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Originally published In Press as doi:10.1074/jbc.M003549200 on August 30, 2000

J. Biol. Chem., Vol. 275, Issue 46, 36124-36133, November 17, 2000
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Leptin Regulates Prothyrotropin-releasing Hormone Biosynthesis

EVIDENCE FOR DIRECT AND INDIRECT PATHWAYS*

Eduardo A. NillniDagger §, Charles VasletDagger , Mark Harris, Anthony Hollenberg, Christian Bjørbæk, and Jeffrey S. Flier

From the Dagger  Division of Endocrinology, Brown University School of Medicine, Rhode Island Hospital, Providence, Rhode Island 02903 and the  Division of Endocrinology, Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215

Received for publication, April 25, 2000, and in revised form, August 4, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The hypothalamic-pituitary-thyroid axis is down-regulated during starvation, and falling levels of leptin are a critical signal for this adaptation, acting to suppress preprothyrotropin-releasing hormone (prepro-TRH) mRNA expression in the paraventricular nucleus of the hypothalamus. This study addresses the mechanism for this regulation, using primary cultures of fetal rat hypothalamic neurons as a model system. Leptin dose-dependently stimulated a 10-fold increase in pro-TRH biosynthesis, with a maximum response at 10 nM. TRH release was quantified using immunoprecipitation, followed by isoelectric focusing gel electrophoresis and specific TRH radioimmunoassay. Leptin stimulated TRH release by 7-fold. Immunocytochemistry revealed that a substantial population of cells expressed TRH or leptin receptors and that 8-13% of those expressing leptin receptors coexpressed TRH. Leptin produced a 5-fold induction of luciferase activity in CV-1 cells transfected with a TRH promoter and the long form of the leptin receptor cDNA. Although the above data are consistent with a direct ability of leptin to promote TRH biosynthesis through actions on TRH neurons, addition of alpha -melanocyte-stimulating hormone produced a 3.5-fold increase in TRH biosynthesis and release, whereas neuropeptide Y treatment suppressed pro-TRH biosynthesis ~3-fold. Furthermore, the melanocortin-4 receptor antagonist SHU9119 partially inhibited leptin-stimulated TRH release from the neuronal culture. Consequently, our data suggest that leptin regulates the TRH neurons through both direct and indirect pathways.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Food deprivation in animals and humans results in endocrine and metabolic changes that include decreases in circulating levels of thyroid hormones (1, 2). Previous work in starved rats has shown that this is associated with a decrease in hypothalamic, but not thalamic, reticular prepro-TRH1 mRNA, supporting the concept that the hypothyroidism of starvation is of hypothalamic origin (1). Leptin is a recently discovered peptide hormone that is synthesized and released by adipose tissue (3-6). Serum leptin levels decrease during starvation, and leptin has been proposed to be a major regulator of the central nervous system-mediated adaptation to starvation (2). Absence of leptin is responsible for the obese phenotype of ob/ob mice, and administration of this hormone to these animals reverses many of the endocrine defects (3-6).

It was recently suggested that leptin has an important role in the neuroendocrine regulation of the HPT axis (2, 7, 8). During prolonged fasting in rats, low levels of triiodothyronine and thyroxine are observed, and TSH is in the low to normal range. This is due in part to fasting-induced suppression of prepro-TRH gene expression in the paraventricular nucleus (PVN) of the hypothalamus neurons. Since the decrease in thyroid hormone levels is blunted in fasted mice and rats by systemic administration of leptin (2, 9), it has been proposed that the decrease in leptin during fasting alters the set point for feedback inhibition by thyroid hormones on prepro-TRH mRNA biosynthesis (8). The mechanism by which leptin regulates energy expenditure through the HPT axis is unknown. Leptin has direct actions on cell bodies in the arcuate nucleus, positively regulating pro-opiomelanocortin (POMC) and thus alpha -MSH and negatively regulating the appetite-stimulating peptide neuropeptide Y (NPY) and the agouti-related peptide (AgRP) (10). NPY afferents neurons on TRH neurons are proposed to be inhibitory. Thus, the effect of leptin on TRH could be indirect, mediated by those arcuate neurons. In this study, using primary cultures of hypothalamic neurons that express high levels of endogenous pro-TRH (7, 11), we examined the hypothesis that leptin can regulate pro-TRH biosynthesis and TRH release by a direct action on TRH neurons. To this end, we determined the colocalization of pro-TRH with the leptin receptor (ObR), investigated the ability of leptin to activate the prepro-TRH promoter, and demonstrated that leptin induces pro-TRH biosynthesis and release. To support an additional indirect regulatory role of leptin in pro-TRH, we determined the effects of alpha -MSH and NPY on the biosynthesis of pro-TRH.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals

The normal and timed-pregnant female Harlan Sprague-Dawley rats used in these studies were purchased from Charles River Laboratories (Wilmington, MA and Kingston, NY).

Tissue Culture

Primary Cultures of Hypothalamic Neurons-- Fetal rat hypothalamic cells (day 17) were prepared as described earlier by us (11). In brief, timed-pregnant female rats on day 17 of gestation were anesthetized with pentobarbital (60 mg/kg). The diencephala from fetuses were removed and dispersed enzymatically with neutral proteases (100 units/dl) for 2 h. The dispersed cells were then plated (5 × 106 cells/ml) in tissue culture flasks for radiolabeling and release studies. All cells were plated in wells precoated with 20 µg/ml poly-D-lysine (Sigma). The cells were maintained in bicarbonate-buffered Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (Life Technologies, Inc.) at 37 °C in an atmosphere of 5% CO2 and 95% humidity. To induce differentiation of neuronal cells and prepro-TRH biosynthesis, the cells were cultured with 50 µM bromodeoxyuridine during the first 4 days as described previously (11). For immunocytochemistry experiments, the cells were plated on four-chamber glass slides (LabTek, Nunc Inc., Naperville, IL) at low density (5 × 105 cells/ml).

CV-1 Cells with the TRH Promoter-- The human TRH promoter and RSV180 were both cloned into the vector pA3luc as described previously (12). The ObRb and STAT3 constructs have been described previously (13). CV-1 cells were grown as described previously (14). The cells were seeded in six-well plates and transfected in triplicate using the calcium phosphate method. Each well received 1.7 µg of reporter, 0.8 µg of STAT3, and 0.8 µg of ObRb expression vectors. In addition, each well received 20 ng of a cytomegalovirus-beta -galactosidase expression vector to control for transfection efficiency. Total amounts of DNA were held constant. Eighteen hours after transfection, the cells were washed and placed in normal medium. Thirty hours after transfection, the cells were incubated with serum-free medium. Forty hours after transfection, 100 nM leptin was added or not. The cells were assayed for luciferase and beta -galactosidase enzyme activities.

CHO Cells Expressing the Leptin Receptor-- CHO cells stably expressing the short form of the leptin receptor (ObRa) were grown and generated as described earlier (13).

Mouse Corticotropic AtT-20 Cells-- AtT-20 were grown in 75-cm2 flasks at 37 °C in an atmosphere of 5% CO2, 95% air, and 90% humidity (15). Each flask was plated with 5 × 106 cells, and cultures were maintained for 6 days in Dulbecco's modified Eagle's essential medium (Life Technologies, Inc.) containing 10% fetal calf serum as described previously (15). The culture medium was removed every 2 days and replaced with fresh medium. Experiments were performed at a confluency corresponding to 20-30 × 106 cells.

Quantitative 32P Reverse Transcription-PCR of ObRb mRNA from Fetal Hypothalamic Neurons

ObRb mRNA was quantified as described (16), except for a few changes. Briefly, total RNA was isolated, and 0.20 µg were subjected to cDNA synthesis (100 µl total) with or without reverse transcriptase. For amplification of rat beta -actin cDNA, the following primers were used: upstream primer, 5'-TTGTAACCAACTGGGACGATATGG-3'; and downstream primer, 5'-GATCTTGATCTTCATGGTGCTAGG-3'. The following primers were used for specific PCR amplification of rat ObRb cDNA: upstream primer, 5'-TATGTCATTGTACCGATAATTATT-3'; and downstream primer, 5'-CCCCTTGTGGAATCTGGAGTGG-3'. Each 50-µl PCR was carried out with 5.0 µl of cDNA as template and 0.50 µl of [alpha -32P]dCTP (29.6 TBq/mmol, 370 MBq/ml; PerkinElmer Life Sciences). The samples were subjected to 18 cycles of amplification for beta -actin and 30 cycles for ObRb. Five microliters of the reaction were run on a denaturing urea-acrylamide gel. The gel was then dried and finally subjected to 32P PhosphorImager analysis (Molecular Dynamics, Inc., Sunnyvale, CA).

Radiolabeling Experiments

After 12 days in culture, hypothalamic neurons (5 × 106/flask) were stimulated with increasing concentrations of leptin for a period of 6 h in low leucine, serum-deprived Dulbecco's modified Eagle's essential medium. The cells were pulsed for the entire 6 h with 0.3 mCi of ]3,4,5-3H]leucine (156 Ci/mmol) for pro-TRH and processing product radiolabeling and 0.3 mCi of [L-2,3,4,5-3H]proline (100 Ci/mmol) for TRH radiolabeling. For long-term labeling, we utilized 90% (9 volumes) leucine-free medium mixed with 1 volume of regular medium. After the incubations, the medium was removed, and the cells were washed three times. After the last wash, the cells were rapidly cooled on ice, and 2 ml of 2 N acetic acid containing 2 mM EDTA, 2 mM EGTA, and enzyme inhibitors (phenylmethylsulfonyl fluoride, aprotinin, bacitracin, bestatin, and pepstatin, each at 0.1%) were added. The cells were scraped and heated to 95 °C for 10 min prior to sonication. One-hundred microliters of sample were removed for protein assay. The remainder of the cell extract was centrifuged at 15,000 rpm for 30 min. The supernatant was then lyophilized and held at -20 °C until analyzed by polyacrylamide gel electrophoresis (PAGE). For TRH analysis, the medium from these cultures was immunoprecipitated with anti-TRH antibodies, followed by isoelectric focusing gel electrophoresis (IEFGE) (see below).

Immunoprecipitation

An immunoprecipitation protocol was carried out as described previously (17). Briefly, lyophilized cell extracts were resuspended in 10 µl of 0.2% bovine serum albumin and 200 µl of hypotonic buffer A (10 mM NaPO4 (pH 7.2), 1 mM EDTA, and 0.1% Triton X-100). Following resuspension, cell extracts were incubated for 24 h at 4 °C with a 1:500 dilution of protein G-purified anti-pro-TRH-(115-151) antibody (15). Then, a 1:1000 dilution of goat anti-rabbit IgG was added along with 75 µl of buffer B (500 mM KCl, 50 mM NaH2PO4 (pH 7.4), 5 mM NaEDTA, and 0.25% Triton X-100). Samples were further incubated for 4 h at 4 °C. Immunoprecipitates of cell extracts were washed once with buffer B and once with buffer C (10 mM NaH2PO4 (pH 7.2) and 15 mM NaCl), which removes EDTA and Triton X-100. The immunoprecipitates were then resuspended in sample buffer (0.0625 M Tris (pH 6.8), 1% SDS, 15% glycerol, and 15 mM dithiothreitol) and boiled for 4 min prior to SDS-PAGE. The release medium obtained from proline-labeled cultures was immunoprecipitated with anti-TRH antibodies as described above and resuspended in sample buffer prior to IEFGE.

SDS-PAGE

Radioactive or unlabeled samples were fractionated by mobility by loading onto a discontinuous Tricine/SDS-PAGE system for separation of low molecular mass peptides (18). A stacking gel was made to 3% cross-linking (acrylamide/bisacrylamide solution), and the separating gel was made to 6% cross-linking (acrylamide/bisacrylamide solution). Gels were run in the Protean 16-cm cell system (Bio-Rad). Following electrophoresis, gels were cut into 1-mm slices in a gel slicer (Hoefer Scientific Instruments, San Francisco, CA) and prepared for either counting or radioimmunoassay. For tritium analysis, immunoprecipitated peptides were extracted from gel slices by incubation in 1 ml of 1 N acetic acid for 24 h at 4 °C. Scintillation fluid (Bio Safe II, Research Product International Corp., IL) was added, and samples were counted in a scintillation counter. Preparation for radioimmunoassay (RIA) included the same acetic acid extraction as described above, but following incubation, gel slices were removed. Samples were then lyophilized and resuspended in the appropriate RIA buffer. Recovery of peptides from gel slices has been shown to be ~90% as determined by RIA prior to and following the electrophoresis (19). To identify the apparent molecular masses of fractionated peptides on SDS-PAGE, a series of molecular mass markers was used: prestained bovine serum albumin, 80.0 kDa; ovalbumin, 49.5 kDa; carbonic anhydrase, 32.5 kDa; soybean trypsin inhibitor, 27.5 kDa; lysozyme, 18.5 kDa (Bio-Rad); trypsin inhibitor, 20.4 kDa; myoglobin, 16.95 kDa; myoglobin fragment IV, 14.4 kDa; myoglobin fragment III, 8.16 kDa; myoglobin fragment II, 6.2 kDa; and myoglobin fragment I, 2.5 kDa (Diversified Biotech, Newton, MA).

IEFGE

Immunoprecipitated [3H]proline-labeled TRH samples with anti-TRH antibodies were fractionated on an IEFGE according to estimated isoelectric points for TRH. This was based on theoretical calculations using MacVector software for protein analysis (pI ~7.00) and using the Swiss Protein Annotated Protein Sequence Database, the TrEMBL Computer-annotated supplement to the Swiss Protein Database (pI ~6.8-7.00). Samples dissolved in sample buffer (6 M urea, 1% CHAPS, 80 mM dithiothreitol, 1.6% Pharmalyte 5-8, and 0.4% Pharmalyte 2.5-5) were run in 30% acrylamide and 1% bisacrylamide gel tubes in buffer containing 6 M urea and 1% CHAPS. To generate a pH 4.6-8.00 gradient, 1% Pharmalyte 2.5-5 and 4% Pharmalyte 5-8 (Amersham Pharmacia Biotech) were added to the solution. The pH gradient was pre-established by subjecting the gel tubes to 100 V for 15 min and 200 V for another 15 min utilizing 100 mM NaOH in the upper chamber and 10 mM H3PO4 in the lower chamber. After the prerun, samples were loaded and run for 2.5 h at 400 V. After the run, each gel tube was sliced (0.5 cm), put in scintillation vials containing 2 N acetic acid, keep at 4 °C for 3 days, and counted. The pH gradient was determined by measuring the pH in each slice. After the run, a gel tube without sample was incubated in 2 ml of 50 mM KCl under vacuum overnight, and the pH was measured with a pH meter and using Bio-Rad IEFGE standards, a mixture of nine natural proteins with isoelectric points ranging from 4.45 to 9.0.

TRH Radioimmunoassays

RIA for TRH was performed in triplicate samples removed from nonradioactive neuronal cells previously exposed to increasing concentrations of leptin. The protocol for TRH RIA is standard in our laboratory and has been published elsewhere (19).

Double-label Immunocytochemistry

Hypothalamic neurons (3 × 105) from 12-day-old cultures were fixed with 4% paraformaldehyde in phosphate-buffered saline and subjected to an immunocytochemistry protocol as described previously (11, 18). CHO cells transfected with an empty vector or expressing the short form of the leptin receptor were subjected to immunostaining using an antibody against the common extracellular domain of the leptin receptor. Generation of this polyclonal antibody was done as described earlier (20).

Immunoreaction of the primary anti-ObR antibody with the hypothalamic neurons was performed at 4 °C for 24 h. Goat anti-rabbit immunoglobulin conjugated with fluorescein isothiocyanate was used as the fluorescence marker. A wide range of dilutions for the primary and secondary antibodies was tested. The optimal dilutions were found to be 1:1000 for the primary antibody and 1:2000 for the secondary antibody, with incubation times of 24 h at 4 °C for the primary antibody and 2 h at room temperature for the secondary antibody. Control experiments, including the incubation of cells without primary antibody or preimmune serum and the blocking of the primary antibody with synthetic peptides against which the antibody was generated, were performed and did not show any positive staining. For colocalization experiments, cells previously stained with anti-ObR antibody followed by fluorescein isothiocyanate probe (green color) were then incubated with anti-pAV37-Texas Red or anti-pST10-Texas Red antibody for 24 h at 4 °C. Anti-pAV37 antibody recognizes the TRH prohormone, and anti-pST10 antibody recognizes the end product of processing prepro-TRH-(160-169) (11). The conjugation of Texas Red (red color) to these antibodies, as well as their ability to obtain successful colocalization with other proteins, was described previously by us in experiments demonstrating the colocalization of pro-TRH with the prohormone convertase-1 (18).

Statistics

TRH RIA values were calculated from release media denied from controls and treated cells. Samples for each condition were taken in triplicate. Protein assay results were used to correct for minor variations in total cell number. Data are displayed as ng/ml. Analysis of variance, followed by a multiple comparison (Tukey-Kramer test), was employed when appropriate.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Leptin Stimulates Pro-TRH Biosynthesis in Primary Cultures of Hypothalamic Neurons-- We have previously used primary cultures of fetal rat hypothalamic neurons to study pro-TRH biosynthesis and processing (11, 21). Using this primary cell system, we examined the effect of leptin on the biosynthesis of pro-TRH. After 12 days in culture, primary cultures of hypothalamic neurons were stimulated with leptin for 6 h in the presence of radioactive leucine (leucine is present 21 times in the pro-TRH sequence). After treatment, radioactive peptides were acid-extracted and subjected to double immunoprecipitation with an antibody, previously characterized by us (18), directed against prepro-TRH-(116-151) (anti-pAV37 antibody) that recognizes the TRH precursor (26 kDa) and an intermediate form of its processing of ~16 kDa. The immunoprecipitates were then subjected to separation on a Tricine/SDS-PAGE system, followed by slicing and radioactive counting. A typical SDS-PAGE profile of immunoprecipitated pro-TRH and the intermediate form of ~16 kDa during leptin treatment as compared with untreated controls is depicted in Fig. 1A.


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  		Fig. 1.   Leptin stimulates pro-TRH biosynthesis in hypothalamic neurons. A depicts the stimulation of pro-TRH biosynthesis by leptin in three independent experiments. In each experiment, 3H-radiolabeled peptides from primary cultures of hypothalamic neurons were immunoprecipitated with anti-pAV37 antibody. Immunoprecipitates were then resolved on a SDS-polyacrylamide gel, and the radioactivity in individual gel slices was counted. B depicts a statistical representation of three independent experiments. The data presented were calculated based on three identical experimental conditions using the Tukey-Kramer test for multiple comparisons (p < 0.02 for all conditions).

Fig. 1B depicts the profile of pro-TRH (26 kDa) biosynthesis in three independent experiments similar to that shown in Fig. 1A. Since the radioactive incorporation into proteins and the SDS-PAGE separation profile vary from one experiment to the next, the values were normalized by calculating the ratio between the pro-TRH radioactive peak in cells treated with leptin versus the pro-TRH peak in untreated controls. Those values were also corrected against the total [3H]leucine incorporated by the cells for each experiment as measured by trichloroacetic acid precipitation. Molecular masses of the identified peaks are indicated based on the migration of standards. The results show an ~10-fold increase in pro-TRH biosynthesis in cells treated with 10 nM leptin as compared with untreated controls. These data indicate that leptin can stimulate the biosynthesis of pro-TRH.

Leptin Stimulates TRH Release in Primary Cultures of Hypothalamic Neurons-- Having determined that leptin stimulates the synthesis of pro-TRH, we wanted to determine whether leptin increases TRH release. We first evaluated the release of TRH synthesized de novo by subjecting the cells treated with leptin to a pulse with [3H]proline (which is present in the TRH molecule). Even though proline is a nonessential amino acid and may not have the advantages of an essential amino acid for radiolabeling, a dramatic increase in radiolabeling in proline-rich proteins has been reported in the rat parathyroid gland (22, 23). More than 90% of the [3H]proline found in the parathyroid gland was incorporated into proline-rich proteins.

Radiolabeled TRH in the release medium was then immunoprecipitated with anti-TRH antibodies and subjected to IEFGE (pH gradient ~4.5-8). We have recently developed the conditions for the identification of TRH utilizing the isoelectric focusing system. The approximate isoelectric point for TRH was ~7.00 based on theoretical calculations (see "Materials and Methods"), and our experimental results are presented in Fig. 2A. The results depicted in Fig. 2A represent a typical IEFGE profile from media samples collected after treatment of hypothalamic cells with leptin for 6 h in the presence of [3H]proline. The data show that treatment of hypothalamic neurons with leptin increased the release of TRH in a dose-dependent fashion from 1 to 10 nM. Ten nanomolar leptin showed an effect on TRH release of ~5-fold as compared with untreated controls. AtT-20 cells, which do not carry endogenous pro-TRH, were used as a control for the IEFGE system. As expected, immunoprecipitation of [3H]proline-radiolabeled release peptides in these cells showed background levels of radioactivity (Fig. 2A).


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  		Fig. 2.   Leptin stimulates TRH release from hypothalamic neurons. The stimulation of pro-TRH biosynthesis by leptin in three independent experiments is depicted in A and B. A, in each experiment, [3H]proline-labeled peptides from primary cultures of hypothalamic neurons were immunoprecipitated with anti-TRH antibody. Immunoprecipitates were then resolved on an isoelectric focusing (IEF) polyacrylamide gel, and the radioactivity in individual gel slices was counted. The pH gradient was determined by measuring the pH of each slice in a parallel gel and using pH standards (Bio-Rad). The formula depicted in A represents the calculation done on the data to bring it to a linear form. B, media samples derived from hypothalamic cultures incubated for 6 h with leptin were analyzed by specific RIA against TRH. The data presented for TRH RIA were calculated based on six identical experimental conditions using the Tukey-Kramer test for multiple comparisons (p < 0.02 for all conditions).

We next wanted to determine whether the increase in TRH release by leptin, in addition to inducing a new biosynthesis of pro-TRH and release of TRH (Fig. 2A), is also due to an action of leptin as a secretagogue. To this end, the release medium was collected from cultured cells exposed to increasing concentrations of leptin for 6 h and compared with that from untreated control cells. The samples were analyzed by specific RIA against TRH as described previously (18). As shown in Fig. 2B, the release of TRH increased in a dose-dependent manner, with the maximum response being observed at 10 nM leptin (15-fold). The big difference in release measured by the two approaches indicates that leptin not only induces the activation of prepro-TRH gene expression to ultimately translate more pro-TRH into protein, but also acts as a secretagogue. The 10-fold difference observed between TRH measurements by radiolabeling versus RIA indicates that leptin also induces the release of TRH stored in mature secretory granules near the plasma membrane of the cell, mechanisms that are independent of gene activation. Since the samples were collected after 6 h, the total amount of TRH measured by RIA may account for the newly synthesized plus TRH molecules accumulated in mature secretory granules. In our previous study (24), we showed that the newly formed pro-TRH needs at least 120 min to be processed and deliver its end products of post-translational processing to mature secretory granules. Studies looking at short-time leptin stimulation (<120 min) are under way to further quantify the amount of TRH release by leptin as secretagogue alone.

ObRb mRNA Expression and Colocalization of Leptin Receptors with Pro-TRH in Primary Cultures of Fetal Rat Hypothalamus-- To further investigate whether leptin might have direct effects on TRH neurons, we first wanted to determine whether the long form of the leptin receptor (ObRb) is expressed in primary cultures of dissociated rat diencephalic neurons by comparing it with the level in whole rat hypothalamic RNA. By applying semiquantitative 32P reverse transcription-PCR using a limiting number of PCR cycles of equal amounts of total RNA from the hypothalamic culture and from rat hypothalamus, we found that ObRb mRNA was highly expressed in the hypothalamic culture (Fig. 3). The levels were only ~4-fold less than those in the rat hypothalamus. As expected, we did not detect any ObRb mRNA in an RNA sample from the cultured neurons subjected to cDNA synthesis without the presence of reverse transcriptase (fourth lane), nor did we detect any ObRb mRNA under these experimental conditions in the same amount of RNA isolated from rat lung tissue (data not shown).


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  		Fig. 3.   ObRb mRNA is highly expressed in fetal hypothalamic neurons. Equal amounts of total RNA from fetal hypothalamic neurons and from a whole rat hypothalamus were subjected to quantitative 32P reverse transcription-PCR using a limiting number of PCR cycles (see "Methods and Methods"). Two independent samples of fetal hypothalamic neurons (first and second lanes) were amplified and run in parallel with a rat hypothalamic sample (third lane) and an RNA sample from fetal hypothalamic neurons that did not receive the reverse transcriptase enzyme during cDNA synthesis (no- RT Control; fourth lane). Shown is a PhosphorImager image of the radioactive PCR products. The upper panel shows the ObRb results, and the lower panel shows the beta -actin (b-Actin) results. This experiment was done twice. ObRb mRNA was not detected in the same amount of RNA from rat lung tissue (not shown).

We then used primary cultures of dissociated rat diencephalic neurons to determine whether pro-TRH colocalizes with leptin receptors. We performed double-label immunocytochemistry with antibodies directed against either pro-TRH-derived peptides or the extracellular domain of the leptin receptor to identify possible colocalization of the ObR and pro-TRH. Single immunostaining in non-permeabilized hypothalamic neurons with anti-ObR antibody revealed, as expected, that a substantial proportion of these neurons expressed the ObR. As expected, the distribution of the ObR in the cells was mostly within the cell bodies and dendrites (Fig. 4A). Statistical analysis of >100 fields revealed that ~30-40% of the hypothalamic cells were positive for the leptin receptor. Of these, ~8-13% also expressed TRH (Fig. 4B; see also insets in A and B for the colocalization of the leptin receptor and pro-TRH). The specificity of anti-ObR antibody was tested using the same immunocytochemistry conditions used in Fig. 4 on CHO cells transfected or not with leptin receptor cDNA. Parental CHO cells transfected with empty vector did not exhibit specific fluorescence (Fig. 5A), whereas CHO cells expressing leptin receptors exhibited a population of cells with distinct staining (Fig. 5B).


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  		Fig. 4.   Colocalization of pro-TRH and the ObR by double immunocytochemistry staining of primary cultures of hypothalamic neurons. Neuronal cells were cultured for up to 12 days and fixed with 4% paraformaldehyde. Fluorescein isothiocyanate-conjugated goat anti-rabbit globulin was used to visualize ObR immunostaining (green). Anti-pAV37 antibody recognizes the TRH prohormone, and anti-pST10 antibody recognizes the end product of processing prepro-TRH-(160-169) (18). Anti-pAV37 and anti-pST10 antibodies were conjugated with Texas Red (red) as described previously (18). A shows non-permeabilized hypothalamic neurons positively stained for the ObR. The inset in A shows the colocalization of anti-ObR and anti-pST10 antibodies; shown are a cell body with positive staining for the ObR (green, arrows) and positive staining in the axonal processes of the same cell (below) using anti-pST10 antibody (red, arrowheads). B shows colocalization of the ObR and pro-TRH (detected with anti-pAV37 antibody); see insets for the ObR (green) and pro-TRH (red) indicated by arrows. The combined figures of the same cells are shown above (yellow). B also shows a cell positive only for pro-TRH (red, arrowhead) and a cell positive only for the ObR (long arrow, green).


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  		Fig. 5.   Specificity of anti-ObR antibodies. A shows the immunocytochemistry results for the ObR in CHO cells transfected with an empty vector. B shows CHO cells expressing the short form of the leptin receptor. The leptin receptor was detected using an antibody against the common extracellular domain of the leptin receptor (13).

Leptin Signaling Positively Regulates the Human TRH Promoter-- Having established the presence of leptin receptors in the TRH neurons, we next wanted determine whether the leptin signaling pathway is capable of directly regulating pro-TRH at the transcriptional level. To pursue this, we used the human TRH promoter (positions -900 to +55) fused to the luciferase reporter and assayed the effect of leptin receptor signaling. To determine the specificity of leptin signaling, we employed a heterologous system whereby we cotransfected the ObRb, the human TRH promoter, and STAT3 in the presence or absence of leptin in the mammalian CV-1 cell line. As depicted in Fig. 6, cells stimulated with 100 nM leptin (similar results were seen with 10 nM leptin) for 6 h showed almost a 5-fold increase in TRH promoter activity. Leptin had no effect in the absence of transfected leptin receptors. Furthermore, the leptin signaling system had no effect on a control luciferase reporter driven by the first 180 base pairs of the Rous sarcoma virus long terminal repeat promoter (data not shown). Thus, these results demonstrate that the leptin signaling pathway directly activates the TRH promoter in a heterologous system.


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  		Fig. 6.   Activation of the TRH promoter by leptin signaling in a heterologous transfection system. CV-1 cells were transiently transfected with the TRH promoter-luciferase construct with or without ObRb expression vectors. Forty-eight hours post-transfection, including serum starvation for 12 h, cells were treated or not with 10 nM leptin for 6 h. Measurements of luciferase activities are shown. The data presented were calculated based on four identical experimental conditions using the Tukey-Kramer test for multiple comparisons (p < 0.02 for all conditions).

alpha -MSH Stimulates Pro-TRH Biosynthesis and TRH Release, whereas NPY Has an Opposite Effect in Primary Cultures of Hypothalamic Neurons-- Although the forgoing data support the hypothesis that leptin can regulate TRH expression through direct effects on the TRH neurons, we recently showed the first preliminary evidence in support of the hypothesis that the action of leptin on TRH neurons may occur also through an indirect pathway of signaling (7). This signaling may involve regulatory peptides produced in the arcuate nucleus of the hypothalamus (7, 8, 25, 26). Recent in vivo data have also indicated a stimulatory role of central melonocortinergic mechanisms on the HPT axis, as intracerebroventricular infusion alpha -MSH prevented the fasting-induced suppression of pre pro-TRH mRNA content in the PVN and partially reversed the fall in thyroide hormone levels (53). In addition, other studies showed that melanocortin antagonist treatment in normal animals caused a suppression of the HPT axis (27). Two candidate neuropeptides for this role are NPY and alpha -MSH, which have been shown to be leptin-responsive (28) and to project nerve fibers to the PVN, where the hypophysiotropic neurons producing pro-TRH are located. Therefore, using our primary cultures of fetal rat hypothalamic neurons, we investigated whether these peptides affect the biosynthesis of pro-TRH in TRH neurons. Fig. 7A demonstrates that 10 nM alpha -MSH for 4 h resulted in a 3.5-fold increase in the biosynthesis of pro-TRH as determined by double immunoprecipitation of radiolabeled samples with anti-pAV37 antibody, followed by SDS-PAGE analysis. Concentrations ranging from 1 to 100 nM showed a dose-dependent increase in pro-TRH biosynthesis (data not shown).


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  		Fig. 7.   alpha -MSH stimulates the biosynthesis of pro-TRH, and NPY inhibits it. These graphs depict an electrophoretic separation of anti-pAV37 antibody-immunoprecipitated [3H]leucine-labeled samples derived from hypothalamic neurons treated with alpha -MSH as compared with untreated controls. The data show an ~3-fold increase in pro-TRH biosynthesis. The 26-kDa peak represents the pro-TRH polypeptide. The graphs shown in A and B are representative of three independent experiments.

Measurement of TRH release by RIA under the same experimental conditions described above showed that TRH release increased 4-fold as compared with untreated controls (4.5 ± 0.04 pmol treated versus 1.2 ± 0.01 pmol untreated). Using the same conditions, we also evaluated the effect of NPY on the biosynthesis of pro-TRH. Fig. 7B shows that incubation of 5 nM NPY for 4 h produced an almost 3-fold decrease in the biosynthesis of pro-TRH as measured by double immunoprecipitation of [3H]leucine-radiolabeled peptides, followed by SDS-PAGE analysis. The release of [3H]proline-labeled TRH was also inhibited 2.8-fold as compared with untreated controls as measured by IEFGE (data not shown). These results demonstrate that these two leptin-responsive peptides regulate the biosynthesis of pro-TRH, supporting the hypothesis that pro-TRH biosynthesis is also regulated by leptin through an indirect pathway.

Direct Stimulation of TRH Neurons by Leptin-- In the experiments described above, we showed that in primary cultures of hypothalamic neurons, under the same experimental conditions, leptin increased the biosynthesis of pro-TRH 10-fold, whereas the alpha -MSH peptide had only a 3-fold effect. This suggests that this difference in pro-TRH biosynthesis caused by leptin versus alpha -MSH might be attributed to a direct action of leptin on TRH. To further clarify the direct involvement of leptin in the biosynthesis of pro-TRH and the release of its end product (TRH) from these cells, we stimulated the cells with leptin, the SHU9119 compound (a melanocortin-4 receptor (MC4R) antagonist), or the alpha -MSH peptide. Then, we monitored the release of TRH by double immunoprecipitation, followed by IEFGE as described above. Fig. 8A depicts a typical profile of three independent experiments. The data show that the SHU9119 compound inhibited the leptin effect on TRH release by 25% (Fig. 8B). This is consistent with the hypothesis that leptin has a direct action on TRH neurons through its receptor, and the 25% inhibition may represent the activation of TRH through the melanocortin signaling pathway. Furthermore, the data show that stimulation of cells with the alpha -MSH peptide resulted in TRH release that was lower (43%) than the effect of leptin (Fig. 8B), consistent with the results presented in Figs. 1 and 7.


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  		Fig. 8.   Direct stimulation of leptin on TRH hypothalamic neurons. A depicts the stimulation of TRH release by leptin and a combination of treatments with SHU9119 (MC4R antagonist) and the alpha -MSH peptide. The graph is a representation of three independent experiments. In each experiment, [3H]proline-labeled peptides from primary cultures of hypothalamic neurons were immunoprecipitated with anti-TRH antibody. Immunoprecipitates were then resolved on an isoelectric focusing polyacrylamide gel, and the radioactivity in individual gel slices was counted. The pH gradient was determined by measuring the pH of each slice in a parallel gel and using pH standards. The formula depicted represents the calculation done on the data to bring it to a linear form. B depicts the calculated percentage of TRH releases from A. The 100% release value was arbitrarily set for leptin.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Leptin is a hormone produced principally in adipose tissue whose central physiologic role is to provide information on energy stores and energy balance to brain centers that regulate appetite, energy expenditure, and neuroendocrine function (2-6). To effectively deliver this information, leptin must reach its central targets and engage receptors on specific hypothalamic neurons (29-31). The output of these neurons is then integrated with other signals, ultimately engaging final effector pathways. When leptin signaling is deficient, due to mutation of either the leptin hormone or leptin receptor genes, severe obesity results in both rodents and humans (3, 30, 32-34), underscoring the fundamental role of leptin in physiology.

Thyroid hormone, the peripheral end product of the HPT axis, is an important regulator of energy expenditure (35). This regulation involves release of hypophysiotropic TRH to stimulate pituitary TSH, which, in turn, stimulates thyroid hormone production and release. TRH neurons in the medial and periventricular parvocellular subdivisions of the PVN are also subjected to negative feedback regulation by circulating levels of thyroid hormone (36). When plasma levels of thyroid hormone fall, the biosynthesis and secretion of TRH from these neurons increase, raising the threshold for feedback inhibition by thyroid hormone on anterior pituitary thyrotrophs and thus increasing TSH secretion. Conversely, elevations in plasma concentrations of thyroid hormone suppress the biosynthesis and secretion of PVN TRH, causing a reduced threshold for feedback regulation by thyroid hormone on thyrotrophs and thus suppressing TSH secretion (36). During fasting, however, this regulatory system is altered, such that decreased circulating thyroid hormone levels are associated with a reduction in the biosynthesis of TRH and the secretion of TSH (1, 37). By creating a transient state of central hypothyroidism, the resulting reduction of thyroid thermogenesis may serve as an important energy conservation mechanism until re-feeding occurs.

Previous studies demonstrated that leptin can prevent the fall of thyroid hormone (thyroxine and triiodothyronine) with starvation in rodents (2, 9) and that this is accompanied by a maintained expression of TRH mRNA in the PVN (8). This underscores the importance of TRH neurons in the regulation of thyroxine/triiodothyronine by leptin. Thus, hypophysiotropic TRH neurons in the PVN are potential targets for leptin. The data presented in this study demonstrate the presence of leptin receptors in pro-TRH-producing neurons. Since our hypothalamic cultures contained an array of different neurons, including those producing TRH that do not belong to the HPT axis, we cannot at this time demonstrate that the TRH neurons carrying ObRb are those related to the HPT axis. However, since in vivo leptin treatment of starved rats only affects TRH mRNA expression within the PVN, and not extra-PVN TRH neurons, it is likely that this is the case.

Consistent with a direct action of leptin on pro-TRH neurons, we demonstrated that the transfected prepro-TRH promoter was activated by ObRb in a heterologous cell system. This effect of leptin signaling to activate the prepro-TRH promoter is consistent with the possibility that pro-TRH biosynthesis may also be positively affected. Utilizing our primary cultures of hypothalamic neurons, we clearly showed that leptin increased the biosynthesis of endogenous pro-TRH in a dose-response fashion consistent with a receptor-ligand response. The greatest increase was observed at 10 nM leptin, an ~10-fold increase over untreated control cells. Consistent with this increase in pro-TRH biosynthesis, we also showed that the amount of newly synthesized TRH released was ~5 times more in leptin-treated than untreated cells, as monitored by a newly developed IEFGE assay for [3H]proline-labeled TRH. In addition, TRH RIA analysis of non-radiolabeled neuronal cultures that were treated with increasing concentrations of leptin revealed that leptin also induced the release of TRH stored in mature secretory granules near the plasma membrane of the cell, mechanisms that are independent of gene activation. In summary, these combined results strongly support a direct regulation of pro-TRH biosynthesis at the transcriptional, translational, and release peptide levels.

Indirect regulation of pro-TRH through other peptide messengers has also been proposed. Using rats with arcuate nucleus lesions induced by monosodium glutamate, it has been suggested that this regulation may, at least in part, be indirect, requiring input from the arcuate nucleus (26). TRH neurons in the PVN are located in a region where they can be regulated by a large number of afferent neuroendocrine inputs. In addition to TRH neurons being densely innervated by NPY neurons, which originate in the arcuate nucleus (7), it was recently shown that alpha -MSH- and AgRP-expressing neurons have nerve fibers that project from the arcuate nucleus to the PVN. These projections are in close proximity to pro-TRH-expressing neurons (25, 39). Since NPY, AgRP, POMC, and cocaine- and amphetamine-regulated transcript (CART) expression in the arcuate nucleus is regulated by leptin in vivo (40-44), it is possible that these neuropeptides could play a role in modulating the regulation of TRH neurons by leptin. With the results presented here, we were able to demonstrate that alpha -MSH can positively regulate pro-TRH biosynthesis and TRH release in a dose-dependent manner, whereas NPY negatively regulates pro-TRH biosynthesis. The signaling mechanisms by which alpha -MSH and NPY regulate pro-TRH biosynthesis are unknown. We are currently pursuing these questions. In this study, we demonstrated that 75% of the leptin action on pro-TRH biosynthesis in hypothalamic neurons was through a direct action on the ObRb receptors in TRH neurons, whereas only 25% of the effect was through the melanocortin signaling pathway (see Fig. 8).

We have shown that ObRb mRNA is highly expressed in specific hypothalamic neurons in the arcuate nucleus that are involved in the regulation of food intake (28, 31). The neurons expressing ObRb mRNA include those expressing NPY, AgRP, POMC, and CART (40-44, 46). A coherent view of the hypothalamic circuits regulated by leptin is beginning to emerge. The current paradigm envisions direct action by leptin on specific neural populations within the mediobasal hypothalamus, especially the arcuate nucleus. One population of arcuate neurons coexpresses NPY and AgRP, and expression of these mRNAs is suppressed by leptin (Fig. 9). These neurons also express ObRb, and leptin acts directly on these neurons, as suggested by our finding of rapid induction of SOCS-3 (suppressor of cytokine signaling-3) mRNA in these cells in response to leptin (20, 28). Substantial evidence indicates that arcuate nucleus-derived NPY and AgRP promote feeding and may induce obesity. A distinct subpopulation of arcuate neurons coexpresses POMC and CART as well as ObRb. POMC and CART mRNAs are positively regulated by leptin, once again through direct activation, as suggested by leptin-induced SOCS-3 and c-Fos expression in these cells (28). Substantial physiologic and genetic evidence indicates that the CART peptide and POMC-encoded alpha -MSH mediate anorexigenic and very likely other actions of leptin (42-44). alpha -MSH exerts its effects primarily through MC4Rs (47-49), mutation of which causes obesity in mice and humans (50-52). AgRP antagonizes alpha -MSH binding and signaling via the MC4R (45) (Fig. 9).


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  		Fig. 9.   Hypothetical model of leptin action on TRH neurons. This schematic represents the proposed model for activation of the thyroid axis by leptin through actions on the pro-TRH neuron in the PVN of the hypothalamus. Two mechanisms of leptin action on pro-TRH neurons are proposed in this study. In one (indirect regulation), leptin regulates arcuate (ARC) neurons expressing POMC, which give rise to alpha -MSH through post-translational processing (induced by leptin), and arcuate neurons expressing AgRP (suppressed by leptin), which then project their nerve fibers into close contact with pro-TRH neurons. These peptides influence TRH expression by the antagonistic actions of alpha -MSH (stimulatory) and AgRP (inhibitory) on MC4Rs. Leptin inhibits the expression of appetite-stimulating NPY, which then projects its nerve fibers into close contact with pro-TRH neurons, producing an inhibitory action of pro-TRH neurons. We propose that leptin also acts directly on TRH neurons through leptin receptors on these cells (stimulatory). In the absence of leptin signaling, reduced negative feedback on the TRH neuron by low levels of thyroxine (T4)/triiodothyronine (T3), acting on the thyroid receptor (TR), fails to produce compensatory increases in TRH expression.

Taken together, the data presented here suggest that leptin may act at two levels to regulate pro-TRH biosynthesis and TRH release: a direct action through TRH neurons and an indirect action through the arcuate nucleus via alpha -MSH, AgRP, and NPY. This regulation may include a combination of positive and negative activation of the prepro-TRH gene as follows: (a) an inhibitory action of leptin on NPY and AgRP release from the arcuate nucleus, leading to a reduced inhibitory effect of these peptides on TRH expression in the PVN; (b) a stimulatory action of leptin on alpha -MSH release from the arcuate nucleus, resulting in stimulation of TRH release from the PVN; and (c) a direct positive action of leptin on TRH neurons in the PVN. In support of the direct pathway of signaling for leptin to the PVN, the following observations are worth noting. Using in situ hybridization of rat brain sections, we previously showed that ObRb mRNA is expressed within the PVN (31). We have reported that peripheral administration of leptin to rodents activates SOCS-3 mRNA in neurons located in the PVN (20). In addition, in this study, we have shown that leptin can directly regulate pro-TRH promoter activity via ObRb in a heterologous system. The signaling pathways and mechanisms by which leptin directly regulates pro-TRH mRNA and TRH biosynthesis are unknown, and these are key issues we are currently investigating. Fig. 9 depicts our current model of leptin regulation of pro-TRH neurons from the HPT axis.

It is presently unknown how leptin reaches TRH neurons in the PVN from the circulation. We have earlier published data demonstrating that high mRNA levels encoding the short form of the leptin receptor (ObRa) are present in rat brain microvessels, which comprise the blood-brain barrier (16). Consistent with this, we have demonstrated that this receptor has the capacity to mediate transport of intact leptin across a monolayer of polarized Madin-Darby canine kidney cells (38). This suggests that leptin may first enter the brain interstitial fluid via receptor-mediated transcellular transport at the blood-brain barrier, followed by diffusion to finally reach ObRb receptors expressed on TRH neurons in the PVN. Further studies are clearly required to demonstrate this directly.

This study provides the first direct evidence for leptin-mediated regulation of prepro-TRH mRNA expression and TRH prohormone biosynthesis. Although anatomical observations have implicated NPY, alpha -MSH, and AgRP peptides in the regulation of the HPT axis, the stimulation of TRH neurons by alpha -MSH is also shown for the first time in this study. Taken together, these results suggest that leptin may affect TRH neurons via both indirect pathways (alpha -MSH and NPY) and direct pathways via leptin receptors expressed on TRH neurons in the PVN, leading to regulation of both TRH expression and processing.

    FOOTNOTES

* This work was supported by National Science Foundation Grant IBN-9507952 and National Institutes of Health Grant 1 RO1 DK58148-01 (to E. A. N.).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.

§ To whom correspondence and reprint requests should be addressed: Div. of Endocrinology, Rhode Island Hospital, 55 Claverick St., Providence, RI 02903. Tel.: 401-444-5733; Fax: 401-444-6964; E-mail: Eduardo_Nillni@Brown.edu.

Published, JBC Papers in Press, August 30, 2000, DOI 10.1074/jbc.M003549200

    ABBREVIATIONS

The abbreviations used are: TRH, thyrotropin-releasing hormone; HPT, hypothalamic-pituitary-thyroid; TSH, thyrotropin; PVN, paraventricular nucleus; POMC, pro-opiomelanocortin; alpha -MSH, alpha -melanocyte-stimulating hormone; NPY, neuropeptide Y; AgRP, agouti-related peptide; ObR, leptin receptor; STAT, signal transducer and activator of transcription; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; IEFGE, isoelectric focusing gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; RIA, radioimmunoassay; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MC4R, melanocortin-4 receptor; CART, cocaine- and amphetamine-regulated transcript.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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