Inverse Shift in Circulating Corticosterone and Leptin Levels Elevates Hypothalamic Deiodinase Type 2 in Fasted Rats
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内分泌学杂志 2005年第6期
Department of Obstetrics and Gynecology and Reproductive Sciences, Yale University School of Medicine (A.C., S.D.), New Haven, Connecticut 06510; and Department of Experimental Pharmacology, University of Naples Federico II (R.M.), 80131 Naples, Italy
Address all correspondence and requests for reprints to: Dr. Sabrina Diano, Department of Obstetrics and Gynecology and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, FMB 339, New Haven, Connecticut 06510. E-mail: sabrina.diano@yale.edu.
Abstract
During food deprivation, plasma T4 and T3 levels are decreased. Under this metabolic condition, hypothalamic deiodinase type 2 (D2) activity and mRNA levels are elevated, whereas TRH mRNA levels are suppressed. Systemic T4 administration does not reverse these hypothalamic changes. The mechanism(s) that underlies this paradoxical regulation of D2 during fasting is unknown. We hypothesize that leptin and/or glucocorticoids play a role in these mechanisms, and their interactions may be an important regulator of the hypothalamic-pituitary-thyroid axis. Thus, we assessed the effects of these hormones on D2 activity levels of food-deprived as well as fed animals using enzyme activity measurements. In food-deprived animals, corticosterone replacement reversed the inhibitory effect of adrenalectomy (ADX) on D2 induction, whereas ADX and ADX plus corticosterone replacement did not significantly affect D2 activity levels in rats fed ad libitum. Leptin administration to fed animals did not change D2 activity, whereas in fasted rats, leptin decreased D2 activity by reducing corticosterone plasma levels. When leptin was administered to fasted animals that were either ADX or ADX plus corticosterone treated at a high dose, D2 activity did not increase. Our results show that during fasting, diminishing leptin levels play a permissive role to enable glucocorticoid-induced up-regulation of D2. Thus, our observations suggest that appropriate induction of D2 activity during negative energy balance is dependent upon both leptin and glucocorticoid signaling.
Introduction
T4, PRODUCED BY THE thyroid gland, is considered to be the inactive form of the thyroid hormone. Its activation to T3 is required for it to function peripherally as well as centrally. Deiodination represents the most important pathway for the activation as well as the deactivation of thyroid hormone. To date, three deiodinases have been cloned and studied (1, 2, 3, 4, 5, 6, 7). Deiodinase type 1 (D1) is the only enzyme that is capable of deiodinating T4 in both its outer as well as inner rings (for review, see Ref. 8). It is widely expressed mainly in peripheral tissues, such as the thyroid gland, liver, and kidney.
Deiodinase type 2 (D2) is responsible for outer ring deiodination, transforming its preferred substrate, T4, to T3. This activating enzyme is localized in peripheral tissues, such as brown adipose tissue, as well as in the central nervous system and pituitary (9, 10, 11, 12, 13). Centrally, D2 has been found to be highly expressed in the hypothalamus (14, 15, 16), specifically in the arcuate nucleus and the region surrounding the third ventricle. Most recently, we demonstrated (17) that the cell types that produce D2 are glial cells, astrocytes, and tanycytes. Finally, deiodinase type 3 (D3) is capable of deiodinating only the inner ring, converting its preferred substrate, T3, to 3,3'T2 and therefore is considered to be an inactivating enzyme. Like D2, D3 is localized peripherally as well as centrally (18, 19, 20, 21). In the central nervous system, D3 is mainly expressed in the cerebral cortex and, unlike D2, is only expressed in neuronal cells (21).
Deiodinases are regulated by circulating thyroid hormone levels (8). In particular, D2 mRNA and activity are increased when circulating T4 levels are decreased and vice versa. In our previous study we found that during food deprivation, D2 mRNA and activity are elevated in the hypothalamus (22). As expected, this increase coincides with a decrease in the plasma levels of T4 as well as T3. When T4 was replaced in these fasted animals, surprisingly we found that D2 mRNA and activity levels were not restored to lower levels. This finding suggested that a signal, other than that from the thyroid, is responsible for this differential regulation of D2.
During food deprivation, circulating levels of the adrenal hormone, corticosterone, rise while levels of adipose-derived leptin decrease. Leptin replacement in fasted rats reverses the rise in corticosterone levels (23) as well as the effect of fasting on TRH (24). Thus, we hypothesized a relationship between changing corticosterone and leptin levels in the up-regulation of hypothalamic D2 during fasting.
Materials and Methods
Animal treatments
Seventy Sprague Dawley male rats from Taconic Farms, Inc. (Germantown, NY; 200–250 g) were used in this study. All procedures were approved by the institutional animal care and use committee of Yale University.
Animals were divided into 14 experimental groups (n = 5 for each group): group 1, intact rats with food ad libitum; group 2, sham-operated rats with food ad libitum; group 3, adrenalectomized (ADX), ad libitum-fed rats; group 4, ADX, low corticosterone-treated ad libitum-fed rats; group 5, ADX, high corticosterone-treated, ad libitum-fed rats; group 6, leptin-treated, ad libitum-fed rats (L); group 7, intact rats fasted for 2 d (48 h); group 8, 2-d fasted, sham-operated rats; group 9, 2-d fasted and ADX rats; group 10, 2-d fasted, ADX, and low corticosterone-treated rats (ADX+C); group 11, 2-d fasted, ADX, and high corticosterone-treated rats [ADX+C(h)]; group 12, 2-d fasted and leptin-treated rats (L); group 13, 2-d fasted, high corticosterone and leptin-treated rats [L+C(h)]; and group 14, 2-d fasted, ADX, and leptin-treated rats. Bilateral adrenalectomy (ADX) or a sham operation was performed under general anesthesia 1 wk before starting the experiments. Immediately after surgery, ADX rats received drinking water containing 0.9% (wt/vol) NaCl.
Corticosterone treatments
Corticosterone treatment was initiated at the time of surgery. Corticosterone (low dose, 0.2 mg/ml; high dose, 0.6 mg/ml; Sigma-Aldrich Corp., St. Louis, MO) (25) was dissolved in ethanol, and this solution was added to the water, yielding a final concentration of 2% ethanol. All of the other groups not treated with corticosterone were given similar drinking water without the hormone. To confirm that the daily water, and thus corticosterone intake, reached the estimated amount and to ensure that the corticosterone supplementation did not alter drinking, we measured water consumption over 24 h.
Leptin treatment
At the time of initiation of food deprivation (1 wk after ADX), all groups were implanted sc with Alzet micro-osmotic pumps (1 μl/h, 3 d; model 1003D, Alza Corp., Palo Alto, CA) filled with either PBS (as control in groups 1–5 and 7–11) or 500 ng/μl leptin (groups 6 and 12–14; recombinant rat leptin; PeproTech, Inc., Rocky Hill, NJ).
D2 assay
The animals were killed, and the mediobasal hypothalamus (defined rostrally by the optic chiasma, caudally by the mamillary bodies, laterally by the optic tract, and superiorly by the apex of the third ventricle) was dissected and stored at –80 C before D2 activity measurements. D2 activity was measured based on the release of radioiodide from the 125I-labeled substrate.
Tissue homogenates were prepared in 0.25 mM sucrose and 20 nM Tris-HCl, pH 7.6, containing 5 mM dithiothreitol and 1.2 mM EDTA (26). The substrate [125I]T4 was obtained from PerkinElmer Life Sciences (Boston MA; specific activity, 1000 μCi/μg) and purified by chromatography using Sephadex LH-20 (Sigma-Aldrich Corp.) before use (26).
Using 100,000 cpm L-[125I]T4 for each sample, incubation mixtures containing 100 μg tissue homogenate in 0.1 M potassium phosphate buffer (pH 7.0), 1 mM EDTA in the presence of 5 nM T4, 30 mM dithiothreitol, 1 mM propylthiouracil (a D1 inhibitor), and 1 μM T3 (to inhibit the inner ring deiodination of T4 due to the presence of D3 activity) were incubated in duplicate at 37 C for 1 h. The reactions were stopped by the addition of 50 μl ice-cold 5% BSA, followed by 200 μl 20% ice-cold trichloroacetic acid and were centrifuged at 4000 x g for 20 min. The supernatant was further purified by cation exchange chromatography on 1.6 ml Dowex 50 W-X2 (100–200 μm pore size mesh; Sigma-Aldrich Corp.). The iodide was then eluted twice with 1 ml 10% glacial acetic acid and counted in a -counter. Enzymatic deiodination was corrected for nonenzymatic 125I production, as determined in blank incubations without homogenates, and multiplied by 2 to account for the random labeling and deiodination for the 3' and 5' positions in labeled T4. Enzymatic activity is expressed as femtomoles of 125I released per hour per milligram of protein (26).
For the determination of proteins, the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL) was employed.
Hormone measurements
Plasma total T4 (TT4), free T4 (FT4), T3, and T4-binding capacity (TBC) were measured with a microparticle enzyme immunoassay, using the AxSym System at Yale-New Haven Hospital (22). Plasma leptin and corticosterone were measured using RIA kits [from Linco Research, Inc. (St. Charles, MO) for leptin and from Diagnostic Products Corp. (Los Angeles, CA) for corticosterone] according to the manufacturers’ protocols. Due to the high number of samples measured in duplicate, assays conducted in our laboratory were run at different times. However, to ensure the consistency of the results, samples from fed and fasted intact rats were always included in every assay as a control.
Statistical analysis
Homogeneity of variance was performed for each group using the F test. For groups that showed heterogeneity, statistical analysis was performed on log-transformed values on the base 10 scale, but untransformed values are given in tables. Means were compared between experimental groups using one-way ANOVA, with mean comparisons by the Student-Newman-Keuls method. A level of confidence of P < 0.05 was used to determine significant differences.
Results
Hormone measurements
Plasma levels of corticosterone, leptin, TT4, FT4, T3, and TBC are shown in Table 1 for fasted animals and in Table 2 for ad libitum-fed rats.
TABLE 1. Effects of ADX, corticosterone replacement at low dose (ADX+C; 0.2 mg/ml in drinking water) or high dose (ADX+C(h); 0.6 mg/ml in drinking water), and/or leptin administration (L; 500 ng/μl leptin in PBS in Alzet microosmotic pumps, 1 μl/h, 3 d) on hormone levels of food-deprived animals
TABLE 2. Effects of ADX, ADX plus corticosterone replacement at low dose (ADX+C; 0.2 mg/ml in drinking water) or high dose [ADX+C(h); 0.6 mg/ml in drinking water], and leptin administration (L; 500 ng/μl leptin in Alzet microosmotic pumps 1 μl/h, 3 d) on hormone levels of ad libitum-fed animals
The 48-h fast increased corticosterone levels in the intact rats (see Table 1) compared with fed controls (see Table 2). ADX with or without leptin replacement in fasted rats resulted in undetectable levels of corticosterone in their plasma (Table 1). Corticosterone administration at low doses (ADX+C) to fasted rats significantly increased circulating corticosterone to physiological levels (not significantly different from levels in intact fed rats in Table 2). The levels of corticosterone in fasted ADX+C(h) and L+C(h) rats were comparable to the levels in intact fasted rats. Leptin administration to fasted animals reduced corticosterone to the levels in intact fed rats.
In ad libitum-fed animals (Table 2), ADX was successfully performed, as shown by the undetectable levels of corticosterone. Corticosterone replacement to physiological levels was conducted successfully, as was corticosterone replacement to fasted levels. Leptin administration to fed animals reduced corticosterone, as previously reported (23).
Fasting induced a significant decrease in plasma leptin levels (Table 1) compared with ad libitum-fed rats (Table 2). ADX of fed animals significantly decreased leptin levels (Table 2), although they were statistically higher than the leptin levels in fasted rats. Corticosterone administration at low and high doses increased leptin levels in fed ADX animals (Table 2). Only high doses of corticosterone significantly increased plasma leptin (Table 1). Leptin levels in fasted ADX+L and L groups (Table 1) were comparable to those in the intact fed group (Table 2).
Total T4 as well as free T4 levels in fasted intact rats were significantly lower compared with those in fed intact animals. Moreover, none of the treatments in any of the fasted groups could elevate TT4 and FT4 levels to those found in fed intact rats.
Fasted rats with high levels of circulating corticosterone [intact, ADX+C(h), and L+C(h)] showed significantly lower levels of TT4 compared with animals with no detectable or just physiological levels of corticosterone (Table 1). In contrast, no statistically significant values were observed in the circulating levels of free T4 between any of the groups of fasted rats.
In fed animals, TT4 as well as FT4 showed a significant decrease in the group treated with the high dose of corticosterone compared with all other groups with undetectable, low, or physiological levels of corticosterone (Table 2).
T3 levels in fasted animals also showed significant decreases compared with fed rats. In fasted animals, plasma T3 levels in ADX+C(h) and leptin-treated rats were similar to levels in intact fed rats. In addition, ADX rats treated with corticosterone to physiological levels showed an increase in circulating T3 compared with intact fasted rats. This increase was significantly lower than that observed in leptin-treated and ADX+C(h) groups (Table 1).
Fasted rats showed a TBC that, although slightly higher, was not significantly different from that observed in fed animals. Moreover, undetectable levels of corticosterone resulted in a significant increase in TBC in both fasted and fed animals, although ADX fed rats with physiological levels of corticosterone showed an elevated TBC compared with the intact fed group.
D2 enzymatic activity
As previously reported by our group (22), fasting in intact animals (94.33 ± 2.69 fmol/mg protein·h) increased D2 enzymatic activity compared with ad libitum-fed rats (80.5 ± 4.95 fmol/mg protein·h; Fig. 1).
FIG. 1. The bar graph shows the results of hypothalamic D2 activity expressed in femtomoles of iodide per hour per milligram of protein in intact ad libitum-fed rats (n = 5) and intact fasted rats (n = 5). Using one-way ANOVA, results are expressed as the mean ± SEM. *, P < 0.05 compared with fed intact rats.
In ad libitum-fed rats, D2 activity (Fig. 2A) in intact and sham-operated animals (80.5 ± 4.95 and 78.76 ± 1.23 fmol/mg protein·h, respectively) did not show any significant changes compared with values in ADX rats (86.39 ± 0.99 fmol/mg protein·h) or ADX rats treated with corticosterone at either low (85.45 ± 1.87 fmol/mg protein·h) or high (85.63 ± 3.08 fmol/mg protein·h) doses.
FIG. 2. A, Bar graph showing hypothalamic D2 activity measurements in all ad libitum-fed rats (n = 5 for each group) either left intact or sham operated or in ADX+C or ADX+C(h) rats. No significant differences were observed between these groups. B, Bar graph showing hypothalamic D2 activity measurements in all fasted animals. Results are expressed as mean ± SEM. *, P < 0.05 compared with intact, sham, and ADX+C(h). #, P < 0.05 compared with ADX rats.
In contrast, in fasted animals (Fig 2B), ADX significantly reduced D2 activity (78.67 ± 3.35 fmol/mg protein·h) compared with intact and sham-operated rats (94.33 ± 2.69 and 98.33 ± 2.87 fmol/mg protein·h, respectively). When corticosterone was replaced in ADX fasted rats, D2 activity showed a dose-dependent increase, with a greater elevation at the high dose of corticosterone, that mimics the serum level in intact fasted rats (88.25 ± 0.87 and 96.27 ± 1.46 fmol/mg protein·h for low and high dose groups, respectively). Indeed, at the high dose of corticosterone, D2 activity did not significantly differ from that in intact fasted animals (96.27 ± 1.46 fmol/mg protein·h in corticosterone-treated rats compared with 94.33 ± 2.69 fmol/mg protein·h in intact rats).
This difference in the effect of corticosterone on D2 activity in fed compared with fasted rats led us to analyze the consequences of leptin administration in the same paradigm (Fig. 3). When leptin was administered to fed animals, no changes were observed between this group and their control fed rats (80.5 ± 4.95 and 78.42 ± 4.21 fmol/mg protein·h). In contrast, leptin administration to fasted animals decreased D2 activity (76.52 ± 1.61 fmol/mg protein·h) compared with their control intact fasted rats (94.33 ± 2.69 fmol/mg protein·h; Fig. 4) and reduced D2 activity to the levels of fed control rats (80.5 ± 4.95 fmol/mg protein·h; Fig. 3). Leptin treatment of fasted rats also reduced plasma corticosterone levels, supporting the idea that during negative energy balance the decrease in plasma leptin and the simultaneous increase in corticosterone levels are necessary for the elevation of D2 activity.
FIG. 3. Graph showing hypothalamic D2 activity measurements in intact ad libitum-fed rats (n = 5), intact fed+L (n = 5), intact fasted animals (n = 5), and intact fasted+L (n = 5). Note that although leptin treatment of fed animals does not affect hypothalamic D2 activity, leptin replacement of fasted rats induces a significant decrease compared with intact fasted rats in D2 measurement, suggesting a critical role for leptin in D2 activity during negative energy balance. Results are expressed as the mean ± SEM. *, P < 0.05 compared with intact fed, intact fed+L, and intact fasted+L animals.
FIG. 4. Graph showing hypothalamic D2 activity measurements in all fasted animals. Intact and ADX+C(h) rats show a statistically significant elevation of D2 activity (*, P < 0.05) compared with intact+L, ADX, ADX+L, and L+C(h). Results are expressed as the mean ± SEM.
We examined the effects of administration of leptin, corticosterone, or both on D2 activity of fasted rats (Fig. 4) and compared the following groups: intact fasted, intact fasted plus leptin, ADX fasted, ADX fasted plus leptin, and ADX fasted plus high corticosterone levels. As described above, leptin administration to fasted animals restored D2 activity to the level in intact fed rats. ADX rats fasted for 48 h and ADX fasted rats treated with leptin did not show any differences in D2 activity compared with the intact rats given leptin. When we administered high doses of corticosterone to fasted ADX rats, D2 activity was statistically elevated to the levels of intact fasted rats, and when the animals with C at high dose were treated with leptin to physiological levels, we did not observe the increase in D2 activity that was seen without leptin treatment (Fig. 4).
Discussion
The results of our study demonstrate that inverse changes in circulating levels of the stress hormone, corticosterone, and the metabolic signal, leptin, are critical triggers for increased D2 activity in the hypothalamus during food deprivation. In fasted rats, ADX significantly reduces hypothalamic D2 activity to the level of animals given food ad libitum. Conversely, corticosterone administration to ADX fasted rats increases D2 activity in a dose-dependent manner. When leptin was administered to fasted rats, D2 activity was reduced to the level of intact fed animals. A comparison of plasma leptin and corticosterone levels showed, in fact, that the leptin-treated fasted group had hormone values similar to intact fed animals. Moreover, leptin administration to ADX fasted rats with or without corticosterone replacement showed no changes in the levels of D2 compared with fasted ADX animals, indicating that the decrease in plasma leptin allows a corticosterone-dependent increase in D2 activity during fasting.
In addition, our study shows that the elevation of circulating glucocorticoids induces a reduction in plasma T3 levels. In fact, intact as well as L+C(h) fasted rats show a significant decrease in circulating T3 compared with intact fed animals. These data are in agreement with previous reports (27, 28) showing that glucocorticoids induced a decrease in extrathyroidal T3 production by lowering deiodinase type 1 (D1) activity in the liver. In contrast, in our study, fasted ADX animals with physiological and high corticosterone levels showed an increase in plasma T3 compared with fasted rats. This may indicate a stimulatory effect of glucocorticoids on peripheral deiodinase activity similar to that reported in a study by Davies and collaborators (29), in which they showed an increase in D1 mRNA and activity after dexamethasone treatment using cultured rat liver and kidney cells. These contradictory data on the effect of glucocorticoids on D1 activity need additional examination, especially in light of more recent reports that show an important stimulatory effect of leptin on peripheral (thyroid gland and liver) D1 and D2 (brown adipose tissue) activities (30, 31). Therefore, the possibility cannot be excluded that interaction between leptin and corticosterone is important for the regulation of peripheral D1 as well as D2 activities.
Food deprivation is known to decrease thyroid function. During fasting, low levels of circulating T4 and T3 coincide with suppressed hypothalamic TRH production and release (25, 32). We previously showed that during food deprivation, D2 mRNA and activity are elevated in the arcuate nucleus-median eminence region of the hypothalamus, which heavily projects to neuroendocrine TRH cells in the paraventricular nucleus of the hypothalamus (16, 22). These elevations in D2 production and activity were not affected by T4 administration, implying that a signal, originating from a site other than the thyroid gland, is responsible for D2 elevations, and the suppressed circulating levels of thyroid hormone are not the cause but, rather, are the result of the suppressed TRH production and release. Similar conclusions were drawn from the work of van Haasteren and collaborators (25), in which they showed that the starvation-induced increase in corticosterone secretion is part of the mechanism responsible for the reduction in TRH synthesis and release during food deprivation. When they replaced corticosterone to physiological levels in ADX fasted rats, they could prevent pro-TRH mRNA reduction by food deprivation in sham-operated rats (25). Our results show that in fasted ADX rats, when corticosterone is replaced to physiological levels, D2 activity is significantly lower than levels in fasted intact, fasted sham-operated, as well as ADX fasted rats with high dose corticosterone replacement. Although an indirect effect of corticosterone on TRH levels via D2 activation might be hypothesized, there is direct evidence that glucocorticoids affect TRH synthesis through the presence of glucocorticoid receptors on TRH-producing cells (33) and the existence of the glucocorticoid-responsive element in the promoter region of the pro-TRH gene (34).
The present results clearly indicate that circulating leptin and corticosterone levels play a key regulatory role in D2 activity in fasted animals. It is also clear, however, that neither of these hormones is critical for baseline expression of D2 activity, because neither of them appeared to affect hypothalamic D2 levels in fed animals.
When leptin is administered to fasted animals, it induces an increase in TRH mRNA to the level in intact animals (24). Therefore, it has been suggested that leptin may directly act on TRH neurons, because the long form of leptin receptors is widely distributed in the hypothalamus and has been found to be expressed in TRH cells (35). However, we propose that leptin-restored TRH mRNA levels in the hypothalamic paraventricular nucleus might be due, at least in part, to its effect in lowering corticosterone levels and, consequently, D2 activity in the arcuate nucleus/median eminence region. The lower D2 activity might, in turn, affect the synthesis and release of arcuate nucleus neuropeptides, such as neuropeptide Y (NPY), agouti-related protein (AgRP), and -MSH that heavily project onto TRH neurons in the paraventricular nucleus. These neuropeptides exert opposite effects on TRH neurons: NPY and AgRP (that are colocalized in the arcuate nucleus) have an inhibitory function (36), whereas -MSH acts as a stimulatory peptide on TRH neurons (37).
In addition to leptin and corticosterone, other peripheral signals may play a role in regulating D2 activity during negative energy balance. For example, ghrelin as well as insulin are two possible candidates, the first being up-regulated, and the second being down-regulated during negative energy homeostasis. Moreover, other hypothalamic peptides may be involved in the regulation of D2 activity, such as hypocretin/orexin, which, like the NPY/AgRP system, is stimulated by food intake (38), glucocorticoids (39), and ghrelin (40). However, opposite effects occur in response to insulin, which increases hypocretin/orexin mRNA while inhibiting that of NPY/AgRP (41, 42). Therefore, additional investigation into the roles of these hormones and peptides on D2 activity is warranted.
The exact manner by which leptin and glucocorticoids act on glial cells producing D2 requires additional study. There is evidence that glucocorticoids directly target glial cells. Indeed, glucocorticoid receptors have been found to be localized in glial cells as well as in neurons with an intense immunoreactivity in the hypothalamus (43). In addition, we and others (44, 45, 46) have found that leptin receptors are abundantly expressed in the hypothalamus in both neuronal and glial elements.
In conclusion, our study reveals the existence of a differential regulation of D2 activity during negative energy metabolism vs. normal metabolic conditions. Although D2 activity does not seem to be affected by either leptin or corticosterone in the normal metabolic state, such as in ad libitum-fed animals, during food deprivation, changes in glucocorticoids and leptin induce an elevation in D2 activity in the hypothalamus. Specifically, this D2 elevation seems to be determined by the inverse shift in circulating leptin and corticosterone (low and high, respectively). In support of this, leptin administration to fasted rats treated with high corticosterone levels did not induce the increase in D2 activity seen in ADX fasted rats treated with corticosterone alone. In addition, leptin administration to fasted rats prevented the increase in D2 activity, although this is probably due to the effect of leptin to reduce plasma corticosterone to the levels of fed animals. This indicates that the diminished plasma leptin resulting from food deprivation plays a permissive role by allowing the increased corticosterone levels to elevate D2 activity.
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Address all correspondence and requests for reprints to: Dr. Sabrina Diano, Department of Obstetrics and Gynecology and Reproductive Sciences, Yale University School of Medicine, 333 Cedar Street, FMB 339, New Haven, Connecticut 06510. E-mail: sabrina.diano@yale.edu.
Abstract
During food deprivation, plasma T4 and T3 levels are decreased. Under this metabolic condition, hypothalamic deiodinase type 2 (D2) activity and mRNA levels are elevated, whereas TRH mRNA levels are suppressed. Systemic T4 administration does not reverse these hypothalamic changes. The mechanism(s) that underlies this paradoxical regulation of D2 during fasting is unknown. We hypothesize that leptin and/or glucocorticoids play a role in these mechanisms, and their interactions may be an important regulator of the hypothalamic-pituitary-thyroid axis. Thus, we assessed the effects of these hormones on D2 activity levels of food-deprived as well as fed animals using enzyme activity measurements. In food-deprived animals, corticosterone replacement reversed the inhibitory effect of adrenalectomy (ADX) on D2 induction, whereas ADX and ADX plus corticosterone replacement did not significantly affect D2 activity levels in rats fed ad libitum. Leptin administration to fed animals did not change D2 activity, whereas in fasted rats, leptin decreased D2 activity by reducing corticosterone plasma levels. When leptin was administered to fasted animals that were either ADX or ADX plus corticosterone treated at a high dose, D2 activity did not increase. Our results show that during fasting, diminishing leptin levels play a permissive role to enable glucocorticoid-induced up-regulation of D2. Thus, our observations suggest that appropriate induction of D2 activity during negative energy balance is dependent upon both leptin and glucocorticoid signaling.
Introduction
T4, PRODUCED BY THE thyroid gland, is considered to be the inactive form of the thyroid hormone. Its activation to T3 is required for it to function peripherally as well as centrally. Deiodination represents the most important pathway for the activation as well as the deactivation of thyroid hormone. To date, three deiodinases have been cloned and studied (1, 2, 3, 4, 5, 6, 7). Deiodinase type 1 (D1) is the only enzyme that is capable of deiodinating T4 in both its outer as well as inner rings (for review, see Ref. 8). It is widely expressed mainly in peripheral tissues, such as the thyroid gland, liver, and kidney.
Deiodinase type 2 (D2) is responsible for outer ring deiodination, transforming its preferred substrate, T4, to T3. This activating enzyme is localized in peripheral tissues, such as brown adipose tissue, as well as in the central nervous system and pituitary (9, 10, 11, 12, 13). Centrally, D2 has been found to be highly expressed in the hypothalamus (14, 15, 16), specifically in the arcuate nucleus and the region surrounding the third ventricle. Most recently, we demonstrated (17) that the cell types that produce D2 are glial cells, astrocytes, and tanycytes. Finally, deiodinase type 3 (D3) is capable of deiodinating only the inner ring, converting its preferred substrate, T3, to 3,3'T2 and therefore is considered to be an inactivating enzyme. Like D2, D3 is localized peripherally as well as centrally (18, 19, 20, 21). In the central nervous system, D3 is mainly expressed in the cerebral cortex and, unlike D2, is only expressed in neuronal cells (21).
Deiodinases are regulated by circulating thyroid hormone levels (8). In particular, D2 mRNA and activity are increased when circulating T4 levels are decreased and vice versa. In our previous study we found that during food deprivation, D2 mRNA and activity are elevated in the hypothalamus (22). As expected, this increase coincides with a decrease in the plasma levels of T4 as well as T3. When T4 was replaced in these fasted animals, surprisingly we found that D2 mRNA and activity levels were not restored to lower levels. This finding suggested that a signal, other than that from the thyroid, is responsible for this differential regulation of D2.
During food deprivation, circulating levels of the adrenal hormone, corticosterone, rise while levels of adipose-derived leptin decrease. Leptin replacement in fasted rats reverses the rise in corticosterone levels (23) as well as the effect of fasting on TRH (24). Thus, we hypothesized a relationship between changing corticosterone and leptin levels in the up-regulation of hypothalamic D2 during fasting.
Materials and Methods
Animal treatments
Seventy Sprague Dawley male rats from Taconic Farms, Inc. (Germantown, NY; 200–250 g) were used in this study. All procedures were approved by the institutional animal care and use committee of Yale University.
Animals were divided into 14 experimental groups (n = 5 for each group): group 1, intact rats with food ad libitum; group 2, sham-operated rats with food ad libitum; group 3, adrenalectomized (ADX), ad libitum-fed rats; group 4, ADX, low corticosterone-treated ad libitum-fed rats; group 5, ADX, high corticosterone-treated, ad libitum-fed rats; group 6, leptin-treated, ad libitum-fed rats (L); group 7, intact rats fasted for 2 d (48 h); group 8, 2-d fasted, sham-operated rats; group 9, 2-d fasted and ADX rats; group 10, 2-d fasted, ADX, and low corticosterone-treated rats (ADX+C); group 11, 2-d fasted, ADX, and high corticosterone-treated rats [ADX+C(h)]; group 12, 2-d fasted and leptin-treated rats (L); group 13, 2-d fasted, high corticosterone and leptin-treated rats [L+C(h)]; and group 14, 2-d fasted, ADX, and leptin-treated rats. Bilateral adrenalectomy (ADX) or a sham operation was performed under general anesthesia 1 wk before starting the experiments. Immediately after surgery, ADX rats received drinking water containing 0.9% (wt/vol) NaCl.
Corticosterone treatments
Corticosterone treatment was initiated at the time of surgery. Corticosterone (low dose, 0.2 mg/ml; high dose, 0.6 mg/ml; Sigma-Aldrich Corp., St. Louis, MO) (25) was dissolved in ethanol, and this solution was added to the water, yielding a final concentration of 2% ethanol. All of the other groups not treated with corticosterone were given similar drinking water without the hormone. To confirm that the daily water, and thus corticosterone intake, reached the estimated amount and to ensure that the corticosterone supplementation did not alter drinking, we measured water consumption over 24 h.
Leptin treatment
At the time of initiation of food deprivation (1 wk after ADX), all groups were implanted sc with Alzet micro-osmotic pumps (1 μl/h, 3 d; model 1003D, Alza Corp., Palo Alto, CA) filled with either PBS (as control in groups 1–5 and 7–11) or 500 ng/μl leptin (groups 6 and 12–14; recombinant rat leptin; PeproTech, Inc., Rocky Hill, NJ).
D2 assay
The animals were killed, and the mediobasal hypothalamus (defined rostrally by the optic chiasma, caudally by the mamillary bodies, laterally by the optic tract, and superiorly by the apex of the third ventricle) was dissected and stored at –80 C before D2 activity measurements. D2 activity was measured based on the release of radioiodide from the 125I-labeled substrate.
Tissue homogenates were prepared in 0.25 mM sucrose and 20 nM Tris-HCl, pH 7.6, containing 5 mM dithiothreitol and 1.2 mM EDTA (26). The substrate [125I]T4 was obtained from PerkinElmer Life Sciences (Boston MA; specific activity, 1000 μCi/μg) and purified by chromatography using Sephadex LH-20 (Sigma-Aldrich Corp.) before use (26).
Using 100,000 cpm L-[125I]T4 for each sample, incubation mixtures containing 100 μg tissue homogenate in 0.1 M potassium phosphate buffer (pH 7.0), 1 mM EDTA in the presence of 5 nM T4, 30 mM dithiothreitol, 1 mM propylthiouracil (a D1 inhibitor), and 1 μM T3 (to inhibit the inner ring deiodination of T4 due to the presence of D3 activity) were incubated in duplicate at 37 C for 1 h. The reactions were stopped by the addition of 50 μl ice-cold 5% BSA, followed by 200 μl 20% ice-cold trichloroacetic acid and were centrifuged at 4000 x g for 20 min. The supernatant was further purified by cation exchange chromatography on 1.6 ml Dowex 50 W-X2 (100–200 μm pore size mesh; Sigma-Aldrich Corp.). The iodide was then eluted twice with 1 ml 10% glacial acetic acid and counted in a -counter. Enzymatic deiodination was corrected for nonenzymatic 125I production, as determined in blank incubations without homogenates, and multiplied by 2 to account for the random labeling and deiodination for the 3' and 5' positions in labeled T4. Enzymatic activity is expressed as femtomoles of 125I released per hour per milligram of protein (26).
For the determination of proteins, the bicinchoninic acid assay (Pierce Chemical Co., Rockford, IL) was employed.
Hormone measurements
Plasma total T4 (TT4), free T4 (FT4), T3, and T4-binding capacity (TBC) were measured with a microparticle enzyme immunoassay, using the AxSym System at Yale-New Haven Hospital (22). Plasma leptin and corticosterone were measured using RIA kits [from Linco Research, Inc. (St. Charles, MO) for leptin and from Diagnostic Products Corp. (Los Angeles, CA) for corticosterone] according to the manufacturers’ protocols. Due to the high number of samples measured in duplicate, assays conducted in our laboratory were run at different times. However, to ensure the consistency of the results, samples from fed and fasted intact rats were always included in every assay as a control.
Statistical analysis
Homogeneity of variance was performed for each group using the F test. For groups that showed heterogeneity, statistical analysis was performed on log-transformed values on the base 10 scale, but untransformed values are given in tables. Means were compared between experimental groups using one-way ANOVA, with mean comparisons by the Student-Newman-Keuls method. A level of confidence of P < 0.05 was used to determine significant differences.
Results
Hormone measurements
Plasma levels of corticosterone, leptin, TT4, FT4, T3, and TBC are shown in Table 1 for fasted animals and in Table 2 for ad libitum-fed rats.
TABLE 1. Effects of ADX, corticosterone replacement at low dose (ADX+C; 0.2 mg/ml in drinking water) or high dose (ADX+C(h); 0.6 mg/ml in drinking water), and/or leptin administration (L; 500 ng/μl leptin in PBS in Alzet microosmotic pumps, 1 μl/h, 3 d) on hormone levels of food-deprived animals
TABLE 2. Effects of ADX, ADX plus corticosterone replacement at low dose (ADX+C; 0.2 mg/ml in drinking water) or high dose [ADX+C(h); 0.6 mg/ml in drinking water], and leptin administration (L; 500 ng/μl leptin in Alzet microosmotic pumps 1 μl/h, 3 d) on hormone levels of ad libitum-fed animals
The 48-h fast increased corticosterone levels in the intact rats (see Table 1) compared with fed controls (see Table 2). ADX with or without leptin replacement in fasted rats resulted in undetectable levels of corticosterone in their plasma (Table 1). Corticosterone administration at low doses (ADX+C) to fasted rats significantly increased circulating corticosterone to physiological levels (not significantly different from levels in intact fed rats in Table 2). The levels of corticosterone in fasted ADX+C(h) and L+C(h) rats were comparable to the levels in intact fasted rats. Leptin administration to fasted animals reduced corticosterone to the levels in intact fed rats.
In ad libitum-fed animals (Table 2), ADX was successfully performed, as shown by the undetectable levels of corticosterone. Corticosterone replacement to physiological levels was conducted successfully, as was corticosterone replacement to fasted levels. Leptin administration to fed animals reduced corticosterone, as previously reported (23).
Fasting induced a significant decrease in plasma leptin levels (Table 1) compared with ad libitum-fed rats (Table 2). ADX of fed animals significantly decreased leptin levels (Table 2), although they were statistically higher than the leptin levels in fasted rats. Corticosterone administration at low and high doses increased leptin levels in fed ADX animals (Table 2). Only high doses of corticosterone significantly increased plasma leptin (Table 1). Leptin levels in fasted ADX+L and L groups (Table 1) were comparable to those in the intact fed group (Table 2).
Total T4 as well as free T4 levels in fasted intact rats were significantly lower compared with those in fed intact animals. Moreover, none of the treatments in any of the fasted groups could elevate TT4 and FT4 levels to those found in fed intact rats.
Fasted rats with high levels of circulating corticosterone [intact, ADX+C(h), and L+C(h)] showed significantly lower levels of TT4 compared with animals with no detectable or just physiological levels of corticosterone (Table 1). In contrast, no statistically significant values were observed in the circulating levels of free T4 between any of the groups of fasted rats.
In fed animals, TT4 as well as FT4 showed a significant decrease in the group treated with the high dose of corticosterone compared with all other groups with undetectable, low, or physiological levels of corticosterone (Table 2).
T3 levels in fasted animals also showed significant decreases compared with fed rats. In fasted animals, plasma T3 levels in ADX+C(h) and leptin-treated rats were similar to levels in intact fed rats. In addition, ADX rats treated with corticosterone to physiological levels showed an increase in circulating T3 compared with intact fasted rats. This increase was significantly lower than that observed in leptin-treated and ADX+C(h) groups (Table 1).
Fasted rats showed a TBC that, although slightly higher, was not significantly different from that observed in fed animals. Moreover, undetectable levels of corticosterone resulted in a significant increase in TBC in both fasted and fed animals, although ADX fed rats with physiological levels of corticosterone showed an elevated TBC compared with the intact fed group.
D2 enzymatic activity
As previously reported by our group (22), fasting in intact animals (94.33 ± 2.69 fmol/mg protein·h) increased D2 enzymatic activity compared with ad libitum-fed rats (80.5 ± 4.95 fmol/mg protein·h; Fig. 1).
FIG. 1. The bar graph shows the results of hypothalamic D2 activity expressed in femtomoles of iodide per hour per milligram of protein in intact ad libitum-fed rats (n = 5) and intact fasted rats (n = 5). Using one-way ANOVA, results are expressed as the mean ± SEM. *, P < 0.05 compared with fed intact rats.
In ad libitum-fed rats, D2 activity (Fig. 2A) in intact and sham-operated animals (80.5 ± 4.95 and 78.76 ± 1.23 fmol/mg protein·h, respectively) did not show any significant changes compared with values in ADX rats (86.39 ± 0.99 fmol/mg protein·h) or ADX rats treated with corticosterone at either low (85.45 ± 1.87 fmol/mg protein·h) or high (85.63 ± 3.08 fmol/mg protein·h) doses.
FIG. 2. A, Bar graph showing hypothalamic D2 activity measurements in all ad libitum-fed rats (n = 5 for each group) either left intact or sham operated or in ADX+C or ADX+C(h) rats. No significant differences were observed between these groups. B, Bar graph showing hypothalamic D2 activity measurements in all fasted animals. Results are expressed as mean ± SEM. *, P < 0.05 compared with intact, sham, and ADX+C(h). #, P < 0.05 compared with ADX rats.
In contrast, in fasted animals (Fig 2B), ADX significantly reduced D2 activity (78.67 ± 3.35 fmol/mg protein·h) compared with intact and sham-operated rats (94.33 ± 2.69 and 98.33 ± 2.87 fmol/mg protein·h, respectively). When corticosterone was replaced in ADX fasted rats, D2 activity showed a dose-dependent increase, with a greater elevation at the high dose of corticosterone, that mimics the serum level in intact fasted rats (88.25 ± 0.87 and 96.27 ± 1.46 fmol/mg protein·h for low and high dose groups, respectively). Indeed, at the high dose of corticosterone, D2 activity did not significantly differ from that in intact fasted animals (96.27 ± 1.46 fmol/mg protein·h in corticosterone-treated rats compared with 94.33 ± 2.69 fmol/mg protein·h in intact rats).
This difference in the effect of corticosterone on D2 activity in fed compared with fasted rats led us to analyze the consequences of leptin administration in the same paradigm (Fig. 3). When leptin was administered to fed animals, no changes were observed between this group and their control fed rats (80.5 ± 4.95 and 78.42 ± 4.21 fmol/mg protein·h). In contrast, leptin administration to fasted animals decreased D2 activity (76.52 ± 1.61 fmol/mg protein·h) compared with their control intact fasted rats (94.33 ± 2.69 fmol/mg protein·h; Fig. 4) and reduced D2 activity to the levels of fed control rats (80.5 ± 4.95 fmol/mg protein·h; Fig. 3). Leptin treatment of fasted rats also reduced plasma corticosterone levels, supporting the idea that during negative energy balance the decrease in plasma leptin and the simultaneous increase in corticosterone levels are necessary for the elevation of D2 activity.
FIG. 3. Graph showing hypothalamic D2 activity measurements in intact ad libitum-fed rats (n = 5), intact fed+L (n = 5), intact fasted animals (n = 5), and intact fasted+L (n = 5). Note that although leptin treatment of fed animals does not affect hypothalamic D2 activity, leptin replacement of fasted rats induces a significant decrease compared with intact fasted rats in D2 measurement, suggesting a critical role for leptin in D2 activity during negative energy balance. Results are expressed as the mean ± SEM. *, P < 0.05 compared with intact fed, intact fed+L, and intact fasted+L animals.
FIG. 4. Graph showing hypothalamic D2 activity measurements in all fasted animals. Intact and ADX+C(h) rats show a statistically significant elevation of D2 activity (*, P < 0.05) compared with intact+L, ADX, ADX+L, and L+C(h). Results are expressed as the mean ± SEM.
We examined the effects of administration of leptin, corticosterone, or both on D2 activity of fasted rats (Fig. 4) and compared the following groups: intact fasted, intact fasted plus leptin, ADX fasted, ADX fasted plus leptin, and ADX fasted plus high corticosterone levels. As described above, leptin administration to fasted animals restored D2 activity to the level in intact fed rats. ADX rats fasted for 48 h and ADX fasted rats treated with leptin did not show any differences in D2 activity compared with the intact rats given leptin. When we administered high doses of corticosterone to fasted ADX rats, D2 activity was statistically elevated to the levels of intact fasted rats, and when the animals with C at high dose were treated with leptin to physiological levels, we did not observe the increase in D2 activity that was seen without leptin treatment (Fig. 4).
Discussion
The results of our study demonstrate that inverse changes in circulating levels of the stress hormone, corticosterone, and the metabolic signal, leptin, are critical triggers for increased D2 activity in the hypothalamus during food deprivation. In fasted rats, ADX significantly reduces hypothalamic D2 activity to the level of animals given food ad libitum. Conversely, corticosterone administration to ADX fasted rats increases D2 activity in a dose-dependent manner. When leptin was administered to fasted rats, D2 activity was reduced to the level of intact fed animals. A comparison of plasma leptin and corticosterone levels showed, in fact, that the leptin-treated fasted group had hormone values similar to intact fed animals. Moreover, leptin administration to ADX fasted rats with or without corticosterone replacement showed no changes in the levels of D2 compared with fasted ADX animals, indicating that the decrease in plasma leptin allows a corticosterone-dependent increase in D2 activity during fasting.
In addition, our study shows that the elevation of circulating glucocorticoids induces a reduction in plasma T3 levels. In fact, intact as well as L+C(h) fasted rats show a significant decrease in circulating T3 compared with intact fed animals. These data are in agreement with previous reports (27, 28) showing that glucocorticoids induced a decrease in extrathyroidal T3 production by lowering deiodinase type 1 (D1) activity in the liver. In contrast, in our study, fasted ADX animals with physiological and high corticosterone levels showed an increase in plasma T3 compared with fasted rats. This may indicate a stimulatory effect of glucocorticoids on peripheral deiodinase activity similar to that reported in a study by Davies and collaborators (29), in which they showed an increase in D1 mRNA and activity after dexamethasone treatment using cultured rat liver and kidney cells. These contradictory data on the effect of glucocorticoids on D1 activity need additional examination, especially in light of more recent reports that show an important stimulatory effect of leptin on peripheral (thyroid gland and liver) D1 and D2 (brown adipose tissue) activities (30, 31). Therefore, the possibility cannot be excluded that interaction between leptin and corticosterone is important for the regulation of peripheral D1 as well as D2 activities.
Food deprivation is known to decrease thyroid function. During fasting, low levels of circulating T4 and T3 coincide with suppressed hypothalamic TRH production and release (25, 32). We previously showed that during food deprivation, D2 mRNA and activity are elevated in the arcuate nucleus-median eminence region of the hypothalamus, which heavily projects to neuroendocrine TRH cells in the paraventricular nucleus of the hypothalamus (16, 22). These elevations in D2 production and activity were not affected by T4 administration, implying that a signal, originating from a site other than the thyroid gland, is responsible for D2 elevations, and the suppressed circulating levels of thyroid hormone are not the cause but, rather, are the result of the suppressed TRH production and release. Similar conclusions were drawn from the work of van Haasteren and collaborators (25), in which they showed that the starvation-induced increase in corticosterone secretion is part of the mechanism responsible for the reduction in TRH synthesis and release during food deprivation. When they replaced corticosterone to physiological levels in ADX fasted rats, they could prevent pro-TRH mRNA reduction by food deprivation in sham-operated rats (25). Our results show that in fasted ADX rats, when corticosterone is replaced to physiological levels, D2 activity is significantly lower than levels in fasted intact, fasted sham-operated, as well as ADX fasted rats with high dose corticosterone replacement. Although an indirect effect of corticosterone on TRH levels via D2 activation might be hypothesized, there is direct evidence that glucocorticoids affect TRH synthesis through the presence of glucocorticoid receptors on TRH-producing cells (33) and the existence of the glucocorticoid-responsive element in the promoter region of the pro-TRH gene (34).
The present results clearly indicate that circulating leptin and corticosterone levels play a key regulatory role in D2 activity in fasted animals. It is also clear, however, that neither of these hormones is critical for baseline expression of D2 activity, because neither of them appeared to affect hypothalamic D2 levels in fed animals.
When leptin is administered to fasted animals, it induces an increase in TRH mRNA to the level in intact animals (24). Therefore, it has been suggested that leptin may directly act on TRH neurons, because the long form of leptin receptors is widely distributed in the hypothalamus and has been found to be expressed in TRH cells (35). However, we propose that leptin-restored TRH mRNA levels in the hypothalamic paraventricular nucleus might be due, at least in part, to its effect in lowering corticosterone levels and, consequently, D2 activity in the arcuate nucleus/median eminence region. The lower D2 activity might, in turn, affect the synthesis and release of arcuate nucleus neuropeptides, such as neuropeptide Y (NPY), agouti-related protein (AgRP), and -MSH that heavily project onto TRH neurons in the paraventricular nucleus. These neuropeptides exert opposite effects on TRH neurons: NPY and AgRP (that are colocalized in the arcuate nucleus) have an inhibitory function (36), whereas -MSH acts as a stimulatory peptide on TRH neurons (37).
In addition to leptin and corticosterone, other peripheral signals may play a role in regulating D2 activity during negative energy balance. For example, ghrelin as well as insulin are two possible candidates, the first being up-regulated, and the second being down-regulated during negative energy homeostasis. Moreover, other hypothalamic peptides may be involved in the regulation of D2 activity, such as hypocretin/orexin, which, like the NPY/AgRP system, is stimulated by food intake (38), glucocorticoids (39), and ghrelin (40). However, opposite effects occur in response to insulin, which increases hypocretin/orexin mRNA while inhibiting that of NPY/AgRP (41, 42). Therefore, additional investigation into the roles of these hormones and peptides on D2 activity is warranted.
The exact manner by which leptin and glucocorticoids act on glial cells producing D2 requires additional study. There is evidence that glucocorticoids directly target glial cells. Indeed, glucocorticoid receptors have been found to be localized in glial cells as well as in neurons with an intense immunoreactivity in the hypothalamus (43). In addition, we and others (44, 45, 46) have found that leptin receptors are abundantly expressed in the hypothalamus in both neuronal and glial elements.
In conclusion, our study reveals the existence of a differential regulation of D2 activity during negative energy metabolism vs. normal metabolic conditions. Although D2 activity does not seem to be affected by either leptin or corticosterone in the normal metabolic state, such as in ad libitum-fed animals, during food deprivation, changes in glucocorticoids and leptin induce an elevation in D2 activity in the hypothalamus. Specifically, this D2 elevation seems to be determined by the inverse shift in circulating leptin and corticosterone (low and high, respectively). In support of this, leptin administration to fasted rats treated with high corticosterone levels did not induce the increase in D2 activity seen in ADX fasted rats treated with corticosterone alone. In addition, leptin administration to fasted rats prevented the increase in D2 activity, although this is probably due to the effect of leptin to reduce plasma corticosterone to the levels of fed animals. This indicates that the diminished plasma leptin resulting from food deprivation plays a permissive role by allowing the increased corticosterone levels to elevate D2 activity.
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