in the LacZ adenovirus-treated mice but was expressed in the (human) SRC-1 adenovirus-treated mice (Fig. 2Go, left). Endogenous (mouse) SRC-1 was expressed in all treatment groups (Fig. 2Go, right).


Figure 2
View larger version (21K):
[in this window]
[in a new window]
  		FIG. 2. SRC-1 expression in mice receiving either a LacZ- or human SRC-1-expressing adenovirus followed by LPS or saline. Mice received either the LacZ- or human SRC-1-expressing adenovirus by tail vein injection, followed 2 d later by ip injection of either LPS or saline (Sal). The mice were killed 2 d later. Livers were harvested for measurements of human SRC-1, mouse SRC-1, and Gapdh RNA by real-time RT-PCR. The y-axis represents the level of expression of each SRC-1 mRNA normalized to Gapdh, as described in Materials and Methods. Human SRC-1 was not detectable in the LacZ adenovirus mice. Each bar is the mean ± SE for six mice.

 
D1 mRNA expression was 35% lower in the LacZ adenovirus mice treated with LPS vs. saline (Fig. 3AGo, left two bars), as expected in the NTIS. In contrast, D1 mRNA was not diminished in the SRC-1 adenovirus mice treated with LPS vs. saline (Fig. 3AGo, right two bars). Measurements of D1 enzyme activity paralleled those of D1 mRNA. Thus, LPS treatment resulted in a 33% decline in D1 enzyme activity in control adenovirus mice (Fig. 3BGo, left two bars), but LPS treatment was not associated with a decline in D1 activity in SRC-1 adenovirus mice (Fig. 3BGo, right two bars). It should be noted that infection with the SRC-1 adenovirus appeared to increase D1 mRNA expression. This was not an artifact of normalization, because Gapdh was unchanged by any of the treatments, and a second control mRNA, phosphoglycerate kinase 1, also was unchanged (data not shown). Furthermore, D1 enzyme activity normalized per microgram protein correlated with mRNA expression. Perhaps the simplest explanation is that SRC-1 induces D1.


Figure 3
View larger version (17K):
[in this window]
[in a new window]
  		FIG. 3. Hepatic D1 expression is decreased by LPS administration to mice, but previous administration of a SRC-1-expressing adenovirus prevents this decrease. Mice received either a LacZ- or SRC-1-expressing adenovirus by tail vein injection, followed 2 d later by ip injection of either LPS or saline (Sal). The mice were killed 2 d later. A, Hepatic D1 and Gapdh expression was measured by real-time RT-PCR. B, D1 enzyme activity in liver homogenates was measured. Each bar is the mean ± SE for six mice. Statistical analysis is by one-tailed t test, with the null hypothesis being that D1 is not decreased by LPS.

 
LPS induced the NTIS as expected in the control adenovirus mice, manifest by a 34% decline in plasma total T3 (Fig. 4AGo, left two bars). In contrast, no decline in plasma total T3 was observed when SRC-1 adenovirus mice were treated with LPS (Fig. 4AGo, right two bars). Measurements of plasma free T3 yielded similar conclusions; in the control adenovirus group, the free T3 was 42% lower in the LPS-treated mice than the saline-treated mice, whereas the corresponding difference in the SRC-1 adenovirus group was only 5.6% (Fig. 4BGo). Thus, the SRC-1 adenovirus prevented the NTIS in LPS-treated mice.


Figure 4
View larger version (20K):
[in this window]
[in a new window]
  		FIG. 4. LPS causes a decline in plasma T3 in LacZ adenovirus-treated mice but does not decrease plasma T3 in SRC-1 adenovirus-treated mice. Mice received either a LacZ- or SRC-1-expressing adenovirus by tail vein injection, followed 2 d later by ip injection of either LPS or saline (Sal). Blood was collected 2 d later, at the time mice were killed, for measurements of plasma total T3 (A), free T3 (B), and total T4 (C). Each bar represents data from six mice (SE could not be calculated for free T3; see Materials and Methods). Statistical analysis is by one-tailed t test, with the null hypothesis being that thyroid hormone levels are not decreased by LPS.

 
In contrast to the decline in T3, control adenovirus mice that received LPS had T4 levels that were statistically similar to those in saline-treated mice (Fig. 4CGo, left two bars), indicating that the drop in circulating T3 was not because of a drop in the D1 substrate, T4. Perhaps more important, plasma T4 declined marginally in the SRC-1 adenovirus mice that received LPS (Fig. 4BGo, right two bars), making the lack of decline in T3 in these mice all the more striking.

In principle, the decrease in plasma T3 in the NTIS could be a result of induction of the inner ring deiodinase D3, in addition to the decrease in D1. Therefore we measured D3 mRNA expression by real-time RT-PCR in the same liver samples that were used to quantify D1 mRNA. We found no detectable D3 expression in any of the samples using cDNA from 10 ng RNA and 35 cycles of real-time PCR. In contrast, cDNA from 10 ng of d-14 whole-mouse embryo RNA reached threshold at 28 cycles. Thus, induction of hepatic D3 is unlikely to be a major contributor to the NTIS in this experimental paradigm.

Adenovirus injection per se does not cause the NTIS
A separate series of experiments was undertaken to determine whether administration of the control (LacZ) adenovirus itself can induce the NTIS. Mice received either LacZ adenovirus or saline by tail vein injection. Two days later, they received saline ip (no LPS), and 2 d after that they were killed for measurements of plasma thyroid hormones and liver D1. The total T3 was 0.94 ± 0.07 ng/ml in the saline mice and 0.92 ± 0.05 ng/ml in the adenovirus mice (mean ± SE, six mice per group). The free T3 values in the saline and adenovirus groups were 5.05 ± 0.42 and 4.32 ± 0.48 pg/ml (P = 0.29); the total T4 values were 19.5 ± 1.4 and 18.2 ± 2.2 ng/ml; and D1 enzyme activities in liver extracts were 2.20 ± 0.18 and 2.38 ± 0.27 pmol iodide/µg protein·h. Thus, the control adenovirus by itself did not significantly decrease plasma T3 or T4, or liver D1, and hence by itself did not induce the NTIS.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Severe medical illness of any etiology is associated with the NTIS, the hallmark of which is a decreased level of circulating T3 (1, 2). The serum TSH is generally normal, although it can be low in the most severe illnesses, and it often rises transiently above the normal range if the patient recovers from the illness (13).

A substantial body of data suggests that cytokines play an integral role in the development of the NTIS (7, 8) and that the decline in circulating T3 is largely a result of decreased T4 to T3 conversion in peripheral organs (4). For example, the administration of endotoxin (LPS) to cattle causes the NTIS, and this is associated with a 45% decrease in hepatic D1 enzyme activity (14). Experimental illness (LPS, Listeria monocytogenes, or turpentine administration) results in a smaller decline in serum T3 and hepatic D1 in IL-6–/– mice vs. control mice (15). Administration of IL-6 to healthy humans results in a decline in serum T3 (16), and a study of hospitalized patients demonstrated an inverse correlation between serum IL-6 and T3 levels (17). IL-1 administration results in a decline in hepatic D1 in mice (18). IL-1 (19) and TNF-{alpha} (20) inhibited D1 activity in HepG2 hepatocarcinoma cells in culture, and our previous data indicate that both IL-1 and IL-6 impair the T3 induction of D1 in cultured rat hepatocytes (9).

Data from patients are limited. A study of deceased intensive care unit patients demonstrated decreased hepatic D1 activity and mRNA levels (21), consistent with the hypothesis that decreased D1 plays an important role in the abnormal circulating thyroid hormone levels in humans with the NTIS. Although D1 is the major source of circulating T3 in rodents, recent evidence suggests that expression of the type 2 deiodinase (D2) in human skeletal muscle may result in this enzyme contributing more to the plasma T3 than D1 in healthy humans (22). D2 was undetectable in skeletal muscle from the deceased intensive care unit subjects, raising the possibility that reduced skeletal muscle D2 may contribute to the NTIS in humans. Interestingly, D3, which degrades T3 by inner ring deiodination, was expressed in the livers and skeletal muscle of these patients even though it apparently is not present in these organs in normal individuals. Thus, induction of hepatic and muscle D3 also might contribute to the NTIS. Indeed, it is likely that a response to illness such as the NTIS is multifactorial in etiology. Alterations in nuclear receptors (23, 24), activation of nuclear factor-{kappa}B (20), and at least in principle, abnormalities in thyroid hormone transporters (25) are among the additional factors that could contribute.

The present study focuses on the role of impaired hepatic D1 and the importance of SRC-1 in this syndrome. Our results confirm the previous finding (9) that cytokines such as IL-1 impair the ability of T3 to induce D1 expression in primary cultures of rat hepatocytes. This converts hepatic T3 production from a euthyroid to a hypothyroid level, which further serves to decrease D1 expression and T3 levels. We now show that forced expression of SRC-1 overcomes the inhibitory effect of IL-1 on the endogenous Dio1 gene in cultured rat hepatocytes, indicating that SRC-1 is rate limiting for T3 induction in the presence of IL-1 even though it is not rate limiting in the absence of cytokines. SRC-1 becomes rate limiting probably not because of a decrease in its expression but more likely because of a functional deficiency. Cytokines induce a large number of genes, and it is possible that limiting amounts of SRC-1 are partitioned between those genes and thyroid hormone-responsive genes such as Dio1. Alternatively, the NTIS may diminish the functional activity of SRC-1, perhaps by phosphorylation or other posttranslational changes. In any case, it is important that these cell culture results correctly predict the effects of SRC-1 in an in vivo model of the NTIS. We found that forced expression of SRC-1 via an adenoviral vector prevents the decreases in hepatic D1 mRNA and enzyme activity, as well as the decrease in circulating T3, in LPS-treated mice. Although the lack of a fall in plasma T3 after LPS in the SRC-1 adenovirus mice is most plausibly ascribed to the lack of a fall in hepatic D1, it is possible that other (unknown) effects of forced SRC-1 expression also contribute to this result. It should be noted that tail vein injections of adenovirus infect mouse liver at least 10-fold more efficiently than other organs, but numerous organs do take up the virus (26).

We observed that expression of endogenous SRC-1 mRNA was higher in the LacZ adenovirus/saline mice than in the other groups (Fig. 2Go). The reason for this is not known. Perhaps homeostatic mechanisms down-regulate endogenous SRC-1 when exogenous (human) SRC-1 is expressed, and perhaps LPS administration (i.e. the NTIS) also causes a modest decrease in endogenous SRC-1 expression.

Clinical interest in the NTIS is driven in part by repeated observations that mortality rates correlate with the severity of the syndrome (27, 28, 29, 30), raising the question of whether the NTIS might be causally related to severe illness and poor prognosis. However, trials of T4 (31) or T3 (32) therapy in severely ill humans with the NTIS have failed to demonstrate any benefit. There are several possible explanations for this failure. The NTIS may simply be a marker for severe illness. The studies to date might not have had sufficient power to detect a real benefit; only 23 and 28 subjects, respectively, were enrolled in the above investigations. It is possible that therapy might have potential benefit in only a subset of NTIS subjects, yet there is no current way of identifying who those subjects might be. Finally, it is possible that therapy could be of benefit but that the previously used treatment paradigms with T3 or T4 are not appropriate.

Our current data are relevant to these issues, because they suggest that the low circulating T3 is at least in part a result of a defect in nuclear receptor coactivator function and that this defect can be overcome by forced expression of SRC-1. SRC-1 is a coactivator not just for thyroid hormone receptors but also for nuclear receptors in general (33) and other transcription factors such as nuclear factor-{kappa}B (34), activator protein-1 (35) and serum response factor (36). Thus, a defect in SRC-1 function would potentially affect gene activation by many transcription factors in the NTIS. This concept fits with our previous observation that cytokine treatment of rat hepatocytes impairs glucocorticoid induction of an appropriate reporter construct and that forced expression of SRC-1 overcomes this effect (9). If one presumes that at least some genes induced by cytokines in the NTIS are beneficial, then it is possible that the defect in SRC-1 limits the induction of these genes and contributes to the adverse outcome in severe nonthyroidal illness. In this case, treatment with thyroid hormone might be harmful, rather than beneficial, because it would partition limiting amounts of functional SRC-1 away from cytokine-responsive, adaptive genes. Thus, instead of treating the NTIS with T3 or T4, it might be more appropriate to treat in a way that restores coactivator function. Such a therapy would restore circulating thyroid hormone levels by treating the underlying cause of the deficiency. Treatments along these lines currently are not feasible because the nature of the defect in coactivator function is not known. The defect may be functional rather than quantitative, because cytokines do not decrease the physical mass of SRC-1 in cultured rat hepatocytes (9). Additional research will be required to better understand the underlying defect and to address if, what, and when therapy of the NTIS should be instituted.


   Footnotes
 
This work was supported by National Institutes of Health Grant R01 DK44155 and the Cell and Molecular Biology Core of the Michigan Diabetes Research and Training Center Grant P60 DK20572.

Disclosure: J.Y. and R.J.K. have nothing to declare.

First Published Online April 6, 2006

Abbreviations: D1, Type 1 iodothyronine deiodinase; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; LPS, lipopolysaccharide; NTIS, nonthyroidal illness syndrome; SRC-1, steroid receptor coactivator 1.

Received November 14, 2005.

Accepted for publication March 27, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Chopra IJ 1997 Euthyroid sick syndrome: is it a misnomer. J Clin Endocrinol Metab 82:329–334[Free Full Text]
  2. McIver B, Gorman CA 1997 Euthyroid sick syndrome: an overview. Thyroid 7:125–132[Medline]
  3. Docter R, Krenning EP, de Jong M, Hennemann G 1993 The sick euthyroid syndrome: changes in thyroid hormone serum parameters and hormone metabolism. Clin Endocrinol (Oxf) 39:499–518[Medline]
  4. Kaptein EM, Robinson WJ, Grieb DA, Nicoloff JT 1982 Peripheral serum thyroxine, triiodothyronine and reverse triiodothyronine kinetics in the low thyroxine state of acute nonthyroidal illnesses. A noncompartmental analysis. J Clin Invest 69:526–535[Medline]
  5. Fliers E, Guldenaar SEF, Wiersinga WM, Swaab DF 1997 Decreased hypothalamic thyrotropin-releasing hormone gene expression in patients with nonthyroidal illness. J Clin Endocrinol Metab 82:4032–4036[Abstract/Free Full Text]
  6. Fekete C, Gereben B, Doleschall M, Harney JW, Dora JM, Bianco AC, Sarkar S, Liposits Z, Rand W, Emerson C, Kacskovics I, Larsen PR, Lechan RM 2004 Lipopolysaccharide induces type 2 iodothyronine deiodinase in the mediobasal hypothalamus: implications for the nonthyroidal illness syndrome. Endocrinology 145:1649–1655[Abstract/Free Full Text]
  7. Papanicolaou DA 2000 Euthyroid sick syndrome and the role of cytokines. Rev Endocr Metab Disord 1:43–48[CrossRef][Medline]
  8. Bartalena L, Bogazzi F, Brogioni S, Grasso L, Martino E 1998 Role of cytokines in the pathogenesis of the euthyroid sick syndrome. Eur J Endocrinol 138:603–614[CrossRef][Medline]
  9. Yu J, Koenig RJ 2000 Regulation of hepatocyte thyroxine 5'-deiodinase by T3 and nuclear receptor coactivators as a model of the sick euthyroid syndrome. J Biol Chem 275:38296–38301[Abstract/Free Full Text]
  10. Toyoda N, Zavacki AM, Maia AL, Harney JW, Larsen PR 1995 A novel retinoid X receptor-independent thyroid hormone response element is present in the human type 1 deiodinase gene. Mol Cell Biol 15:5100–5112[Abstract]
  11. Boelen A, Kwakkel J, Alkemade A, Renckens R, Kaptein E, Kuiper G, Wiersinga WM, Visser TJ 2005 Induction of type 3 deiodinase activity in inflammatory cells of mice with chronic local inflammation. Endocrinology 146:5128–5134[Abstract/Free Full Text]
  12. Toyoda N, Harney JW, Berry MJ, Larsen PR 1994 Identification of critical amino acids for 3,5,3'-triiodothyronine deiodination by human type 1 deiodinase based on comparative function-structural analyses of the human, dog and rat enzymes. J Biol Chem 269:20329–20334[Abstract/Free Full Text]
  13. Bacci V, Schussler GC, Kaplan TB 1982 The relationship between serum triiodothyronine and thyrotropin during systemic illness. J Clin Endocrinol Metab 54:1229–1235[Abstract/Free Full Text]
  14. Kahl S, Elsasser TH, Blum JW 2000 Effect of endotoxin challenge on hepatic 5'-deiodinase activity in cattle. Domest Anim Endocrinol 18:133–143[CrossRef][Medline]
  15. Boelen A, Maas MAW, Lowik C, Platvoet MC, Wiersinga WM 1996 Induced illness in interleukin-6 (IL-6) knock-out mice: a causal role of IL-6 in the development of the low 3,5,3'-triiodothyronine syndrome. Endocrinology 137:5250–5254[Abstract]
  16. Torpy DJ, Tsigos C, Lotsikas AJ, Defensor R, Chrousos GP, Papanicolaou DA 1998 Acute and delayed effects of a single-dose injection of interleukin-6 on thyroid function in healthy humans. Metabolism 47:1289–1293[CrossRef][Medline]
  17. Boelen A, Platvoet-Ter Schiphorst MC, Wiersinga WM 1993 Association between serum interleukin-6 and serum 3,5,3'-triiodothyronine in nonthyroidal illness. J Clin Endocrinol Metab 77:1695–1699[Abstract]
  18. Boelen A, Platvoet-Ter Schiphorst MC, Bakker O, Wiersinga WM 1995 The role of cytokines in the lipopolysaccharide-induced sick euthyroid syndrome in mice. J Endocrinol 146:475–483[Abstract/Free Full Text]
  19. Jakobs TC, Mentrup B, Schmutzler C, Dreher I, Kohrle J 2002 Proinflammatory cytokines inhibit the expression and function of human type I 5'-deiodinase in HepG2 hepatocarcinoma cells. Eur J Endocrinol 146:559–566[Abstract]
  20. Nagaya T, Fujieda M, Otsuka G, Yang JP, Okamoto T, Seo H 2000 A potential role of activated NF-{kappa}B in the pathogenesis of euthyroid sick syndrome. J Clin Invest 106:393–402[Medline]
  21. Peeters RP, Wouters PJ, Kaptein E, van Toor H, Visser TJ, den Berghe GV 2003 Reduced activation and increased inactivation of thyroid hormone in tissues of critically ill patients. J Clin Endocrinol Metab 88:3202–3211[Abstract/Free Full Text]
  22. Maia AL, Kim BW, Huang SA, Harney JW, Larsen PR 2005 Type 2 iodothyronine deiodinase is the major source of plasma T3 in euthyroid humans. J Clin Invest 115:2524–2533[CrossRef][Medline]
  23. Beigneux AP, Moser AH, Shigenaga JK, Grunfeld C, Feingold KR 2003 Sick euthyroid syndrome is associated with decreased TR expression and DNA binding in mouse liver. Am J Physiol Endocrinol Metab 284:E228–E236
  24. Ghose R, Zimmerman TL, Thevananther S, Karpen SJ 2004 Endotoxin leads to rapid subcellular re-localization of hepatic RXR{alpha}: a novel mechanism for reduced hepatic gene expression in inflammation. Nucl Recept 2:4[CrossRef][Medline]
  25. Peeters RP, van der Geyten S, Wouters PJ, Darras VM, van Toor H, Kaptein E, Visser TJ, Van den Berghe G 2005 Tissue thyroid hormone levels in critical illness. J Clin Endocrinol Metab 90:6498–6507[Abstract/Free Full Text]
  26. Smith TA, Mehaffey MG, Kayda DB, Saunders JM, Yei S, Trapnell BC, McClelland A, Kaleko M 1993 Adenovirus mediated expression of therapeutic plasma levels of human factor IX in mice. Nat Genet 5:397–402[CrossRef][Medline]
  27. Iervasi G, Landi P, Raciti M, Ripoli A, Scarlattini M, L’Abbate A, Donato L 2003 Low-T3 syndrome: a strong prognostic predictor of death in patients with heart disease. Circulation 107:708–713[Abstract/Free Full Text]
  28. Pingitore A, Landi P, Taddei MC, Ripoli A, L’Abbate A, Iervasi G 2005 Triiodothyronine levels for risk stratification of patients with chronic heart failure. Am J Med 118:132–136[CrossRef][Medline]
  29. Scoscia E, Baglioni S, Eslami A, Iervasi G, Monti S, Todisco T 2004 Low triiodothyronine (T3) state: a predictor of outcome in respiratory failure? Results of a clinical pilot study. Eur J Endocrinol 151:557–560[Abstract]
  30. Chinga-Alayo E, Villena J, Evans AT, Zimic M 2005 Thyroid hormone levels improve the prediction of mortality among patients admitted to the intensive care unit. Intens Care Med 31:1356–1361[CrossRef][Medline]
  31. Brent GA, Hershman JM 1986 Thyroxine therapy in patients with severe nonthyroidal illnesses and low serum thyroxine concentration. J Clin Endocrinol Metab 63:1–8[Abstract/Free Full Text]
  32. Becker RA, Vaughan GM, Ziegler G, Seraile LG, Goldfarb IW, Mansour EH, McManus WF, Pruitt Jr BA, Mason Jr AD 1982 Hypermetabolic low triiodothyronine syndrome of burn injury. Crit Care Med 10:870–875[Medline]
  33. Oñate SA, Tsai SY, Tsai MJ, O’Malley BW 1995 Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 270:1354–1357[Abstract/Free Full Text]
  34. Na SY, Lee SK, Han SJ, Choi HS, Im SY, Lee JW 1998 Steroid receptor coactivator-1 interacts with the p50 subunit and coactivates nuclear factor {kappa}B-mediated transactivations. J Biol Chem 273:10831–10834[Abstract/Free Full Text]
  35. Lee SK, Kim HJ, Na SY, Kim TS, Choi HS, Im SY, Lee JW 1998 Steroid receptor coactivator-1 coactivates activating protein-1-mediated transactivations through interaction with the c-Jun and c-Fos subunits. J Biol Chem 273:16651–16654[Abstract/Free Full Text]
  36. Kim HJ, Kim JH, Lee JW 1998 Steroid receptor coactivator-1 interacts with serum response factor and coactivates serum response element-mediated transactivations. J Biol Chem 273:28564–28567[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Eur Respir JHome page
F. Laghi, N. Adiguzel, and M. J. Tobin
Endocrinological derangements in COPD
Eur. Respir. J., October 1, 2009; 34(4): 975 - 996.
[Abstract] [Full Text] [PDF]

Home page
 Physiol. GenomicsHome page
M. T. Flowers, M. P. Keller, Y. Choi, H. Lan, C. Kendziorski, J. M. Ntambi, and A. D. Attie
Liver gene expression analysis reveals endoplasmic reticulum stress and metabolic dysfunction in SCD1-deficient mice fed a very low-fat diet
Physiol Genomics, May 1, 2008; 33(3): 361 - 372.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
J Kwakkel, O Chassande, H C van Beeren, W M Wiersinga, and A Boelen
Lacking thyroid hormone receptor {beta} gene does not influence alterations in peripheral thyroid hormone metabolism during acute illness
J. Endocrinol., April 1, 2008; 197(1): 151 - 158.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
E. L. Olivares, M. P. Marassi, R. S. Fortunato, A. C. M. da Silva, R. H. Costa-e-Sousa, I. G. Araujo, E. C. Mattos, M. O. Masuda, M. A. Mulcahey, S. A. Huang, et al.
Thyroid Function Disturbance and Type 3 Iodothyronine Deiodinase Induction after Myocardial Infarction in Rats A Time Course Study
Endocrinology, October 1, 2007; 148(10): 4786 - 4792.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
J Kwakkel, W M Wiersinga, and A Boelen
Interleukin-1{beta} modulates endogenous thyroid hormone receptor {alpha} gene transcription in liver cells
J. Endocrinol., August 1, 2007; 194(2): 257 - 265.
[Abstract] [Full Text] [PDF]

Home page
 J. Pharmacol. Exp. Ther.Home page
E. S. Tien, K. Matsui, R. Moore, and M. Negishi
The Nuclear Receptor Constitutively Active/Androstane Receptor Regulates Type 1 Deiodinase and Thyroid Hormone Activity in the Regenerating Mouse Liver
J. Pharmacol. Exp. Ther., January 1, 2007; 320(1): 307 - 313.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yu, J.
Right arrow Articles by Koenig, R. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yu, J.
Right arrow Articles by Koenig, R. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals