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. 2008 Jan 24;149(5):2484–2493. doi: 10.1210/en.2007-1697

Induction of Type 2 Iodothyronine Deiodinase in the Mediobasal Hypothalamus by Bacterial Lipopolysaccharide: Role of Corticosterone

Edith Sánchez 1, Praful S Singru 1, Csaba Fekete 1, Ronald M Lechan 1
PMCID: PMC2329263  PMID: 18218695

Abstract

To determine whether endotoxin-induced activation of type 2 iodothyronine deiodinase (D2) in the mediobasal hypothalamus is dependent on circulating levels of corticosterone, the effect of bacterial lipopolysaccharide (LPS) on D2 gene expression was studied in adrenalectomized, corticosterone-clamped adult, male, Sprague Dawley rats. In sham-adrenalectomized animals, LPS (250 μg/100 g body weight) increased circulating levels of corticosterone and IL-6, as well as tanycyte D2 mRNA in the mediobasal hypothalamus. Adrenalectomized, corticosterone-clamped animals showed no significant rise in corticosterone after LPS, compared with saline-treated controls but increased IL-6 levels and tanycyte D2 mRNA similar to LPS-treated sham controls. To further clarify the potential role of corticosterone in the regulation of D2 gene expression by LPS, animals were administered high doses of corticosterone to attain levels similar to that observed in the LPS-treated group. No significant increase in D2 mRNA was observed in the mediobasal hypothalamus with the exception of a small subpopulation of cells in the lateral walls of the third ventricle. These data indicate that the LPS-induced increase in D2 mRNA in the mediobasal hypothalamus is largely independent of circulating corticosterone and indicate that mechanisms other than adrenal activation are involved in the regulation of most tanycyte D2-expressing cells by endotoxin.

TYPE 2 IODOTHYRONINE DEIODINASE (D2) is the principal enzyme in the central nervous system that catalyzes the intracellular generation of T3 from its less potent precursor, T4 (1,2). Although expressed in several regions of brain where it functions to maintain constant, local tissue levels of T3 (1,2), in the hypothalamus, D2 may function primarily as a regulatory signal by increasing or decreasing T3 levels under the appropriate physiological situations (3).

Unique to the hypothalamus is the synthesis of D2 by a specialized type of glial cell, the tanycyte, that lines the floor and infralateral walls of the third ventricle between the rostral-caudal limits of the median eminence and the infundibular recess (4,5,6). These cells are characterized by apical villi-like evaginations with phagocytic properties that extend into the cerebrospinal fluid (CSF), and a single, basal process that ramifies into the underlying neuropil of the hypothalamic arcuate nucleus and median eminence, surrounding blood vessels in numerous end feet processes including those of the fenestrated capillaries of the primary portal plexus (7,8,9,10). Thus, these cells are in a strategic position to extract T4 from either the CSF and/or the blood stream and, after conversion to the more biologically active T3, provide a source of T3 to adjacent hypothalamic neurons either by direct release into the arcuate nucleus, retrograde transport from axon terminals in the median eminence or by increasing T3 concentrations in the CSF that could diffuse into the substance of the brain by volume transmission (3,4,6,11).

Recent studies from our laboratories (12,13) and by Boelen et al. (14) have demonstrated that bacterial lipopolysaccharide (LPS) rapidly and markedly increases D2 activity and mRNA in the mediobasal hypothalamus. Because endotoxin administration results in central hypothyroidism by suppressing hypophysiotropic TRH neurons in the hypothalamic paraventricular nucleus (15,16), we raised the possibility that an ensuing hypothalamic-specific increase in T3 concentration contributes to this response by local feedback inhibition of hypophysiotropic TRH gene expression (3,12,13). Indeed, evidence of increased hypothalamic T3 content in association with increased D2 expression in the mediobasal hypothalamus has been reported in the Japanese quail after exposure to long photoperiods (17) and in fasting mice (18). The mechanism whereby D2 is activated by endotoxin in tanycytes, however, is unknown. Because the hypothalamic-pituitary-adrenal axis is markedly up-regulated during infection (19,20,21,22) and corticosterone has been proposed to increase D2 activity in the mediobasal hypothalamus during fasting (23), we tested the hypothesis that increased circulating levels of corticosterone also participates in the regulation of tanycyte D2 expression after LPS administration.

Materials and Methods

Animals

The experiments were carried out on adult, male, intact (n = 27), sham adrenalectomized (n = 18) or adrenalectomized (n = 36), Sprague Dawley rats performed by Taconic Farms (Germantown, NY) through a dorsal approach, weighing 200–250 g. Animals were acclimatized to standard environmental conditions for at least 5 d (light between 0600 and 1800 h, temperature 22 ± 1 C) and received rat chow and tap water ad libitum, except that adrenalectomized rats received 0.5% NaCl in their drinking water. Sham or adrenalectomized rats were housed individually and intact rats were housed three per cage. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at the Tufts-New England Medical Center and Tufts University School of Medicine.

D2 mRNA response to LPS

To determine the time course at which D2 mRNA is maximally increased in tanycytes after LPS administration, LPS [250 μg/100 g body weight (BW) in saline; Sigma O127: B8; Sigma Chemical Co., St. Louis, MO] was injected ip to intact animals (n = 3/group), and the brains were prepared for in situ hybridization histochemistry 3, 6, 9, and 12 h after a single injection as described below. Control animals were administered an equal volume of vehicle ip (250 μl) and prepared for study 6–9 h later.

Tissue processing

Animals were overdosed with pentobarbital (50 mg/kg ip) and perfused transcardially first with 20 ml 0.01 m PBS (pH 7.4), containing 15,000 U/liter heparin sulfate, followed by 150 ml 4% paraformaldehyde in PBS. The brains were removed and postfixed by immersion in the same fixative for 2 h at room temperature. Tissue blocks containing the hypothalamus were cryoprotected in 25% sucrose/PBS at 4 C overnight and then snap frozen on dry ice. Serial 18-μm-thick coronal sections through the rostrocaudal extent of the median eminence were cut on a cryostat (Leica CM3050 S; Leica Microsystems, Nussloch GmbH, Germany) and adhered to Superfrost /Plus glass slides (Fisher Scientific Co., Pittsburgh, PA) to obtain four sets of slides, each set containing every fourth section through the median eminence. The tissue sections were desiccated overnight at 42 C and stored at −80 C until prepared for in situ hybridization histochemistry.

Pellet preparation and implantation

Corticosterone pellets were prepared as reported by Strack et al. (24). A combination of corticosterone (Sigma; catalog no. C-2505) and cholesterol (Steraloids, Inc., Wilton, NH; catalog no. C6760) or cholesterol alone, were melted under sterile conditions. The material was poured into 100-mg mold wells (Ted Pella, Inc., Redding, CA), avoiding the formation of bubbles, and allowed to solidify.

Pellets containing 30 or 45% corticosterone were implanted into adult rats 7 d after adrenalectomy. Animals were anesthetized with ketamine (80 mg/kg BW ip; Phoenix Pharmaceuticals Inc., St. Joseph, MO) and xylazine (9 mg/kg BW ip; Phoenix Pharmaceuticals) and the pellets placed sc above the sacrum through a 12-mm incision in the skin. Control animals subjected to sham adrenalectomy were implanted sc with a 100% cholesterol pellet. One week after implantation, adrenalectomized and sham control animals were each divided into two groups and administered either LPS (250 μg/100 g BW) or saline ip (250 μl). To determine the efficacy of the LPS response in adrenalectomized animals replaced with corticosterone, IL-6 levels were used as an index of the immunological response (25).

Animals receiving 45% corticosterone pellets were studied 9 h after LPS administration. Because the 30% corticosterone-treated group showed more significant clinical manifestations after endotoxin administration, this group was studied 6 h after LPS administration.

IL-6 determinations

Blood obtained from the inferior vena cava was centrifuged at 3000 rpm × 15 min, and serum aliquots stored at −80 C in polypropylene tubes until assayed. Samples were thawed at room temperature and diluted 1:1 with RD5–16 calibrator diluent and subjected to a rat ELISA system kit for IL-6 (Quantikine; R&D Systems, Inc., Minneapolis, MN). A standard curve was run at the same time by diluting known amounts of rat IL-6 standard with RD5–16 calibrator diluent. The ELISA was performed following the manufacturer’s instructions, and the OD was determined using a microplate reader (Multiskan Ascent; Thermo Labsystems, Milford, MA) set to 450 nm. Wavelength readings were corrected by subtracting readings at 540 nm from the readings at 450 nm. Sensitivity of the assay was 14–36 pg/ml.

Corticosterone injections

To simulate levels of corticosterone attained after LPS administration, a separate group of intact animals (n = 6) was treated with sc injected corticosterone at a dose of 560 μg per rat in 50% ethanol-sterile saline every 2 h for four doses, beginning at 0700 h. Animals were compared with a control group receiving only vehicle (n = 6) or LPS (n = 6). At conclusion of the study, the animals were deeply anesthetized with pentobarbital (50 mg/kg ip) and blood collected from the inferior vena cava before the beginning of the perfusion procedure. Tissues were prepared for in situ hybridization as described above.

Corticosterone measurements

Serum samples stored at −80 C were thawed at room temperature, and 100-μl aliquots were extracted in glass tubes (10 × 75 mm, Fisher Scientific) with 1 ml of ethyl acetate (HPLC grade; Fisher Scientific). The organic phase was transferred into a clean glass tube and the solvent evaporated under a stream of nitrogen gas. The residual was resuspended in 100 μl of diluted extraction buffer (rat ELISA system kit; Neogen Corp., Lexington, KY) and then further diluted 100 times with the same extraction buffer. A standard curve was run simultaneously by diluting known amounts of rat corticosterone standard in duplicates into the diluted extraction buffer. The ELISA was performed after the manufacturer’s instructions and the OD determined using a microplate reader (model 680; Bio-Rad Laboratories, Hercules, CA) set to 450 nm. The sensitivity of the assay was 0.05–5.0 ng/ml.

In situ hybridization histochemistry

Every fourth section through the median eminence was hybridized with an 800-bp single stranded [35S] uridine 5-triphosphate-labeled cRNA probe complementary to the entire coding region of the rat D2 gene as previously described (12). Hybridizations were performed under plastic coverslips in a buffer containing 50% formamide, a 2-fold concentration of standard sodium citrate (2× saline sodium citrate), 10% dextran sulfate, 0.25% BSA, 0.25% Ficoll 400, 0.25% polyvinylpyrolidone 360, 250 mm Tris (pH 8.0), 0.5% sodium dodecyl sulfate, 250 μg/ml denatured salmon sperm DNA, and 5 × 105 cpm of the radiolabeled probe for 16 h at 55 C.

Slides were dipped into Kodak NTB autoradiography emulsion (Eastman Kodak, Rochester, NY) diluted 1:1 in distilled water and the autoradiograms developed after 10 d of exposure at 4 C. The specificity of hybridization was confirmed using sense probes, which resulted in the total absence of specific hybridization signal in the hypothalamus.

Image analysis

Autoradiograms were visualized with an Axioplan 2 imaging microscope (Carl Zeiss Microimaging Inc., Thornwood, NY) under dark-field illumination using a COHU 4912 video camera (COHU, Inc., San Diego, CA) and the images analyzed with a Macintosh G4 computer using Scion Image software (National Institutes of Health, Bethesda, MD). Background was removed by thresholding the image. Integrated density values (density × area) of the mediobasal hypothalamus were measured in five, consecutive, rostrocaudal serial sections through the median eminence in one set of slides for each animal extending from approximately −2.56 to −3.14 mm from the bregma using the rat brain stereotaxic atlas of Paxinos and Watson (26). Nonlinearity of radioactivity in the emulsion was evaluated by comparing density values with a calibration curve created from autoradiograms of known dilutions of the radiolabeled probes, immobilized on glass slides in 1.5% gelatin, fixed with 4% paraformaldehyde, and exposed and developed simultaneously with the in situ hybridization autoradiograms.

Statistical analysis

Results are presented as means ± sem. IL-6 and corticosterone measurements, and in situ hybridization analyses were compared by one-way ANOVA followed by Newman-Keuls post hoc test using Prism 4 software (GraphPad Software, Inc., San Diego, CA). P < 0.05 values were considered statistically significant.

Results

D2 mRNA response to LPS in the mediobasal hypothalamus

Silver grains representing D2 mRNA were distributed in the median eminence and the floor and infralateral walls of the third ventricle in control animals (Fig. 1A). LPS administration resulted in a significant increase in D2 mRNA expression in the mediobasal hypothalamus, first evident at 3 h and reaching a maximal response 6–9 h after treatment (Fig. 1, B–D). By 12 h after LPS, D2 expression began to decrease in all areas of the mediobasal hypothalamus (Fig. 1E).

Figure 1.

Figure 1

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Dark-field photomicrographs showing D2 mRNA in the mediobasal hypothalamus (MBH) after a single dose of LPS (250 μg/100 g BW). A, D2 mRNA (arrows) is present in tanycytes lining the floor and infralateral walls of the third ventricle and increases 3 (B), 6 (C), and 9 h (D) after LPS administration, with the maximal response reached between 6 and 9 h. E, Twelve hours after LPS, the D2 response is beginning to decline. F, Computerized image analysis of D2 mRNA in the MBH after saline and LPS administration. Data represent mean ± sem of three animals per group. **, P < 0.01; ***, P < 0.001. III, Third ventricle; ME, median eminence. Scale bar, 200 μm.

Effect of LPS administration on immune activation in corticosterone clamped animals

To determine the effect of exogenous corticosterone on the immune response after LPS, IL-6 levels were measured in adrenalectomized animals receiving 30 and 45% corticosterone pellets. In sham-adrenalectomized animals implanted with a 100% cholesterol pellet, LPS resulted in a dramatic increase in IL-6 levels when compared with saline-treated controls perfused 6 h (Sham/Chol/Sal vs. Sham/Chol/LPS: 15 ± 5 vs. 548 ± 153, **, P < 0.01) (Fig. 2A) or 9 h after LPS administration (Sham/Chol/Sal vs. Sham/ chol/LPS: 15 ± 3 vs. 450 ± 95; ***, P < 0.001) (Fig. 2B). A similar increase was observed in adrenalectomized animals replaced with 30% corticosterone pellets (Adx/Cort30%/Sal vs. Adx/Cort30%/LPS: 77 ± 38 vs. 540 ± 142; **, P < 0.01) (Fig. 2A). In contrast, adrenalectomized animals treated with 45% corticosterone pellets showed a minimal IL-6 response that did not differ from sham controls treated with saline (Adx/Cort45%/Sal vs. Adx/Cort45%/LPS: 27 ± 5 vs. 32 ± 6, P > 0.05) (Fig. 2B), suggesting immune suppression by the higher corticosterone dose. Accordingly, studies on the effect of corticosterone clamp on the LPS-induced D2 response was performed in animals implanted with 30% corticosterone pellets.

Figure 2.

Figure 2

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Effect of corticosterone replacement on IL-6 levels in LPS-treated animals. A, IL-6 levels rise approximately 7-fold after administration of LPS in both sham and adrenalectomized animals treated with a 30% corticosterone pellet. SH, Sham; CHOL, cholesterol; SAL, saline; ADX, adrenalectomized; CORT, corticosterone. B, In contrast, significant diminution of the response is observed in adrenalectomized animals replaced with a 45% corticosterone pellet. Data represent the mean ± sem of four to eight animals (sham groups) or five to 10 animals (adrenalectomized groups). **, P < 0.01; ***, P < 0.001.

Serum corticosterone levels after LPS administration

To establish that the corticosterone-clamped adrenalectomized animals maintain circulating corticosterone levels substantially lower than sham-adrenalectomized control animals administered with LPS, serum corticosterone levels were measured in all animal groups. A significant increase in serum corticosterone levels was observed in the LPS treated sham-adrenectomized control group, compared with vehicle-treated control (Sham/Chol/Sal vs. Sham/Chol/LPS: 84 ± 8 vs. 404 ± 38 ng/ml; ***, P < 0.001) (Fig. 3). In contrast, levels of corticosterone in the adrenalectomized corticosterone-clamped animals receiving either vehicle or LPS were comparable with sham-adrenalectomized controls receiving vehicle (Adx/Cort/Sal vs. Adx/Cort/LPS: 119 ± 8 vs. 108 ± 3 ng/ml, P > 0.05) and significantly lower than levels in the sham-adrenalectomized animals receiving LPS (Fig. 3).

Figure 3.

Figure 3

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Effect of LPS on circulating corticosterone levels in sham-adrenalectomized controls and adrenalectomized animals treated with a 30% corticosterone pellet. Data represent mean ± sem of animals described in Fig 2. ***, P < 0.001. CHOL, Cholesterol; SAL, saline; ADX, adrenalectomized; CORT, corticosterone.

Effect of LPS administration on D2 mRNA expression in the mediobasal hypothalamus of corticosterone clamped animals

As anticipated, LPS resulted in a substantial increase of D2 mRNA in the median eminence and infralateral walls of the third ventricle of the sham-adrenalectomized control animals 6 h after LPS administration (Fig 4B), which by quantitative analysis, increased approximately 3-fold over vehicle-treated controls [Sham/Chol/Sal vs. Sham/chol/LPS (integrated density units): 45 ± 17 vs. 150 ± 11; **, P < 0.001] (Fig 4E). Adrenalectomized animals replaced with the 30% corticosterone pellet showed a similar distribution and expression of D2 mRNA as sham-adrenalectomized control animals (Fig. 4, A and C). After LPS administration, an increased accumulation of silver grains was observed over the infralateral walls and floor of the third ventricle and in the external zone of the median eminence, similar to that observed in the sham-adrenalectomized, LPS-treated controls (Fig. 4, B and D). D2 increased approximately 3-fold, compared with controls receiving vehicle (Adx/Cort/Sal vs. Adx/Cort/ LPS: 38 ± 6 vs. 137 ± 32; **, P < 0.001) (Fig. 4E).

Figure 4.

Figure 4

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Dark-field photomicrographs showing D2 mRNA in the mediobasal hypothalamus (MBH) of sham-adrenalectomized controls implanted sc with a cholesterol pellet and treated with either saline (A) or LPS (B), compared with adrenalectomized rats replaced with a 30% corticosterone pellet and similarly treated with saline (C) or LPS (D). D2 mRNA is significantly increased after LPS in the walls of the third ventricle (III) and median eminence (ME) in both sham and adrenalectomized animals. E, Computerized image analysis of D2 mRNA in the MBH after saline and LPS administration. SH, Sham; CHOL, cholesterol; SAL, saline; ADX, adrenalectomized; CORT, corticosterone. Data represent mean ± sem of animals described in Fig 2. **, P < 0.01. Scale bar, 200 μm.

Effect of high dose of corticosterone on D2 mRNA expression in the mediobasal hypothalamus

To determine whether repeated administration of high doses of exogenous corticosterone that simulate levels achieved after LPS administration replicate the effect of LPS to increase D2 expression in the mediobasal hypothalamus, a separate group of intact animals was studied. By administering corticosterone at a dose of 560 μg every 2 h for four doses, circulating corticosterone levels were substantially greater than the saline control (Saline vs. High Cort: 98 ± 4 vs. 418 ± 63 ng/ml; **, P < 0.01) and comparable with levels achieved after the administration of LPS (High Cort vs. LPS: 418 ± 63 vs. 375 ± 53 ng/ml) (Fig. 5). Despite achieving high circulating levels of corticosterone, no significant increase in D2 mRNA expression of the mediobasal hypothalamus was observed in the high-dose corticosterone-treated group by quantitative image analysis when compared with the controls [Saline vs. Vehicle vs. High Cort (integrated density units): 180 ± 48 vs. 269 ± 52 vs. 427 ± 87] (Fig. 6A, C, and E). In addition, expression of D2 mRNA in the high corticosterone-treated group was significantly less than the LPS group [High Cort vs. LPS (integrated density units): 427 ± 87 vs. 1145 ± 162, ***, P < 0.001] (Fig. 6, B, D, and E).

Figure 5.

Figure 5

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Circulating corticosterone levels after ip administration of LPS or sc administration of high-dose corticosterone (Cort). Data represent the mean ± sem of four to eight animals (saline control and LPS treated) or six animals [vehicle (50% ethanol in saline) and high dose corticosterone]. **, P < 0.01.

Figure 6.

Figure 6

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Dark-field photomicrographs of D2 mRNA expression in the mediobasal hypothalamus (MBH) 9 h after animals received either ip injections of saline (A) and 250 μg/100 g BW LPS (B) or sc injections of vehicle (50% ethanol in saline) (C) and high-dose corticosterone (D). Note that D2 mRNA expression is significantly increased only in the MBH of the LPS-treated animals, whereas high-dose corticosterone has no significant effect. E, Computerized image analysis of D2 mRNA in the MBH of saline, LPS, vehicle, and high-dose corticosterone (Cort)-treated animals. Data represent mean ± sem of six to eight animals in each group. ***, P < 0.001. III, Third ventricle. Scale bar, 120 μm.

Although quantitative analysis of D2 expression in the mediobasal hypothalamus in the high dose corticosterone-treated animals was not significantly different from controls, visual inspection of the in situ hybridization autoradiograms under dark-field microscopy suggested the possibility of a small increase in hybridization density focally located in a region occupied by a subset of tanycytes (α-tanycytes) lining a portion of the lateral walls of the third ventricle in both high-dose corticosterone- and vehicle-ethanol-treated animals (Fig. 6, C and D). Accordingly, the analysis of D2 mRNA hybridization density in the mediobasal hypothalamus was refined to look exclusively at this region in the third ventricle wall, excluding the median eminence (Fig. 7). This analysis showed a significant difference in D2 mRNA in the lateral walls of the third ventricle in the high dose corticosterone-treated animals, compared with both the LPS- and saline-treated groups [Saline vs. High Cort vs. LPS (integrated density units): 24 ± 5 vs. 185 ± 32 vs. 314 ± 42, *, P < 0.05] but similar to the vehicle-treated group [Vehicle vs. High Cort (integrated density units): 146 ± 69 vs. 185 ± 32] (Fig. 7).

Figure 7.

Figure 7

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A, D2 mRNA expression in the lateral walls of the third ventricle. Groups are as shown in Fig. 6. Cort, Corticosterone. B, In situ hybridization autoradiogram of D2 mRNA showing the region (bracketed) analyzed in A. Data represent mean ± sem. *, P < 0.05; ***, P < 0.001.

Discussion

The tanycyte is emerging as an important regulatory component of the hypothalamic-pituitary-thyroid (HPT) axis. In addition to expression of D2 (4,5), an enzyme that converts T4 into the more biologically active thyroid hormone, T3, tanycytes also express the thyroid hormone monocarboxylate 8 transporter, and in some animal species, the thyroid hormone degrading enzyme, type 3 iodothyronine deiodinase (27,28). It has been proposed, therefore, that tanycytes contribute to the regulation of HPT axis by controlling the local concentration of T3 in the mediobasal hypothalamus (3,6,17,29). Contrary to other regions of the central nervous system in which D2 serves primarily to maintain constant tissue levels of T3 (4,30), tanycyte deiodinases may have a broader function that allows T3 to serve as a regulatory signal in the hypothalamus by increasing or decreasing T3 in discrete neuronal populations. In the Japanese quail, for example, an increase in tanycyte D2 in response to long photoperiods is associated with increased T3 content in the mediobasal hypothalamus, resulting in typical, seasonal, reproductive changes observed in this animal species (17). Changes in T3 levels in the mediobasal hypothalamus may also contribute to appetite regulation and body weight (31) and partly explain the hyperphagic responses observed with thyrotoxicosis (32).

Studies by Fekete et al. (12) and Boelen et al. (14) have raised the possibility that D2 in the tanycytes serves an important regulatory role in suppression of the HPT axis associated with chronic illness by increasing hypothalamic tissue levels of T3. Characteristic of this disorder, commonly referred to as the nonthyroidal illness syndrome in man (33), is a fall in the circulating levels of thyroid hormone levels. In contrast to the increase in TRH gene expression in hypophysiotropic neurons in primary hypothyroidism, however, TRH gene expression is suppressed in the hypothalamic paraventricular nucleus after endotoxin administration, consistent with a central hypothyroid state (15,16,34,35). Presumably reduction of the HPT axis under these circumstances is an important adaptive mechanism to reduce energy expenditure and perhaps conserve nitrogen loses by suppressing hypophysiotropic TRH neurons and anterior pituitary thyrotropes until the aversive stimulus has been removed. Mechanisms by which hypophysiotropic TRH neurons could be suppressed by increased tanycyte T3 production include feedback inhibition by retrograde accumulation of T3 into TRH neurons from their axon terminals in the external zone of the median eminence, direct uptake from T3 from the CSF in the third ventricle (36), or indirect effects of T3 on neurons in the hypothalamic arcuate nucleus that send inhibitory, monosynaptic projections to hypophysiotropic TRH neurons in the paraventricular nucleus (6,37,38). The mechanisms whereby D2 is activated by endotoxin in tanycytes, however, are not understood.

Recent studies by Diano et al. (39) and Coppola et al. (23) have demonstrated that D2 mRNA and enzymatic activity increase in the mediobasal hypothalamus in association with fasting and that elevated glucocorticoid levels are crucial to activate D2 under this condition. Namely, the fasting-associated increase in mediobasal D2 activity is prevented in adrenalectomized animals and the response could be restored to normal in these animals by glucocorticoid replacement but not leptin or thyroid hormone administration (23). Other evidence for an effect of glucocorticoids on D2 activation has been given by Darras et al. (40,41), showing that glucocorticoids increase D2 mRNA and enzymatic activity in regions of the embryonic chicken brain and adult amphibian axolotl brain. Because LPS induces a marked activation of the hypothalamic-pituitary-adrenal axis (19,20,25,42), we determined whether the associated rise in circulating corticosterone similarly contributes to increased D2 activity in tanycytes.

As previously recognized (12), endotoxin resulted in a pronounced but gradual increase in tanycyte D2 mRNA expression in both the cell perikarya lining the walls and floor of the third ventricle and in their processes, particularly those extending through the substance of the median eminence and terminating in the external zone. The peak response occurred between 6 and 9 h after LPS administration. This response correlates with the reduction in TRH mRNA in the paraventricular nucleus and TSHβ mRNA in the pituitary observed by Kondo et al. (16) and Boelen et al. (14) after endotoxin administration, supporting the hypothesis that tanycyte D2 activation can contribute to central hypothyroidism.

Despite the importance of glucocorticoids in increasing mediobasal hypothalamic D2 activity in fasting animals (39), no significant effects of glucocorticoids on the increase in D2 gene expression were apparent in this study after endotoxin administration. Adrenalectomized animals clamped with 30% corticosterone pellets that achieved circulating corticosterone levels comparable with that of normal animals did not show a significant difference in their tanycyte D2 response to endotoxin when compared with intact animals. Namely, by quantitative image analysis of the in situ hybridization autoradiograms, D2 mRNA similarly increased approximately 3-fold in both intact animals and adrenalectomized animals replaced with corticosterone. Furthermore, exogenous administration of glucocorticoids that achieved circulating levels of corticosterone simulating the maximal rise observed after LPS administration (16) also had no significant effect on D2 gene expression in the mediobasal hypothalamus.

These studies suggest that endotoxin-induced D2 activation in the mediobasal hypothalamus is independent of changes in levels of circulating corticosterone and that other factors must be of primary importance for this response. Recent studies in our laboratories have focused on the potential role of proinflammatory cytokines, either induced locally by LPS or present in the circulation, as a major cause for D2 activation in tanycytes (12,43). Tanycytes express TNF type 1 receptor (p55) mRNA in response to endotoxin (44) and TNFα has been shown to increase D2 activity in GH3 cells (45). In addition, nuclear factor-κB, which plays a key role in the signaling pathway of TNFα, binds to a specific locus in the promoter of the human D2 gene (dio2) (43). Indeed, cotransfection of p65, an essential component of the nuclear factor-κB complex, together with a human dio2 promoter 5′ flanking region-luciferase construct, results in an approximately 150-fold increase of hdio2 promoter activity (12), supporting the potential importance of this signaling pathway in D2 activation.

Careful inspection of the in situ hybridization autoradiograms in the high-dose corticosterone-treated animals, however, suggested the possibility of a small increase in D2 mRNA in the lateral walls of the third ventricle in the region occupied by α-tanycytes, although significantly less than that observed in LPS-treated animals. This region was previously shown by Diano et al. (39) to be the most sensitive to the effects of fasting-induced D2 up-regulation, compared with that in other regions of the mediobasal hypothalamus (39). Indeed, by image analysis, a statistically significant increase in D2 mRNA was observed in this discrete region of the third ventricle in the high-dose corticosterone-treated animals, compared with saline controls, suggesting a tissue-specific effect of high-dose corticosterone on a subset of tanycytes. Nevertheless, a similar increase in D2 mRNA in this region was also seen in the vehicle-treated control group that contains ethanol.

The role of pharmacologically relevant concentrations of ethanol in the activation of several hypothalamic genes, in particular those from the paraventricular nucleus and the arcuate-median eminence region, is well established (46,47,48,49). Because ethanol is known to activate adenylyl cyclase and D2 activity can be induced by substances that increase cAMP (50) via a cAMP response element in the promoter of the D2 gene (51), one might conclude that the effect of high-dose corticosterone administration on α-tanycytes was nonspecific and due to ethanol. However, circulating corticosterone levels in the vehicle-treated animals were intermediate between that observed in saline-treated controls and LPS-treated animals, suggesting that ethanol administration results in mild stress response. Therefore, we cannot exclude with certainty that a subset of α-tanycytes expressing D2 are regulated by corticosterone. Relatively little is currently known about the physiology of α1-tanycytes, but it can be distinguished from other tanycyte subtypes by its projections to the hypothalamic ventromedial nucleus (9). Similar to β-tanycytes, however, α1-tanycytes are capable of transporting substances extracted from the third ventricle to its basal processes (52).

We conclude that the LPS-induced increase in D2 gene expression in the mediobasal hypothalamus is generally not mediated by the associated increase in glucocorticoids, although a modest effect of glucocorticoids on a subset of tanycytes in the lateral walls of the third ventricle may occur. Other mechanisms, such as an increase in proinflammatory cytokines, may be of primary importance in the D2 response to LPS.

Acknowledgments

The authors thank Dr. Edouard Vannier and Thaddeus John Unger for their valuable technical assistance for the IL-6 and corticosterone ELISA.

Footnotes

This work was supported by Grant DK37021 from the National Institutes of Health.

Disclosure Statement: The authors have nothing to disclose.

First Published Online January 24, 2008

Abbreviations: BW, Body weight; CSF, cerebrospinal fluid; D2, type 2 iodothyronine deiodinase; HPT, hypothalamic-pituitary-thyroid; LPS, lipopolysaccharide.

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