Sick euthyroid syndrome is associated with decreased TR expression and DNA binding in mouse liver

Anne P. Beigneux, Arthur H. Moser, Judy K. Shigenaga, Carl Grunfeld, and Kenneth R. Feingold

Department of Medicine, University of California San Francisco and Metabolism Section, Medical Service, Department of Veterans Affairs Medical Center, San Francisco, California 94121


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Infection is associated with low serum thyroid hormones and thyrotropin levels. Here we demonstrate that infection also reduces thyroid hormone receptor (TR) expression. In gel shift experiments, retinoid X receptor (RXR)/TR DNA binding was reduced in mouse liver by 60 and 77%, respectively, 4 and 16 h after lipopolysaccharide (LPS) administration. Surprisingly, LPS did not decrease either TR-alpha or TR-beta protein levels at 4 h, but by 16 h TR-alpha 1, TR-alpha 2, and TR-beta levels were reduced by 55, 87, and 41%, respectively. We previously reported that LPS rapidly decreases RXR protein levels in liver. Therefore, we added RXR-beta to hepatic nuclear extracts prepared 4 h after LPS treatment, which restored RXR/TR DNA binding to a level comparable to that of controls. A similar experiment conducted on extracts prepared 16 h after LPS administration did not restore RXR/TR DNA binding. We propose that decreased RXR expression is limiting for RXR/TR DNA binding at 4 h, whereas the reduction in both TR and RXR levels results in further decreased binding at 16 h.

acute-phase response; thyroid hormone receptor; retinoid X receptor; lipid metabolism


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE NONTHYROIDAL ILLNESSES, such as infection, inflammation, or trauma, result in what is known as the sick euthyroid syndrome (6, 26, 33). The characteristic feature of this syndrome is low triiodothyronine (T3) plasma levels. If the nonthyroidal illness is severe, low serum thyroxine (T4) levels and an inappropriately low normal to subnormal thyrotropin serum concentration may also occur. The pathogenesis of the sick euthyroid syndrome is poorly understood, but both central (pituitary and hypothalamic) and peripheral defects have been proposed. The central defect manifests itself through a low to normal thyrotropin serum secretion despite low T3 and T4 plasma levels. The most commonly described peripheral defect is the reduced conversion of T4 to T3 because of a decrease in 5'-deiodination in the liver.

Injection of bacterial lipopolysaccharide (LPS), which is known to induce the sick euthyroid syndrome (43), triggers a systemic inflammatory response referred to as the acute-phase response (APR), characterized by alterations in hepatic carbohydrate and lipid metabolism. Hypertriglyceridemia, decreased high-density lipoprotein cholesterol levels, and inhibition of bile acid synthesis are among the alterations in lipid metabolism that occur during the APR (15).

The liver plays an important role in thyroid hormone metabolism, and thyroid hormones, which influence all major metabolic pathways, are also important regulators of lipid and glucose metabolism in the liver. T3 has been shown to stimulate the hepatic expression of genes involved in lipogenesis, such as malic enzyme (41) and Spot14 (17, 23), and in bile acid synthesis, such as cholesterol 7alpha -hydroxylase (Cyp7a; see Ref. 14). Of note, the expression of Cyp7a (12) and of malic enzyme (8) has previously been shown to be decreased during the APR.

T3 exerts its diverse metabolic effects by binding to nuclear thyroid hormone receptors (TRs). TRs are encoded by two homologous but distinct genes, TR-alpha and TR-beta , located on two separate chromosomes. In addition, alternative splicing from each gene generates multiple TR isoforms, resulting in further diversity. To date, three T3-binding isoforms of the TR have been identified, namely TR-alpha 1, TR-beta 1, and TR-beta 2. Furthermore, three non-T3-binding isoforms have been described, the splice variants TR-alpha 2 and TR-alpha 3 and Rev-erbA, which is encoded by the opposite strand of the TR-alpha gene. The TR-beta 1 isoform is widely expressed, but it is particularly abundant in liver and kidney (16). In contrast, the expression of TR-beta 2 is relatively restricted and is most highly expressed in the pituitary. TR-alpha 1 and TR-alpha 2 are abundant in the brain but are also found in most tissues, including heart and liver.

Extensive structural similarities have resulted in the classification of TRs in the type II subfamily of nuclear hormone receptors, which also includes the retinoic acid receptor (RAR), peroxisome proliferator-activated receptors (PPAR), and liver X receptor (LXR; see Ref. 24). These receptors have in common that they heterodimerize with the retinoid X receptor (RXR) for high-affinity binding to DNA. T3-induced gene expression involves binding of RXR/TR heterodimers to specific thyroid hormone response elements (TRE) present in the promoter of the target genes. Three sequence motifs have been shown to function as TRE. These motifs include a direct repeat separated by four nucleotides (DR4), a palindrome called inverted repeat (IR) 0, and an everted repeat separated by six nucleotides (ER6; see Ref. 44).

Several clinical studies evaluating the efficiency of T4 or T3 administration in the sick euthyroid syndrome have reported contradictory results, from no benefit to improvement (6). These results suggested to us that, during the APR, a reduction in TR expression might occur, decreasing thyroid hormone responsiveness, thereby participating in the pathophysiology of the sick euthyroid syndrome. This hypothesis seemed reasonable since our laboratory and others have shown that treatment with LPS or tumor necrosis factor (TNF) decreases the expression of another nuclear hormone receptor, RXR, in hamster liver (3) and rat GH3 cells (37). We also found that the coordinate repression of RXR and PPAR-alpha could represent one mechanism by which hepatic fatty acid oxidation is reduced during the APR (3). Finally, we have previously proposed that the concomitant decrease in RXR and LXR expression participates in the downregulation of Cyp7a in liver after LPS administration (3), thereby leading to the inhibition of bile acid synthesis associated with the APR (12, 27). Thus, to test whether a decrease in TR levels in liver could participate in the alterations in lipid metabolism that are associated with infection, we measured TR DNA binding activity and protein levels in mouse liver after LPS treatment.


    MATERIALS AND METHODS
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Materials. LPS (Escherichia coli 55:B5) was obtained from Difco Laboratories and freshly diluted to a desired concentration in pyrogen-free 0.9% saline. Oligo(dT)-cellulose type 77F was from Amersham Pharmacia Biotech. [alpha -32P]dCTP (3,000 Ci/mmol) and [gamma -32P]dATP (3,000 Ci/mmol) were purchased from NEN Life Science Products.

Animals. All animal studies were conducted in accordance with the highest standards of humane care and according to the principles and procedures described in Guidelines for Care and Use of Experimental Animals. Six-week-old female C57BL/6 mice were purchased from Jackson Laboratory. The animals were maintained in a normal-light-cycle room and were provided with rodent chow and water ad libitum. Anesthesia was induced with halothane. To determine the effect of APR on TR-alpha and TR-beta mRNA levels, mice were injected intraperitoneally with 0.1-100 µg LPS in saline or with saline alone. In all other experiments mice were injected intraperitoneally with 100 µg LPS in saline or with saline alone. Food was withdrawn in all groups at the time of injection, since LPS induces anorexia in rodents (13). In all experiments, animals were killed between 10:00 and 11:00 AM for the 4-, 16-, and 24-h time points and between 3:00 and 4:00 PM for the 8-h time points. The doses of LPS used in this study were nonlethal, since the lethal dose 50% (LD50) for LPS in rodents is ~5 mg/100 g body wt, but they have significant effects on triglyceride and cholesterol metabolism (10, 11).

Measurement of total T3 and total T4 serum levels. Measurements were made on fresh serum, at the time indicated after saline or LPS administration, using Coat-A-Count total T3 and T4 RIA kits (Diagnostic Products).

Preparation of nuclear extracts. Fresh liver (0.5-1 g) was homogenized in 10 mM HEPES (pH 7.9), 25 mM KCl, 0.15 mM spermine, 1 mM EDTA, 2 M sucrose, 10% glycerol, 50 mM NaF, 2 mM sodium metavanadate, 0.5 mM dithiothreitol, and 1% protease inhibitor cocktail (Sigma) 4 and 16 h after LPS or saline treatment. Immediately after homogenization, nuclear proteins were extracted as described previously (3). Nuclear protein content was determined by the Bradford assay (Bio-Rad), and yields were similar in saline- and LPS-treated groups.

Western blot analysis. Denatured nuclear protein extracts (50 µg) were loaded on 10% polyacrylamide precast gels (Bio-Rad) and subjected to electrophoresis. After electrotransfer to a polyvinylidene difluoride membrane (Amersham Pharmacia Biotech), blots were blocked with PBS containing 0.10% Tween (PBS-T) and 5% dry milk for 1 h at room temperature. Blots were then incubated for 1 h at room temperature with polyclonal rabbit antibodies (Affinity BioReagents) raised against human TR-alpha or TR-beta 1 (dilution 1:1,000 in PBS-T). Both antibodies were purified from rabbits that had been immunized with a synthetic peptide whose sequence was derived from the NH2-terminal region of either human TR-alpha or human TR-beta 1 (Affinity BioReagents). This NH2-terminal region is highly divergent between TR-alpha and TR-beta isoforms but is identical to the corresponding rat sequence. Immune complexes were then detected using horseradish peroxidase-linked donkey anti-rabbit IgG (dilution 1:20,000 in PBS-T) according to the enhanced chemiluminescence plus Western blotting kit (Amersham Pharmacia Biotech). Immunoreactive bands obtained by autoradiography were quantified by densitometry.

RNA isolation and Northern blot analysis. Total RNA was isolated from 150 to 400 mg snap-frozen whole tissue using Tri-Reagent (Molecular Research Center). Poly(A)+ RNA was subsequently purified using oligo(dT) cellulose. RNA was quantified by measuring absorption at 260 nm. Poly(A)+ (10 µg) was denatured and electrophoresed on a 1% agarose-formaldehyde gel. The uniformity of sample applications was checked by ultraviolet visualization of the ethidium bromide-stained gel before electrotransfer to a Nytran membrane (Scleicher & Schuell). Prehybridization, hybridization, and washing procedures were performed as described previously (3). Membranes were probed with [alpha -32P]dCTP-labeled cDNAs using the random priming technique. RNA levels were detected by exposure of the membrane to X-ray film and quantified by densitometry. Glyceraldehyde-3-phosphate dehydrogenase was used as a control probe. Human RXR-alpha cDNA was a gift from Dr. Daniel D. Bikle (University of California, San Francisco, CA). Mouse RXR-beta and RXR-gamma cDNAs were kindly provided by Dr. David J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX). The following mouse probes were generated by RT-PCR starting from total RNA from mouse liver (ME, Spot14, TR-beta 1) and heart (TR-alpha ) and using the following primers: malic enzyme, 5'-CCA CCA GCG CGG CTA CCT GCT GAC GCG GGA-3' (upper) and 5'-CCT CTG ACT CGC CGG TGC CGC AGC CCG ATG-3' (lower; see Ref. 34); Spot14, 5'-ATG CAA GTG CTA ACG AAA CGC-3' (upper) and 5'-AGA AGT GCA GGT GGA ACT GGG C-3' (lower; see Ref. 35); TR-beta 1, 5'-GCC TGG GAC AAG CAG AAG CCC CGT-3' (upper) and 5'-AGC GAC ATT CCT GGC ACT GGT TGC G-3' (lower; see Refs. 1 and 42); TR-alpha , 5'-ATG GAA CAG AAG CCA AGC AAG GTG GAG-3' (upper) and 5'-CTG CAG CAG AGC CAC TTC CGT GTC A-3' (lower).

Electrophoretic gel mobility shift assays. Nuclear extract (10 µg) was incubated on ice for 30 min with 6 × 104 counts/min 32P-labeled oligonucleotides in 15 µl binding buffer consisting of 20% glycerol, 25 mM Tris · HCl (pH 7.5), 40 mM KCl, 0.5 mM MgCl2, 0.1 mM EDTA, 1 mM dithiothreitol, 2 µg poly(dI-dC), and 1 µg salmon sperm DNA. Double-stranded oligonucleotide probes were end labeled with T4 polynucleotide kinase in the presence of 50 µCi [gamma -32P]dATP and purified on a Sephadex G-25 column (Amersham Pharmacia Biotech). DNA-protein complexes were separated by electrophoresis (constant voltage of 300 volts) on a 5% nondenaturing polyacrylamide gel in 0.5× Tris-borate-EDTA at 4°C. Gel was dried, exposed to X-ray film, and quantified by densitometry. The following oligonucleotides (upper strand) were used: IR0 (wild type) 5'-GTA CCT CAG GTC ATG ACC TGA C-3' (28); mutated IR0 (mutant) 5'-AGC TTC AGA ACA TGT ACT GAC-3'.

In competition assay, 100-fold molar excess of the specific or mutated unlabeled oligonucleotide was preincubated on ice for 1 h with 10 µg nuclear extract from a control mouse in the binding buffer before the oligonucleotide probe was added.

When indicated, 5 ng-5 µg of recombinant human RXR-beta (Affinity BioReagents) were preincubated on ice for 1 h with binding buffer that contained a 10-µg nuclear extract from an LPS-treated mouse before the labeled oligonucleotide was added.

In supershift studies, nuclear extracts were incubated with 2 µl of anti-RXR-alpha , anti-RXR-beta , anti-RXR-gamma , or anti-rabbit IgG (Santa Cruz) or with 5 µl of anti-TR-beta 1 or anti-TR-alpha (Affinity BioReagents) for 1 h at room temperature after addition of the labeled probe.

Statistical analysis. Data are expressed as means ± SE of experiments from 3-5 animals/group for each time point. Differences between two experimental groups were analyzed using the unpaired t-test. A P value <0.05 was considered significant.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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LPS treatment decreases the expression of TR-beta target genes in mouse liver. Malic enzyme (30, 36) and Spot14 (17, 19, 23, 29) are two well-characterized thyroid hormone-responsive genes in the liver. Studies in TR-beta knockout mice have shown that TR-beta is necessary for thyroid hormone-mediated activation of expression of malic enzyme and Spot14 and for maintaining basal Spot14 mRNA levels (41). We first tested whether our model of acute inflammation was associated with a decrease in the level of expression of malic enzyme and Spot14. As shown in Fig. 1A, 4 h after LPS administration (100 µg) there was a trend for malic enzyme expression levels to decrease; these did not reach significance, but by 8 h we observed a 68% decrease in malic enzyme mRNA levels. A similar reduction (67%) was observed 16 h after LPS treatment, and by 24 h malic enzyme mRNA levels were slowly returning toward normal levels, with a 50% decrease at this time point. Furthermore, LPS administration (100 µg) was also associated with a decrease in Spot14 expression, leading to a rapid 61% reduction in Spot14 mRNA levels within 4 h (Fig. 1B). A similar 58% decrease in Spot14 mRNA levels was observed 8 h after LPS treatment. By 16 h, Spot14 mRNA levels were returning toward normal levels, with a modest 3 and 23% decrease, respectively, 16 and 24 h after LPS administration.


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Fig. 1.   Reduction in malic enzyme (A) and Spot14 (B) mRNA levels in mice liver after lipopolysaccharide (LPS) treatment. Mice were injected ip with either saline () or 100 µg LPS (), and animals were killed at the time indicated (hours) after LPS administration. Poly(A)+ RNA was isolated, and Northern blot analysis was performed as described under MATERIALS AND METHODS. A glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was used to check for RNA integrity. After normalization to GAPDH, data (means ± SE, n = 4) are expressed as a percentage of control. ME, malic enzyme; LPS, lipopolysaccharide. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Therefore, LPS treatment is associated with a time-dependent reduction in malic enzyme and Spot14 mRNA levels in mouse liver. It should be noted that these findings are specific to LPS, and not to the anorectic effect of LPS, since food was withdrawn in all groups at the time of LPS injection in every experiment described in this article. We next examined the effect of LPS administration on TR binding to DNA to further analyze the possible molecular mechanisms underlying the sick euthyroid syndrome.

LPS treatment reduces RXR/TR heterodimers binding to a specific TRE. To determine whether the decrease in malic enzyme and Spot14 mRNA levels could be associated with a reduction in TR binding to DNA, gel shift experiments were carried out on hepatic nuclear extracts prepared from mice treated with LPS (100 µg) or with saline. We used a 32P-labeled oligonucleotide containing a response element more specific for TR, i.e., an inverted repeat (IR) 0 (22, 25) rather than a DR4 (to which LXR and constitutive androstane receptor can also bind) or an ER6 (also recognized by the pregnane X receptor). Although TR can bind to its cognate DNA response element (TRE) as monomers or homodimers, the major form of TR bound to TRE is the heterodimer with RXR (44). As shown in Fig. 2, A and B, using an IR0, we detected a strong shifted band in the samples from saline-treated mice. Competition with cold 100-fold molar excess of the same specific oligonucleotide (wild type) but not of mutated (mutant) oligonucleotide demonstrated the specificity of the binding activity we observed in our samples. Furthermore, at 4 h, LPS administration resulted in a 60% decrease in TR binding activity in mouse liver nuclear extracts (Fig. 2A). At 16 h, the binding was decreased further, with TR binding activity being reduced by 77% in nuclear extracts prepared from mice treated with LPS compared with control mice (Fig. 2B).


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Fig. 2.   Effect of LPS administration on retinoid X receptor (RXR)/thyroid hormone receptor (TR) binding to a specific thyroid hormone response element (TRE). Mice were injected ip with either saline or LPS (100 µg). Four (A) and 16 (B) h after treatment, hepatic nuclear extracts were prepared as described under MATERIALS AND METHODS. Nuclear extract (10 µg) was incubated with a specific radiolabeled oligonucleotide [inverted repeat (IR) 0] for RXR/TR heterodimers. Representative electrophoretic gel mobility shift assays are shown for each time point (A and B). Unlabeled specific [100× wild type (wt)] and nonspecific [100× mutant (mut)] competing oligonucleotides were included at 100-fold molar excess 1 h before the addition of the labeled probe. C: representative electrophoretic gel mobility shift assay using hepatic nuclear extract from control (lanes 1-7) or LPS-treated (lanes 8-14) mice incubated with antibodies raised against RXR-alpha (lanes 2 and 9), RXR-beta (lanes 3 and 10), RXR-gamma (lanes 4 and 11), TR-alpha (lanes 5 and 12), TR-beta 1 (lanes 6 and 13), and rabbit IgG (lanes 7 and 14). Arrows, complexes supershifted by the different antibodies.

To identify the proteins in these complexes, we performed supershift experiments. As shown in Fig. 2C, a portion of the bound probe was supershifted after incubation of the samples with antibodies raised against TR-beta 1, TR-alpha , RXR-alpha , and RXR-beta . In our hands, RXR-gamma antiserum was unable to supershift the bound probe. We also verified that the formation of RXR/TR complexes was not affected by incubation with a nonspecific IgG. As shown in Fig. 2C, our results suggest that RXR-alpha was involved in at least two different complexes, as well as TR-beta 1. TR-alpha 1 and/or TR-alpha 2 were also present in two different complexes. Meanwhile, RXR-beta was only detected in one complex. These results suggest that the broad band we observed in our samples using an IR0 as a probe probably consisted of multiple RXR/TR heterodimers. It should be noted that the intensity of the supershifted bands reflects the overall pattern of expression of these nuclear hormone receptors in the liver, with TR-beta being more abundant than TR-alpha and RXR-alpha being the major RXR isoform in this tissue. Finally, as shown in Fig. 2C, every supershifted complex was affected by LPS treatment, indicating that the APR is associated with an overall reduction in RXR/TR heterodimer DNA binding in mouse liver (Fig. 2C, lanes 8-14).

To determine whether a decrease in circulating thyroid hormone levels could be a determinant factor in the LPS-induced reduction in RXR/TR heterodimer DNA binding in liver, we measured total T3 and total T4 levels in mouse serum 4 and 16 h after LPS administration (100 µg). As previously published (5), LPS treatment was not associated with a significant decrease in total T3 serum levels either at 4 h (48.8 ± 3.5 ng/dl in saline vs. 42.2 ± 1.5 ng/dl in LPS-treated group) or at 16 h (40.4 ± 2.0 ng/dl in saline vs. 39.2 ± 1.6 ng/dl in LPS-treated group). Total T4 levels were not significantly reduced at 4 h (2.58 ± 0.28 µg/dl in saline vs. 1.74 ± 0.34 µg/dl in LPS-treated group), but by 16 h LPS administration was associated with a significant (P < 0.05) decrease in total T4 serum levels (1.74 ± 0.19 µg/dl in saline vs. 1.14 ± 0.06 µg/dl in LPS-treated group). Because this reduction in total T4 levels does not provide a convincing explanation for the marked reduction in RXR/TR heterodimers binding to DNA, we next tested whether LPS treatment leads to a decrease in TR protein levels in mouse liver.

LPS administration decreases TR protein levels at 16 h but not at 4 h in mouse liver. We initially determined the effect of LPS treatment on TR-beta 1 protein levels in mouse liver, since TR-beta 1 is the major TR isoform in liver (16). To examine whether a decrease in TR-beta 1 protein levels could account for the reduction in RXR/TR binding activity as soon as 4 h after LPS administration (100 µg), Western blot analyses were carried out using nuclear extracts prepared from mouse liver. As expected (9), a major band corresponding to a 55-kDa protein was observed with the TR-beta 1 antibody (Fig. 3A). Surprisingly, LPS treatment did not result in a significant decrease in TR-beta 1 protein levels at 4 h, but by 16 h LPS administration was associated with a 41% decrease in TR-beta 1 protein levels (Fig. 3A). We then tested whether the decrease in RXR/TR binding activity we observed 4 h after LPS treatment could result from a decrease in TR-alpha protein levels. As shown in Fig. 3B, TR-alpha 1 (48 kDa) and TR-alpha 2 (58 kDa) protein levels were also not significantly reduced 4 h after LPS administration. On the other hand, 16 h after LPS treatment, there was a 55 and 87% decrease in TR-alpha 1 and TR-alpha 2 protein levels, respectively (Fig. 3B).


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Fig. 3.   Time-dependent decrease in TR-beta 1 (A) and TR-alpha 1 and TR-alpha 2 (B) protein levels in mice liver after LPS administration. Mice were injected ip with either saline or LPS (100 µg) and were killed 4 and 16 h after treatment. Hepatic nuclear extracts were prepared, and Western blot analysis was performed as described under MATERIALS AND METHODS. Blots were exposed to X-ray film for 2 min (TR-beta 1) and 5 min (TR-alpha ), respectively.

Thus our model of inflammation is associated with an overall reduction in RXR/TR heterodimer binding activity in mouse liver at both 4 and 16 h after LPS administration. Furthermore, our results indicate that LPS administration is associated with a time-dependent reduction in TR-beta 1, TR-alpha 1, and TR-alpha 2 protein levels in mouse liver. The decrease in TR-beta 1, TR-alpha 1, and TR-alpha 2 protein levels we observed at 16 h is consistent with the concomitant decrease in RXR/TR binding activity at this time point. However, at 4 h the decrease in RXR/TR binding activity occurred despite normal TR-beta 1 and TR-alpha protein levels. Because we have previously reported that RXR mRNA and protein levels are reduced in rodent liver as early as 4 h after LPS administration (Table 1), we next determined whether RXR repression could play a role in the reduction of RXR/TR binding during the APR.

                              
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Table 1.   Decrease in RXR levels in rodent liver 4 and 16 h after LPS administration

LPS-induced RXR repression participates in the reduction in RXR/TR binding to TRE at 4 h. To determine whether the decrease in RXR levels we previously described (3, 4) 4 h after LPS treatment could account for the reduction in RXR/TR binding at this time point, we next carried out gel shift experiments in which we preincubated liver nuclear extracts from LPS-treated mice with 5, 50, 500, or 5,000 ng recombinant human RXR-beta protein. The nuclear extracts were prepared from mice liver 4 h after injection of LPS (100 µg) or saline alone. As shown in Fig. 4, adding increasing quantities of recombinant RXR-beta protein to nuclear extracts prepared from LPS-treated mice (lanes 5-7) allowed us to dose dependently restore RXR/TR binding up to the level observed in samples prepared from control mice (lane 2). Beyond 500 ng, further addition of recombinant RXR-beta protein resulted in the binding of recombinant RXR-beta protein to the IR0 oligonucleotide as a homodimer (Fig. 4, lane 4). Compared with RXR/TR heterodimers, RXR-beta homodimers exhibited low specificity for the IR0 oligonucleotide, since 100-fold excess of cold wild-type IR0 only poorly competed with the labeled IR0 probe (Fig. 4, lane 14).


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Fig. 4.   Representative electrophoretic gel mobility shift assay using hepatic nuclear extract from control (lanes 2 and 8) or LPS-treated (lanes 3-7 and 9-13) mice. Mice were injected ip with either saline or LPS (100 µg). Four and 16 h after treatment, hepatic nuclear extracts were prepared as described under MATERIALS AND METHODS. Nuclear extracts (10 µg) were incubated with a specific radiolabeled oligonucleotide (IR0) for RXR-TR heterodimers in the presence of recombinant RXR-beta (5, 50, 500, or 5,000 ng). Unlabeled specific (100× wt) and nonspecific (100× mut) competing oligonucleotides were included at 100-fold molar excess 1 h before the addition of the labeled probe (lanes 14 and 15).

The same experiments were carried out on nuclear extracts prepared from mice liver 16 h after injection of LPS (100 µg) or saline alone. As shown in Fig. 4, at this time point adding up to 500 ng recombinant RXR-beta protein to nuclear extracts prepared from LPS-treated mice (lanes 11-13) did not restore RXR/TR binding to the level observed in samples prepared from control mice (lane 8). Addition of higher amounts of recombinant RXR-beta protein mainly resulted in the binding of RXR-beta as a homodimer to the IR0 oligonucleotide (lane 10).

Our results suggest that, at 4 h, the LPS-induced decrease in RXR levels plays a key role in the reduction of RXR/TR binding to IR0, whereas, at 16 h, the reduction both in RXR levels and in TR levels participates in the decrease of RXR/TR binding to IR0.

LPS treatment decreases the expression level of all TR isoforms in mouse liver. To determine whether the decrease in TR protein levels we observed during the APR is because of a reduction in gene expression, we next examined the effect of LPS administration on TR mRNA levels. As shown in Fig. 5A, LPS administration (100 µg) was associated with a time-dependent decrease in TR-beta 1 expression levels in mouse liver. TR-beta 1 mRNA levels were decreased by 45% 4 h after LPS treatment. By 8 h, LPS administration induced a marked 83% reduction in TR-beta 1 mRNA levels, and this effect was maintained for at least 16 h (89% decrease at 16 h after LPS treatment). But by 24 h TR-beta 1 mRNA levels were slowly returning toward normal levels, with LPS treatment leading to a more modest 68% reduction in TR-beta 1 mRNA levels at this time point. Furthermore, at 16 h, the LPS-induced decrease in TR-beta 1 mRNA levels was dose dependent, with a half-maximum effect occurring at ~7 µg LPS (data not shown).


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Fig. 5.   Effect of LPS treatment on TR mRNA levels. A: reduction in TR-beta 1 mRNA levels in mouse liver after LPS treatment. B: reduction in TR-alpha 1 (, ) and TR-alpha 2 (open circle , ) mRNA levels in mouse liver after LPS treatment. In all experiments, mice were injected ip with either saline (, open circle ) or 100 µg LPS (, ), and animals were killed at the time indicated (hours) after LPS administration. Poly(A)+ RNA was isolated, and Northern blot analysis was performed as described under MATERIALS AND METHODS. A GAPDH probe was used to check for RNA integrity. After normalization to GAPDH, data (means ± SE, n = 4) are expressed as a percentage of control. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Because it has been shown that the TR-beta and TR-alpha genes exhibit differential tissue-specific regulation (16), we next tested whether LPS administration could decrease TR-alpha mRNA levels in mouse liver. As described under MATERIALS AND METHODS, we used a mouse cDNA that cross-reacts with TR-alpha 1 (5 kb) and TR-alpha 2 (2.5 kb). LPS treatment (100 µg) was associated with a rapid and time-dependent decrease in TR-alpha 1 and TR-alpha 2 expression levels (Fig. 5B). Within 2 h, LPS administration induced decreases of 72 and 37% in TR-alpha 1 and TR-alpha 2 mRNA levels, respectively. At 4 h, TR-alpha 1 and TR-alpha 2 mRNA levels were further decreased by 87 and 48%, respectively, and after 8 h LPS treatment was associated with a 74 and a 82% reduction in TR-alpha 1 and TR-alpha 2 mRNA levels, respectively. In our hands, a similar decrease was maintained for at least 24 h for both isoforms (81 and 73% decrease at 16 and 24 h, respectively, for TR-alpha 1; 93 and 82% decrease at 16 and 24 h, respectively, for TR-alpha 2). Furthermore, at 16 h, the LPS-mediated reduction in TR-alpha mRNA levels was dose dependent, with the half-maximum effect occurring at ~7 µg (data not shown).

Therefore, the LPS-induced decrease in TR-beta 1 mRNA levels was also associated with a reduced expression in the minor isoform, TR-alpha , in the liver. This overall reduction in TR-beta 1 and TR-alpha mRNA levels occurs at relatively low doses, since the LD50 for LPS in rodents is ~5 mg/100 g body wt. Furthermore, this inhibitory effect was not restricted to the liver but rather constituted a general response to LPS in all peripheral tissue tested. In particular, we found that TR-beta 1 mRNA levels were decreased by 65% (P < 0.001) and TR-alpha 1 mRNA levels by 77% (P < 0.001) in white adipose tissue 16 h after LPS treatment (data not shown). Also, although RXR-alpha and RXR-gamma mRNA levels were below detection by Northern blot analysis, RXR-beta mRNA levels were reduced by 58% (P < 0.001) in this tissue at 16 h (data not shown). Finally, this decrease in TR-beta 1 and TR-alpha mRNA levels precedes the decrease in TR-beta 1 and TR-alpha protein levels in liver.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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REFERENCES

Infection, inflammation, and trauma are known to induce changes in metabolism, which are part of the pathophysiology of the APR. In the liver, the synthesis of certain proteins is increased (positive acute-phase proteins), such as C-reactive protein (which is thought to play a protective role during infection; see Ref. 38), whereas the synthesis of other proteins (negative acute-phase proteins) is decreased (20). Several negative acute-phase proteins are involved in lipid metabolism, and the decrease in expression of these proteins leads to many changes during the APR, including a decrease in hepatic fatty acid oxidation, ketogenesis, and bile acid synthesis (15). During the APR, most of the changes in hepatic protein expression are thought to occur at the transcriptional level. The mechanism by which gene transcription is stimulated during the APR has been studied extensively. Transcription factors such as CCAAT/enhancer binding protein, nuclear transcription factor-kappa B, and members of the signal transducer and activator of transcription family are thought to mediate the increase in expression of most positive acute-phase proteins (2). On the other hand, downregulation of members of the nuclear hormone receptor superfamily, including hepatic nuclear factor 4 (31), PPAR (3), LXR (3), and RAR (7), is thought to be involved in the repression of several negative acute-phase proteins.

We therefore determined whether TR, a member of the nuclear hormone receptor superfamily, could be downregulated during the APR, thereby representing one possible mechanism by which malic enzyme and Spot14 are repressed in our model of inflammation. Our experiments do not rule out the possibility that other molecular mechanisms are involved in the decrease in expression of these two proteins during the APR. Another transcription factor, sterol regulatory element-binding protein-1 (SREBP-1), has been identified as a key activator of malic enzyme and Spot14 during fasting-refeeding treatments (34). However, it is unlikely that SREBP-1 plays a role in the downregulation of these enzymes during the APR, since the proinflammatory cytokine TNF has been shown to actually stimulate the maturation of SREBP-1 in hepatocytes (21). Finally, our experiments do not exclude the possibility that LPS decreases the expression of malic enzyme and Spot14 at the posttranscriptional level. Unfortunately, RNA degradation studies typically use actinomycin D, a compound that dramatically increases LPS toxicity, rendering the interpretation of the results difficult.

Here we show that a time-dependent decrease in TR-beta protein and mRNA levels occurs in mouse liver, suggesting that TR-beta repression is at least in part the result of a decrease in protein synthesis after LPS administration. Furthermore, the LPS-induced decrease in TR-beta , the major isoform in the liver, was not compensated by an upregulation of TR-alpha . On the contrary, TR-alpha protein and mRNA levels were markedly decreased after LPS treatment. We also report a reduction in TR DNA binding in mouse liver during the APR. However, the reduction in TR DNA binding preceded the decrease in TR protein levels.

Of note, the decrease in TR DNA binding even preceded the hereby and previously described (5) LPS-induced decrease in thyroid hormone circulating levels. In our hands, serum T3 levels were unaffected at least for the first 16 h after LPS treatment, whereas serum T4 levels started to decline 16 h after LPS administration.

TR forms heterodimers with RXR for high-affinity binding to DNA (22). Because we have already shown that all three RXR isoforms (RXR-alpha , -beta , and -gamma ) are downregulated at the protein level as early as 4 h after LPS treatment (3), we hypothesized that the early decrease in RXR availability in liver might play an important role in the decrease in TR DNA binding at 4 h. Our results suggest that this might be the case, since adding recombinant RXR-beta to these extracts restored TR DNA binding at least partially at this early time point. These findings are consistent with previous studies (18, 32) in which others showed that PPAR-alpha can inhibit RXR/TR DNA binding by "titering" out RXR. Although the mechanism of reduction is different, since PPAR-alpha is actually downregulated during the APR (3), both their experiments and ours imply that RXR availability could limit TR DNA binding. On the other hand, our findings are in apparent contradiction with observations in hepatocyte-specific RXR-alpha -deficient mice (40). Using a cre/loxP recombination technique, these authors reported that the deletion of RXR-alpha specifically in the liver did not affect malic enzyme basal mRNA levels. However, as suggested by our electrophoretic mobility shift assay experiments, TR also forms heterodimers with RXR-beta . It is conceivable that, in the setting of RXR-alpha deficiency, RXR-beta /TR and/or RXR-gamma /TR heterodimers could play a role in maintaining basal level of key enzymes such as malic enzyme, which constitute a major source of NADPH in hepatocytes (39). In our model of inflammation, however, such compensatory mechanism could not occur, since all three RXR isoforms are repressed in the liver. At the later time point (16 h), the coordinate downregulation of both RXR and TR resulted in further reduction in TR DNA binding, which could not significantly be corrected by an excess amount of recombinant RXR-beta . Therefore, our results suggest that the time-dependent decrease in TR DNA binding that we observed in mouse liver after LPS administration results from a dual mechanism. The early decrease in RXR levels seems to play a critical role in the reduction in binding at 4 h, suggesting that, even in the setting of nonpermissive heterodimers, such as RXR/TR heterodimers, the silent partner RXR could constitute a target for fine regulation of the pathway. In contrast, at the later time point, a decrease in both TR and RXR levels results in a further reduction in TR DNA binding.

Overall, the changes in TR and RXR levels that occur during the APR will limit the impact of T3 in liver. Whether this effect constitutes a physiological or a pathological response to infection and inflammation remains unclear. On one hand, because the APR is associated with an increased metabolic rate, a reduction in T3 activity may improve survival by conserving energy, limiting hepatic proteolysis and catabolism. On the other hand, critically ill patients exhibiting the severe form of sick euthyroid syndrome have a worse prognosis. It would be of interest to determine whether a reduction in TR and/or RXR levels also occurs in the heart during the APR, since such an effect could increase the mortality risk by decreasing cardiac function. In summary, the APR results in a time-dependent decrease in TR protein, mRNA, and DNA binding activity in mouse liver. These changes occur in the early phases of the inflammatory response, providing an additional level of regulation of thyroid hormone action during infection.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Research Service of the Department of Veterans Affairs and by National Institutes of Health Grants DK-49448 and AR-39639.


    FOOTNOTES

Address for reprint requests and other correspondence: A. P. Beigneux, Gladstone Institute of Cardiovascular Disease, P. O. Box 419100, San Francisco, CA 94141-9100 (E-mail: abeigneux{at}gladstone.ucsf.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

September 3, 2002;10.1152/ajpendo.00155.2002

Received 11 April 2002; accepted in final form 28 August 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Endocrinol Metab 284(1):E228-E236