help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-1242
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/5/2516    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Huo, L.
Right arrow Articles by Bjørbæk, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Huo, L.
Right arrow Articles by Bjørbæk, C.
Endocrinology Vol. 145, No. 5 2516-2523
Copyright © 2004 by The Endocrine Society

Role of Signal Transducer and Activator of Transcription 3 in Regulation of Hypothalamic trh Gene Expression by Leptin

Lihong Huo, Heike Münzberg, Eduardo A. Nillni and Christian Bjørbæk

Department of Medicine (L.H., H.M., C.B.), Division of Endocrinology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston Massachusetts 02215; and Division of Endocrinology (E.A.N.), Department of Medicine, Brown Medical School, Rhode Island Hospital, Providence Rhode Island 02903

Address all correspondence and requests for reprints to: Dr. Christian Bjørbæk, Division of Endocrinology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, Massachusetts 02215. E-mail: cbjorbae{at}bidmc.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
During starvation in rodents, the hypothalamic-pituitary-thyroid axis is down-regulated, resulting in low circulating thyroid hormone levels. This involves a reduction in hypothalamic TRH mRNA that is caused in part by a fall in serum leptin levels, which is sensed by neurons within the hypothalamus. The mechanism by which this regulation occurs is not fully understood. Here we show transfection data and in vivo evidence, suggesting that leptin can regulate trh gene expression via activation of intracellular signal transducer and activator of transcription 3 (STAT3) proteins in TRH neurons. In trh promoter assays using transfected cells, functional STAT3 proteins are required for maximal activation of the trh promoter by leptin. Consistent with this, the STAT3-binding site on the leptin receptor is also required for this regulation. Using double immunohistochemistry, we show that peripherally administered leptin rapidly stimulates STAT3 phosphorylation in approximately 40% of TRH neurons in the paraventricular nucleus of the hypothalamus (PVN) in rats. Detailed anatomical analyses reveal that the leptin-responsive TRH neurons are concentrated in the caudal region of the medial and periventricular parvocellular subnucleus of the PVN. Combined, our data show that only a subpopulation of TRH neurons in the PVN is leptin responsive and suggest that stimulation of hypothalamic trh gene expression by leptin involves activation of STAT3 and that this signaling pathway is important for regulation of the hypothalamic-pituitary-thyroid axis by leptin.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE, THE end product of the neuroendocrine hypothalamic-pituitary-thyroid (HPT) axis, is a key stimulator of energy expenditure, largely through increasing basal metabolic rate in cells (1). TRH is released in the median eminence from nerve terminals of neurons located in the medial and periventricular parvocellular subdivisions of the paraventricular nucleus of the hypothalamus (PVN). This stimulates release of TSH from the pituitary, resulting in secretion of thyroid hormone from the thyroid gland (2, 3, 4). The HPT axis is also regulated by negative feedback mechanisms so that circulating thyroid hormone levels are maintained at a relative constant level (5). However, during fasting in rodents, this feedback system is altered such that expression of TRH and secretion of TSH are both reduced, resulting in a dramatic fall in blood thyroid hormone levels (6, 7). This may serve as an important mechanism to conserve energy by reducing thyroid hormone-dependent thermogenesis until refeeding occurs. Down- regulation of hypothalamic TRH mRNA and circulating thyroid levels during fasting can be prevented by administration of leptin, the adipocyte-derived polypeptide, suggesting a physiological role of this hormone in regulation of the HPT axis in rodents (8, 9).

Leptin acts on the brain to regulate appetite, energy expenditure, and neuroendocrine function (8, 10, 11, 12, 13). The hormone is structurally related to cytokines, and leptin receptors belong to the cytokine receptor superfamily (14). Binding of leptin to the long form of the leptin receptor (ObRb) activates receptor-associated Janus tyrosine kinase 2 that phosphorylates ObRb on specific tyrosine residues (15, 16, 17). Cytoplasmic signal transducer and activator of transcription (STAT3) proteins then bind to receptor phosphotyrosines, specifically tyrosine 1138 near the cytoplasmic tail of ObRb, allowing STAT3 to become phosphorylated on residue Y705 by Janus tyrosine kinase 2 (18, 19, 20). Phosphorylated STAT3 (P-STAT3) dimerizes and enters the nucleus, in which it regulates gene transcription by binding to STAT3-responsive DNA elements (18). We recently demonstrated that leptin induces STAT3 phosphorylation in nuclei of proopiomelanocortin (POMC) neurons in the hypothalamus of rats, and showed that leptin can directly activate the proximal pomc promoter in cells expressing the leptin receptor (21). This process requires STAT3 phosphorylation that is mediated by Y1138 on ObRb, suggesting that STAT3 is critical for mediating genomic effects of leptin to regulate pomc gene-expression and the central melanocortin pathway.

Our previous work shows that leptin can directly regulate pro-TRH biosynthesis and TRH secretion in cultured hypothalamic TRH neurons (22), suggesting direct effects of leptin on TRH neurons in the PVN. This possibility is supported by the demonstration of ObRb mRNA expression within the PVN of rats (23) and by showing that TRH neurons express suppressor of cytokine signaling (SOCS)-3 mRNA (which is a marker of direct leptin action) (24) after leptin administration to rats (25). These studies suggest that leptin may directly affect TRH neurons via expression of ObRb on those cells. Other data support indirect pathways to regulate TRH neurons, including synaptic input from leptin-responsive POMC neurons in the arcuate nucleus of the hypothalamus (26, 27). This mechanism involves leptin-dependent release of the POMC-derived peptide, {alpha}MSH, acting on melanocortin receptors expressed in TRH neurons (25, 28, 29). In the present study, we demonstrate activation of STAT3 tyrosine phosphorylation in TRH neurons by leptin in vivo, show that this occurs in a subpopulation of TRH neurons within the PVN, and provide further evidence suggesting a role of the STAT3 transcription factor to regulate the activity of the trh promoter by leptin.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Recombinant murine leptin was obtained from Dr. E. Parlow (National Institute of Diabetes and Digestive and Kidney Diseases and The National Hormone and Pituitary Program, Torrance, CA). The expression vector encoding the long form of the murine leptin receptor (ObRb WT) was described earlier (16), and its mutant with replacement of tyrosine 1138 to serine (ObRb Y1138S) was provided by Dr. M. Myers (Joslin Diabetes Center, Boston, MA). Dr. S. Melmed (University of California, Los Angeles) and Dr. T. Hirano (Osaka University, Osaka, Japan) provided the wild-type STAT3 and dominant-negative (DN) STAT3 (Y705F) expression vectors. A human (–900/+55) trh promoter-luciferase construct was generated earlier by Dr. A. Hollenberg (25). All reagents for transfection were from Invitrogen Life Technologies, Inc. (Carlsbad, CA). Buffer supply for immunohistochemistry (IHC) was purchased from Sigma (St. Louis, MO), avidin biotin complex vectastain was from Vector Laboratories (Burlingame, CA) and diaminobenzidine developing solution was from Roche (Basel, Germany). Phospho-specific-(Y705)-STAT3 antibodies were purchased from New England Biolabs (Beverly, MA), rabbit-anti-pro-TRH antibody was produced by Dr. E. Nillni (22), goat-antirabbit was from Jackson ImmunoResearch Laboratories (West Grove, PA), and normal goat serum was purchased from Invitrogen Life Technologies. Fluorescent goat antirabbit immunoglobulin conjugate was purchased from Molecular Probes (Eugene, OR).

Cell culture and transient transfection
293T cells were grown in DMEM with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (10 mg/ml) added and incubated at 37 C in 5% CO2. After grown to approximately 70% confluence, cells were transfected with LipofectAMINE according to the recommendations by the manufacturer (Invitrogen Life Technologies. All stimulations were done 12–18 h post transfection. Hormone concentrations and stimulation times are indicated in the figure legends.

Luciferase and ß-galactosidase assays
Cells were lysed and aliquots were used for luciferase assay as described earlier (24). Briefly, luciferin (Molecular Probes) and assay buffer were added simultaneously to the cell lysate, and luciferin was measured for 20 sec in a luminometer (LB 9501, EG&G Berthold, Bad Wildbad, Germany). Using Galacton (Tropix Inc., Bedford, MA), ß-Galactosidase activities were determined as described by the manufacturer and samples were measured in the luminometer for 5 sec.

Animals and IHC
Male Sprague Dawley rats (with femoral vein catheter), 6–7 wk of age, were purchased from Charles River laboratories, Inc. (Wilmington, MA). Animal procedures were in accordance with the guidelines and approval of the Harvard Medical School and Beth Israel Deaconess Medical Center Institutional Animal Care and Use Committees. Rats were either fasted for 48 h or fed ad libitum before iv injection with leptin (1.0 mg/kg body weight) or vehicle (PBS). Animals were then deeply anesthetized with Ketamin (100 µg/ kg body weight) and Xylazine (10 µg/kg body weight), the heart uncovered, and the circulation flushed with 0.9% saline for 5 min via the left ventricle, followed by 10% neutral buffered formalin solution for 30 min. After that, the brain was carefully removed, postfixed for 12–15 h in formalin solution, and finally cryoprotected in 20% sucrose solution. Brains were frozen in dry ice and cut in 25-µm-thick coronal sections on a sliding microtome, collected in five series, and stored in 0.02% sodium azide containing PBS at 4 C until further use.

For double IHC, free-floating tissue sections were used. For P-STAT3 IHC, the procedure was generally performed as described before (21). Briefly, the tissue was pretreated with 1% NaOH and 1% H2O2 in H2O for 20 min, 0.3% glycine for 10 min, and 0.03% SDS for 10 min. After that, sections were blocked for 1 h with 3% goat serum in PBS/0.25% TritonX-100/ 0.2% sodium azide. The P-STAT3 antibody was then added (1:3000) and incubated over night at room temperature. On the next day, sections were washed, incubated with biotinylated secondary goat antirabbit antibody for 2 h (1:1,000), and then treated with avidin biotin complex solution for 1 h. Finally, the signal was developed with diaminobenzidine solution, giving a brown precipitate. Consecutively, IHC for TRH was performed by incubating sections in 0.3% H2O2 to block endogenous peroxide, blocked in 3% normal goat serum in PBS/0.25% Triton X-100/0.02% sodium azide for 1 h, and incubated overnight at room temperature with the primary antibody (anti-pro-TRH, 1:50,000). On the next day, sections were washed, incubated with a fluorescent secondary antibody for 1 h (2 µg/ml) generating green fluorescence. Results were visualized using either fluorescence (TRH) or bright-field light (P-STAT3) sources and captured with a digital camera (AxioCam, Carl Zeiss, Thornwood, NY) mounted on a Zeiss microscope (Axioscope2, Carl Zeiss). Using Adobe Photoshop software (Adobe, San Jose, CA), fluorescence and bright-field photographs were combined using RGB channels to visualize double-labeled cells.

Counting of TRH and P-STAT3 positive cells
One of five series obtained from each animal was subjected to double-IHC for TRH and P-STAT3 immunoreactivity as described above. For counting of single- and double-labeled TRH and P-STAT3-positive cells, all brain sections in the series containing regions of the PVN were analyzed (total of 9 ± 1 sections for each animal, Bregma –0.9 mm to –2.1 mm). Cell counts were obtained from both sides of the brain in each section. All sections were carefully examined by dark-field microscopy to assign positive cells to appropriate subdivisions of the PVN using locations of adjacent landmarks. Sections were also organized systematically in a rostral to caudal manner according to the rat brain atlas (30). TRH-positive cells, P-STAT3-positive cells, and double-labeled cells were scored in each PVN section from fed and fasted leptin-treated animals. Estimates of the total number of single- and dual-labeled cells in the entire PVN was obtained by multiplying the cell counts by five to account for the fact that only one of the five series were analyzed from each animal. No P-STAT3 staining was detected in the PVN of PBS-treated rats. Data are shown as means ± SE.

Brown nuclear P-STAT3 stain was easy to distinguish in individual cells. A relative dense staining of TRH fibers within the PVN did, however, make identification of TRH cell bodies somewhat difficult in some areas, but this was overcome by verification at higher magnification of each field. The main source for errors in these analyses is likely double counting of the same cells because some neurons may extend through several sections. Whereas this problem is somewhat reduced by counting only every fifth section through the PVN, the obtained cell counts for the entire PVN may be slight overestimates.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leptin stimulates the activity of the human trh promoter via STAT3 in transfected 293T cells
In vivo (31, 32) and in vitro (16, 17) studies show that STAT3 is activated by the leptin receptor, ObRb. As demonstrated previously, a STAT3-like-response element (TTCCGGAGGA) is present in the proximal (–141 to –132) human trh promoter and mutagenesis of this site results in loss of leptin responsiveness of the promoter in transfected cells (25), suggesting a role of STAT3 in regulation of trh transcription by leptin in TRH neurons. To investigate this possibility further, we transiently transfected reporter plasmids containing proximal sequences of the human (–900 to +55) trh promoter located upstream of the luciferase reporter gene in the promoterless PA3LUC vector into the human embryonic kidney cell line, 293T. Cells were first cotransfected with ObRb expression vectors together with either wild-type (WT) STAT3 or empty vector (pcDNA3). Approximately 16 h following transfection, cells were left untreated or treated with 40 nM leptin for 6 h. As shown in Fig. 1AGo, measurements of luciferase activities in lysates from mock-transfected cells demonstrated stimulation of the human trh promoter by approximately 4-fold, whereas coexpression of STAT3 increased this response to approximately 9-fold. In Fig. 1BGo, cells were cotransfected with ObRb plasmids, together with either WT or DN STAT3 (Y705F) expression vectors, and the cells were treated for 6 h as described above. As shown, expression of DN STAT3 strongly attenuates (>70%) the leptin response. The intracellular domain of the long form of the murine leptin receptor contains three conserved tyrosine residues, in which the C-terminal Y1138 is known to mediate leptin-dependent binding and activation of STAT3 (19, 33, 34). To further support the finding that STAT3 is critical for regulation of trh promoter activity by leptin, we therefore tested the significance of Y1138 for this effect. In Fig. 1CGo, cells were cotransfected with either the WT or mutated (Y1138S) ObRb expression plasmids and treated with leptin for 6 h before measurements of luciferase activities. As shown, cells expressing leptin receptors lacking Y1138 showed a marked reduction (~70%) in promoter activity in response to leptin, compared with cells expressing the WT ObRb. Combined, these results demonstrate that Y1138 of ObRb and functional STAT3 proteins are required for maximal activation of the trh promoter by leptin in 293T cells.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 1. Leptin stimulates the human trh promoter via STAT3 in transfected 293T cells. A, Human embryonic kidney cells (293T) were transfected with ObRb and human trh-luc reporter constructs. Empty vector (pcDNA3) or WT STAT3 plasmids were also cotransfected into the cells. B, Cells were transfected with vectors encoding WT STAT3 or DN STAT3 proteins, together with the human trh-luc reporter construct and ObRb plasmids. C, Cells were transfected with ObRb or ObRb lacking tyrosine 1138 (Y1138S) vectors, together with the human trh-luc reporter construct. In all experiments, cells were left untreated (black bars) or treated with 40 nM leptin for 6 h (open bars). A CMV-lacZ control vector was also cotransfected into the cells to normalize luciferase activities. Transfections and treatments were done three times, each time in triplicates. Shown is one representative experiment. Data are means ± SEM.

 
Leptin rapidly stimulates STAT3 phosphorylation in TRH neurons in the PVN in rats
To further support the suggested role of STAT3 in trh gene regulation by leptin, we next determined whether STAT3 is phosphorylated in hypothalamic TRH neurons of leptin-treated rats. For this purpose, we developed a double-IHC method to colocalize P-STAT3 proteins and pro-TRH peptides. Ad libitum-fed Sprague Dawley rats were given a single iv injection of recombinant leptin (1.0 mg/kg body weight) or vehicle (PBS) and killed 45 min later. Twenty-five- micrometer-thick coronal brain sections were subjected to double-IHC as described above. Brown nuclear staining represents P-STAT3 and green cytoplasmic color represents TRH. Figure 2AGo shows one set of representative low-magnification microphotographs of sections from the PVN of PBS- and leptin-treated rats. Consistent with our earlier report (21), a dense population of cells with nuclear STAT3 staining was seen in the PVN from leptin-treated rats (Fig. 2AGo, bottom left). In striking contrast, very few P-STAT3-positive-cells were found in the PVN from PBS-treated animals (Fig. 2AGo, top left). Dense populations of TRH-containing cell bodies and processes were found in the same fields of both sections (Fig. 2AGo, middle photographs). The merged images of the left and middle panels are shown in the right panels of Fig. 2AGo. High numbers of double-labeled cells were found in the leptin-treated animals. As described further below, many single-labeled P-STAT3 and TRH neurons were also seen within the PVN. Examples of double-labeled neurons and of single-labeled P-STAT3 cells from the leptin-treated animal are shown by high-magnification microphotographs in Fig. 2BGo. To investigate whether starvation influences induction of STAT3 phosphorylation in the PVN by leptin, we also performed IHC in 48-h fasted rats injected with leptin as described above for fed rats. As indicated in Fig. 2CGo, similar levels of P-STAT3 staining was detected in the PVN of fasted (Fig. 2CGo, upper) and fed (Fig. 2CGo, bottom) animals after leptin treatment.



View larger version (73K):
[in this window]
[in a new window]
  		FIG. 2. Leptin rapidly stimulates STAT3 phosphorylation in TRH neurons in the PVN. Fed rats were given a single iv injection of recombinant leptin (1.0 mg/kg) or vehicle (PBS) and killed 45 min later. Coronal brain sections were obtained and subjected to double IHC using anti-P-STAT3 (brown staining) and anti-pro-TRH (green fluorescence staining) antiserum. A, Low magnification. Upper panels are from a PBS-treated rat and the bottom panels from a leptin-treated rat. Left images, P-STAT3 IHC; middle, TRH IHC; right, merged images of the left and middle images. B, High magnification of areas marked in A. Vertical arrows point to single-labeled P-STAT3 cells and horizontal arrows to dual-labeled cells. C, Representative examples of P-STAT3 staining in the PVN from leptin-treated fasted (upper) and fed (bottom) rats. 3v, Third ventricle. Scale bars, 100 µm (A), 10 µm (B),100 µm (C).

 
Leptin-responsive TRH neurons are concentrated in the caudal region of the PVN
Leptin-responsive cells in the PVN have not earlier been characterized in an anatomical and chemical manner. We therefore systematically counted double-labeled P-STAT3 and TRH cells throughout the PVN in four leptin-treated rats. The results from one representative rat are presented in Figs. 3Go and 4Go. Schematic drawings of three sections, ordered in rostral-to-caudal manner, depicting location of single- labeled TRH neurons and TRH neurons containing P-STAT3, are shown in Fig. 3AGo. In these three sections we found that 5, 15, and 75% of TRH neurons, respectively, were positive for P-STAT3. In addition, the leptin-responsive TRH neurons in the PVN were almost exclusively located in the parvocellular region near the third ventricle. Results from all sections (n = 9) that contained TRH neurons in the PVN from this rat are depicted in Fig. 3BGo. A total of 464 cells were TRH positive and 221 of these were colocalized with P-STAT3 in the PVN in the nine sections from this animal. After similar analysis of sections from three additional leptin-treated fed animals and by accounting for the fact that only every fifth section through the PVN was counted in each animal, we estimate that a total of approximately 2200 (± 13%) TRH cells exist in the entire PVN of the rat brain, of which approximately 42% (± 14%) are P-STAT3-positive. However, in the most caudal region of the PVN, nearly 80% were double labeled, whereas only 4–10% doubles were detected in the rostral sections (Fig. 3CGo). A similar rostral-to-caudal distribution and percentage of double-labeled cells were obtained from analysis of sections from a leptin-treated rat that was fasted for 48 h (not shown). Combined, these IHC studies show that STAT3 is rapidly phosphorylated in TRH neurons in vivo in response to exogenous leptin administration. In addition, our data suggest that approximately 40% of TRH neurons are P-STAT3 positive, and these leptin-responsive cells are concentrated in the caudal part of the PVN.



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 3. Leptin-responsive TRH neurons are concentrated in the caudal region of the PVN. Shown are results from the counting of one series of sections (n = 9) of the PVN from one leptin-treated rat in a rostral-to-caudal manner. The animal was treated and brain sections obtained as described in Fig. 2Go. A, Schematic drawings of three of the nine sections ordered in a rostral-to-caudal manner. Filled circles represent P-STAT3-positive TRH neurons, and open circles indicate single-labeled TRH cell bodies. B, Cell counts of TRH and TRH/P-STAT3 dual-labeled neurons for each section encompassing the PVN in the leptin-treated rat from A. Results depicted in C are derived from B and shows the percent of dual- labeled cells in each section. 3v, Third ventricle; f, fornix.

 


View larger version (25K):
[in this window]
[in a new window]
  		FIG. 4. Leptin activates STAT3 phosphorylation in neurons located in the caudal region of the PVN. Shown are results from counting of one series of sections (n = 9) of the PVN from the same leptin-treated rat presented in Fig. 3Go. A, Schematic drawings of three sections ordered in a rostral-to-caudal manner. Filled circles represent P-STAT3-positive TRH neurons, and x indicates single-labeled P-STAT3 neurons. B, Cell counts of P-STAT3 and TRH/P-STAT3 dual-labeled neurons for each section encompassing the PVN in the leptin-treated rat from A. Results in C are derived from B and depicts the percent of dual-labeled cells in each section. 3v, Third ventricle; f, fornix.

 
Schematic drawings of three sections, ordered in a rostral-to-caudal manner, depict the location of P-STAT3-positive neurons and P-STAT3 cells containing TRH cytoplasmic immunoreactivity (Fig. 4AGo). In these three sections, we found that 25, 35, and 84% of P-STAT3-positive neurons were TRH neurons, respectively. P-STAT3 staining was most dense in the parvocellular region near the third ventricle and in the caudal region of the PVN. Analyses of all sections (n = 9) containing P-STAT3-positive neurons in the PVN from one representative animal are shown in Fig. 4Go, B and C. Similar results were obtained from analysis of a leptin-treated fasted animal (not shown). Interestingly, of an estimated approximately 2150 P-STAT3 cells in the entire PVN of leptin-treated rats, only about 43% (± 12%) were TRH neurons, suggesting that other types of neurons in the PVN are activated by leptin.

Leptin-responsive TRH neurons are found only within the PVN of the hypothalamus
In addition to the PVN, TRH neurons exist in several other regions of the hypothalamus such as the lateral hypothalamic area, the dorsomedial hypothalamus, arcuate nucleus, and the preoptic area (35). Because these populations of neurons have no known projections to the median eminence and are not regulated in conjunction with the thyrotropic neurons of the PVN, it is presumed that they do not serve a hypophysiotropic function (36, 37). As we reported earlier (21), leptin-dependent STAT3 phosphorylation is also present in the lateral hypothalamic area the arcuate, and the dorsomedial hypothalamus leaving the possibility that TRH neurons outside the PVN could also be leptin responsive. But as indicated in Fig. 5Go, leptin-responsive (P-STAT3 positive) TRH neurons are uniquely found within the PVN, whereas TRH neurons located elsewhere in the hypothalamus were not activated by leptin as measured by STAT3 phosphorylation.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 5. Leptin-responsive TRH neurons are uniquely located in the PVN. A and B, Schematic drawings of the same section of the hypothalamus from a leptin-treated rat. Open circles indicate TRH cell bodies, x indicates P-STAT3-positive neurons, and closed circles depict double-labeled neurons. Animals were treated and brain sections obtained as described in Fig. 2Go and in Materials and Methods. LH, Lateral hypothalamic area; PaMP, Pa medial parvicellular; f, fornix; opt, optic tract; Arc, arcuate nucleus.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using IHC analyses, we have previously shown that leptin can stimulate phosphorylation of the STAT3 transcription factor in the PVN of rats (21). However, a detailed description of the anatomical location and neurochemical nature of leptin-responsive cells at this site has not been reported. We demonstrate here that leptin can induce STAT3 phosphorylation in TRH neurons located in the parvocellular subdivision of the PVN of rats. Based on the rapid nature of this activation after peripheral leptin administration (<45 min), together with the previously reported expression of ObRb mRNA in the PVN (23) and the known capacity of leptin to stimulate intracellular STAT3 tyrosine phosphorylation in cells expressing ObRb (16), we conclude that leptin is likely to enter the brain from the circulation and act directly on TRH neurons that express ObRb. Furthermore, we show that leptin does not activate STAT3 in TRH neurons located in other regions of the hypothalamus, including the perifornical and lateral hypothalamic areas. These results, together with the well-described contribution of the parvocellular TRH neurons to the tuberoinfundibular system (38, 39, 40, 41), suggest that leptin specifically influences the TRH neurons that are involved in regulation of the neuroendocrine hypothalamic-pituitary-thyroid axis.

Earlier studies suggest that leptin receptor-dependent activation of STAT3 DNA-binding activity plays a critical role in stimulation of the trh promoter by leptin in transfected cell lines (25). Moreover, activated STAT3 proteins present in the nucleus of leptin-treated cells can bind to a specific DNA sequence of the proximal human and murine trh promoter, suggesting that this STAT3-element is important for transcriptional activation of the trh gene by leptin (25). We here extend those findings by demonstrating that expression of DN STAT3 proteins in transfected cells ablates leptin-stimulated activation of a trh promoter reporter plasmid. Furthermore, a mutant of ObRb that lacks its STAT3 binding site (tyrosine 1138) is strongly reduced in its capacity to activate the trh promoter in response to leptin. These studies therefore suggest that STAT3 in required for maximal stimulation of the trh promoter by leptin. Our data showing leptin-dependent activation of STAT3 phosphorylation in nuclei of TRH neurons in vivo support this hypothesis. However, to conclusively prove this, more complex studies are required, including chromosomal-immunoprecipitation experiments demonstrating leptin-induced STAT3 binding to the trh promoter in vivo or generation of transgenic mice overexpressing DN STAT3 proteins in TRH neurons or conditional deletion of the endogenous stat3 gene in TRH cells. We did not find evidence for differences in P-STAT3 activation by leptin in the PVN between fed and fasted rats. This could imply that leptin may stimulate trh transcription in both fed and fasted animals, although this has not been carefully examined. Alternatively, activation of STAT3 in the fed state may not lead to stimulation of TRH mRNA due to increased activities of factors that limit trh gene-expression. Further studies will determine this.

In total, we estimate that approximately 2150 leptin-responsive (STAT3-positive) cells exist in the PVN of the rat. The majority of these were found in the caudal regions of the periventricular parvocellular subnucleus of the PVN, where approximately 80–90% contained cytoplasmic TRH immunoreactivity. This location of TRH-producing neurons is consistent with earlier mapping studies showing that this region has one of the greatest densities of TRH perikarya in the hypothalamus (35, 42). Whereas both leptin-responsive cells and TRH neurons were detected in several other nuclei of the hypothalamus, none of these cells were positive for both P-STAT3 and TRH. Interestingly, approximately 57% of all leptin-responsive cells in the PVN were not TRH neurons, suggesting that leptin can affect the activity of other types of neurons in this hypothalamic nucleus. Putative candidates include CRH-producing cells, but additional double-labeling studies will test this possibility. Moreover, of the estimated 2200 TRH-producing cells in the PVN, about 58% were not responsive to leptin, suggesting that other neuronal and/or hormonal inputs may be important for these cells. In the present studies, we cannot entirely exclude the possibility that additional TRH neurons within the PVN are responsive to leptin, due to either activation at earlier or later time points after leptin injection, compared with the 45 min used in this study or submaximal sensitivity of one of the antibodies. Arguing against this is the fact that we obtained similar percentages of double-labeled cells as early as 20 min after leptin administration (not shown) and that the vast majority of TRH neurons in the most caudal regions were double labeled.

Starvation of rodents is associated with a rapid fall in circulating thyroid hormone concentrations and a reduction in TRH mRNA levels in the PVN, both of which can be reversed by leptin treatment, providing evidence that leptin can regulate the neuroendocrine thyroid axis (8, 26). This regulation by leptin has been suggested to occur via both direct actions of leptin on TRH neurons that express leptin receptors and via indirect signals from leptin-responsive neurons that project to the TRH neurons (43). The data supporting indirect regulation suggest that this is mediated via synaptic input from ObRb-expressing neurons located in the arcuate nucleus of the hypothalamus (26, 27, 44), namely the POMC and neuropeptide Y (NPY) neurons that are directly activated and inhibited by leptin, respectively (21, 45, 46, 47). Anatomical evidence supporting the indirect mechanism includes findings that both NPY receptors and melanocortin-4 receptors (MC4-R) are expressed in TRH neurons (48, 49). The MC4-R is a high-affinity receptor for the key peptide product of the pomc gene, namely {alpha}MSH (50, 51, 52) and is also a receptor for the {alpha}MSH antagonist, agouti-related-peptide (AgRP) (53, 54, 55, 56, 57), which is coexpressed with a proportion of hypothalamic NPY-expressing neurons (58, 59). In addition, ultrastructural analyses suggest that both POMC and NPY/AgRP neurons provide synaptic input to TRH neurons (44). POMC neurons also coexpress cocaine and amphetamine regulate transcript (60) and axon terminals containing this peptide can be detected on TRH cell bodies in the PVN (27). Pharmacological results also support indirect pathways to influence TRH neurons. Specifically, data show that: 1) in transfected cells, the trh promoter is responsive to cAMP, an intracellular signaling mediator of the MC4-R (25); 2) in ex vivo hypothalamic extracts, leptin-induced release of TRH peptides is blocked by AgRP (61); 3) treatment of the same hypothalamic extracts with {alpha}MSH alone stimulates TRH release (62); and 4) in primary hypothalamic neuronal cultures, the synthetic MC4-R antagonist, SHU9119, partially inhibits leptin-stimulated TRH release (22).

On the other hand, consistent with a pathway whereby leptin directly influences TRH neurons are studies showing that: 1) leptin can stimulate the TRH promoter in isolated cells expressing the ObRb (25); 2) ObRb mRNA is present in the PVN of rats (23) and ObR proteins are expressed in TRH neurons in hypothalamic cell cultures (22); 3) leptin can induce mRNA expression of the leptin-responsive marker, SOCS-3, in TRH neurons in the PVN in vivo (25); 4) leptin can rapidly regulate neuronal polarization of neurons in isolated brain slices of the PVN (63); and 5) leptin can stimulate release of TRH peptides from dispersed hypothalamic cultures (22). In addition, we demonstrate here that TRH neurons in the PVN are rapidly responsive to leptin using an assay that detects a known downstream signal (STAT3 phosphorylation) of the leptin receptor. Although it cannot be entirely ruled out from this study that STAT3 may be activated by leptin via other factors that ultimately act on TRH neurons, we consider this less likely based on the above reasons listed in support of the direct pathway and on the fact that hypothalamic STAT3 is activated as quickly as 20 min after leptin administration (not shown) and is activated at sites that overlap well with known sites of ObRb mRNA expression in the rat (23). Demonstration of functional leptin receptor protein expression in TRH neurons in the PVN are required to ultimately prove this, but such analyses have been hampered the by lack of sensitive and specific anti-ObR antibodies suitable for IHC.

The percentage of leptin-responsive TRH neurons (>40%) in the PVN found in this study is substantially higher, compared with earlier estimates. In primary hypothalamic neurons from fetal rats, approximately 10% of TRH neurons were reported to express ObR proteins (22), and 8–13% of TRH neurons in the medial parvocellular division of the PVN were shown to express SOCS-3 mRNA in leptin-treated rats (25). These discrepancies are most likely due to a superior sensitivity of the IHC assay for phosphorylated STAT3 used in the present study vs. in situ hybridization detection for SOCS-3 and TRH mRNA (25). In addition, the percentage of TRH neurons that express leptin receptors in primary hypothalamic cultures may well differ from that found in vivo because we specifically investigated the PVN in adult rats, whereas the primary neuronal cells were grown from whole fetal hypothalami (22).

Important questions that remain include whether functionally different subsets of TRH neurons in the parvocellular region of the PVN exist. Possibilities include one subset that is directly responsive to leptin, a separate subset that is responsive to neuropeptide/neurotransmitter input from other leptin-responsive cells like POMC/cocaine and amphetamine regulate transcript and/or NPY/AgRP neurons and/or a third population of cells that responds to both direct and indirect signals. The relative importance of the direct vs. indirect inputs to hypophysiotropic TRH neurons is yet unknown, and it is intriguing to speculate whether these pathways play specific roles in regulation of the thyroid axis under different physiological conditions. Finally, it is not clear whether all TRH neurons in parvocellular PVN that respond directly or indirectly to leptin regulate the neuroendocrine thyroid axis or whether some TRH neurons subserve other or additional functions, like regulation of appetite or the autonomic nervous system.


   Acknowledgments
 
We thank Dr. A. N. Hollenberg (Boston, MA) and Dr. J. K. Elmquist (Boston, MA) for valuable advice with regard to the manuscript.


   Footnotes
 
This work was supported by National Institutes of Health Grant (RO1 DK60673 to C.B.), National Institutes of Health Grant (RO1 DK58148 to E.A.N.), and an Emmy-Noether grant from the Deutsche Forschungsgemeinschaft (MU 1662/2-1 to H.M.).

L.H. and H.M. contributed equally to this work.

Abbreviations: AgRP, Agouti-related-peptide; DN, dominant negative; HPT, hypothalamic-pituitary-thyroid; IHC, immunohistochemistry; MC4-R, melanocortin-4 receptor; NPY, neuropeptide Y; ObRb, long form of the leptin receptor; POMC, proopiomelanocortin; P-STAT3, phosphorylated STAT3; PVN, paraventricular nucleus of the hypothalamus; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; WT, wild-type.

Received September 18, 2003.

Accepted for publication January 27, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Freake HC, Oppenheimer JH 1995 Thermogenesis and thyroid function. Nutrition 15:263–291
  2. Hollander CS, Mitsuma T, Shenkman L, Woolf P, Gershengorn MC 1972 Thyrotropin-releasing hormone: evidence for thyroid response to intravenous injection in man. Science 175:209–210[Abstract/Free Full Text]
  3. Shenkman L, Mitsuma T, Suphavai A, Hollander CS 1972 Triiodothyronine and thyroid-stimulating hormone response to thyrotrophin-releasing hormone. A new test of thyroidal and pituitary reserve. Lancet 1:111–112[CrossRef][Medline]
  4. Dannies PS, Tashjian Jr AH1974 Pyroglutamyl-histidyl-prolineamide (TRH). A neurohormone which affects the release and synthesis of prolactin and thyrotropin. Isr J Med Sci 10:1294–1304
  5. Toni R, Lechan RM 1993 Neuroendocrine regulation of thyrotropin-releasing hormone (TRH) in the tuberoinfundibular system. J Endocrinol Invest 16:715–753[Medline]
  6. Blake NG, Eckland DJ, Foster OJ, Lightman SL 1991 Inhibition of hypothalamic thyrotropin-releasing hormone messenger ribonucleic acid during food deprivation. Endocrinology 129:2714–2718[Abstract]
  7. Rondeel JM, Heide R, de Greef WJ, van Toor H, van Haasteren GA, Klootwijk W, Visser TJ 1992 Effect of starvation and subsequent refeeding on thyroid function and release of hypothalamic thyrotropin-releasing hormone. Neuroendocrinology 56:348–353[Medline]
  8. Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, Flier JS 1996 Role of leptin in the neuroendocrine response to fasting. Nature 382:250–252[CrossRef][Medline]
  9. Legradi G, Emerson CH, Ahima RS, Flier JS, Lechan RM 1997 Leptin prevents fasting-induced suppression of prothyrotropin-releasing hormone messenger ribonucleic acid in neurons of the hypothalamic paraventricular nucleus. Endocrinology 138:2569–2576[Abstract/Free Full Text]
  10. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM 1994 Positional cloning of the mouse obese gene and its human homologue. Nature 372:425–432[CrossRef][Medline]
  11. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F 1995 Effects of the obese gene product on body weight regulation in ob/ob mice. Science 269:540–543[Abstract/Free Full Text]
  12. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, Rabinowitz D, Lallone RL, Burley SK, Friedman JM 1995 Weight-reducing effects of the plasma protein encoded by the obese gene. Science 269:543–546[Abstract/Free Full Text]
  13. Campfield LA, Smith FJ, Burn P 1998 Strategies and potential molecular targets for obesity treatment. Science 280:1383–1387[Abstract/Free Full Text]
  14. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, Devos R, Richards GJ, Campfield LA, Clark FT, Deeds J, Muir C, Sanker S, Moriarty A, Moore KJ, Smutko JS, Mays GG, Woolf EA, Monroe CA, Tepper RI 1995 Identification and expression cloning of a leptin receptor, OB-R. Cell 83:1263–1271[CrossRef][Medline]
  15. Ghilardi N, Ziegler S, Wiestner A, Stoffel R, Heim MH, Skoda RC 1996 Defective STAT signaling by the leptin receptor in diabetic mice. Proc Natl Acad Sci USA 93:6231–6235[Abstract/Free Full Text]
  16. Bjorbaek C, Uotani S, da Silva B, Flier JS 1997 Divergent signaling capacities of the long and short isoforms of the leptin receptor. J Biol Chem 272:32686–32695[Abstract/Free Full Text]
  17. Baumann H, Morella KK, White DW, Dembski M, Bailon PS, Kim H, Lai CF, Tartaglia LA 1996 The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc Natl Acad Sci USA 93:8374–8378[Abstract/Free Full Text]
  18. Ihle JN 1995 Cytokine receptor signalling. Nature 377:591–594[CrossRef][Medline]
  19. White DW, Kuropatwinski KK, Devos R, Baumann H, Tartaglia LA 1997 Leptin receptor (OB-R) signaling. Cytoplasmic domain mutational analysis and evidence for receptor homo-oligomerization. J Biol Chem 272:4065–4071[Abstract/Free Full Text]
  20. Bjorbaek C, Lavery HJ, Bates SH, Olson RK, Davis SM, Flier JS, Myers Jr MG 2000 SOCS3 mediates feedback inhibition of the leptin receptor via Tyr985. J Biol Chem 275:40649–40657[Abstract/Free Full Text]
  21. Munzberg H, Huo L, Nillni EA, Hollenberg AN, Bjorbaek C 2003 Role of signal transducer and activator of transcription 3 in regulation of hypothalamic proopiomelanocortin gene expression by leptin. Endocrinology 144:2121–2131[Abstract/Free Full Text]
  22. Nillni EA, Vaslet C, Harris M, Hollenberg A, Bjorbaek C, Flier JS 2000 Leptin regulates prothyrotropin-releasing hormone biosynthesis. Evidence for direct and indirect pathways. J Biol Chem 275:36124–36133[Abstract/Free Full Text]
  23. Elmquist JK, Bjorbaek C, Ahima RS, Flier JS, Saper CB 1998 Distributions of leptin receptor mRNA isoforms in the rat brain. J Comp Neurol 395:535–547[CrossRef][Medline]
  24. Bjorbaek C, Elmquist JK, Frantz JD, Shoelson SE, Flier JS 1998 Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol Cell 1:619–625[CrossRef][Medline]
  25. Harris M, Aschkenasi C, Elias CF, Chandrankunnel A, Nillni EA, Bjorbaek C, Elmquist JK, Flier JS, Hollenberg AN 2001 Transcriptional regulation of the thyrotropin-releasing hormone gene by leptin and melanocortin signaling. J Clin Invest 107:111–120[Medline]
  26. Legradi G, Emerson CH, Ahima RS, Rand WM, Flier JS, Lechan RM 1998 Arcuate nucleus ablation prevents fasting-induced suppression of ProTRH mRNA in the hypothalamic paraventricular nucleus. Neuroendocrinology 68:89–97[CrossRef][Medline]
  27. Fekete C, Legradi G, Mihaly E, Huang QH, Tatro JB, Rand WM, Emerson CH, Lechan RM 2000 {alpha}-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression. J Neurosci 20:1550–1558[Abstract/Free Full Text]
  28. Elmquist JK 2001 Hypothalamic pathways underlying the endocrine, autonomic, and behavioral effects of leptin. Int J Obes Relat Metab Disord 25(Suppl 5):S78–S82
  29. Liu H, Kishi T, Roseberry AG, Cai X, Lee CE, Montez JM, Friedman JM, Elmquist JK 2003T transgenic mice expressing green fluorescent protein under the control of the melanocortin-4 receptor promoter. J Neurosci 23:7143–7154
  30. Paxinos G, Watson C 1998 The rat brain in stereotaxic coordinates. San Diego: Academic Press
  31. Vaisse C, Halaas JL, Horvath CM, Darnell Jr JE, Stoffel M, Friedman JM 1996 Leptin activation of Stat3 in the hypothalamus of wild-type and ob/ob mice but not db/db mice. Nat Genet 14:95–97[CrossRef][Medline]
  32. El-Haschimi K, Pierroz DD, Hileman SM, Bjorbaek C, Flier JS 2000 Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity. J Clin Invest 105:1827–1832[Medline]
  33. Banks AS, Davis SM, Bates SH, Myers Jr MG 2000 Activation of downstream signals by the long form of the leptin receptor. J Biol Chem 275:14563–14572[Abstract/Free Full Text]
  34. Lechan RM, Jackson IM 1999 Leptin receptor activation of SH2 domain containing protein tyrosine phosphatase 2 modulates Ob receptor signal transduction. Proc Natl Acad Sci USA 96:9677–9682[Abstract/Free Full Text]
  35. Lechan RM, Jackson IM 1982 Immunohistochemical localization of thyrotropin-releasing hormone in the rat hypothalamus and pituitary. Endocrinology 111:55–65[Abstract]
  36. Ishikawa K, Taniguchi Y, Inoue K, Kurosumi K, Suzuki M 1988 Immunocytochemical delineation of thyrotropic area: origin of thyrotropin-releasing hormone in the median eminence. Neuroendocrinology 47:384–388[Medline]
  37. Nillni EA, Sevarino KA 1999 The biology of pro-thyrotropin-releasing hormone-derived peptides. Endocr Rev 20:599–648[Abstract/Free Full Text]
  38. Lechan RM, Nestler JL, Jacobson S, Reichlin S 1980 The hypothalamic ’tuberoinfundibular’ system of the rat as demonstrated by horseradish peroxidase (HRP) microiontophoresis. Brain Res 195:13–27[CrossRef][Medline]
  39. Wiegand SJ, Price JL 1980 Cells of origin of the afferent fibers to the median eminence in the rat. J Comp Neurol 192:1–19[CrossRef][Medline]
  40. Martin JB, Reichlin S 1972 Plasma thyrotropin (TSH) response to hypothalamic electrical stimulation and to injection of synthetic thyrotropin releasing hormone (TRH). Endocrinology 90:1079–1085[Medline]
  41. Aizawa T, Greer MA 1981 Delineation of the hypothalamic area controlling thyrotropin secretion in the rat. Endocrinology 109:1731–1738[Abstract]
  42. Brownstein MJ, Palkovits M, Saavedra JM, Bassiri RM, Utiger RD 1974 Thyrotropin-releasing hormone in specific nuclei of rat brain. Science 185:267–269[Abstract/Free Full Text]
  43. Bjorbaek C, Hollenberg AN 2002 Leptin and melanocortin signaling in the hypothalamus. Vitam Horm 65:281–311[Medline]
  44. Mihaly E, Fekete C, Tatro JB, Liposits Z, Stopa EG, Lechan RM 2000 Hypophysiotropic thyrotropin-releasing hormone-synthesizing neurons in the human hypothalamus are innervated by neuropeptide Y, agouti-related protein, and {alpha}-melanocyte-stimulating hormone. J Clin Endocrinol Metab 85:2596–2603[Abstract/Free Full Text]
  45. Wang Q, Bing C, Al-Barazanji K, Mossakowaska DE, Wang XM, McBay DL, Neville WA, Taddayon M, Pickavance L, Dryden S, Thomas ME, McHale MT, Gloyer IS, Wilson S, Buckingham R, Arch JR, Trayhurn P, Williams G 1997 Interactions between leptin and hypothalamic neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat. Diabetes 46:335–341[Abstract]
  46. Cheung CC, Clifton DK, Steiner RA 1997 Proopiomelanocortin neurons are direct targets for leptin in the hypothalamus. Endocrinology 138:4489–4492[Abstract/Free Full Text]
  47. Baskin DG, Schwartz MW, Seeley RJ, Woods SC, Porte Jr D, Breininger JF, Jonak Z, Schaefer J, Krouse M, Burghardt C, Campfield LA, Burn P, Kochan JP 1999 Leptin receptor long-form splice-variant protein expression in neuron cell bodies of the brain and colocalization with neuropeptide Y mRNA in the arcuate nucleus. J Histochem Cytochem 47:353–362[Abstract/Free Full Text]
  48. Broberger C, Visser TJ, Kuhar MJ, Hokfelt T 1999 Neuropeptide Y innervation and neuropeptide-Y-Y1-receptor-expressing neurons in the paraventricular hypothalamic nucleus of the mouse. Neuroendocrinology 70:295–305[CrossRef][Medline]
  49. Elmquist JK, Marcus JN 2003 Rethinking the central causes of diabetes. Nat Med 9:645–647[CrossRef][Medline]
  50. Marsh DJ, Hollopeter G, Huszar D, Laufer R, Yagaloff KA, Fisher SL, Burn P, Palmiter RD 1999 Response of melanocortin-4 receptor-deficient mice to anorectic and orexigenic peptides. Nat Genet 21:119–122[CrossRef][Medline]
  51. Oosterom J, Nijenhuis WA, Schaaper WM, Slootstra J, Meloen RH, Gispen WH, Burbach JP, Adan RA 1999 Conformation of the core sequence in melanocortin peptides directs selectivity for the melanocortin MC3 and MC4 receptors. J Biol Chem 274:16853–16860[Abstract/Free Full Text]
  52. Butler AA, Cone RD 2003 Knockout studies defining different roles for melanocortin receptors in energy homeostasis. Ann NY Acad Sci 994:240–245[Abstract/Free Full Text]
  53. Yang Y, Chen M, Lai Y, Gantz I, Yagmurlu A, Georgeson KE, Harmon CM 2003 Molecular determination of agouti-related protein binding to human melanocortin-4 receptor. Mol Pharmacol 64:94–103[Abstract/Free Full Text]
  54. Bagnol D, Lu X-Y, Kaelin CB, Day HEW, Ollmann M, Gantz I, Akil H, Barsh GS, Watson SJ 1999 Anatomy of an endogenous antagonist: relationship between agouti-related protein and proopiomelanocortin in brain. J Neurosci 19:RC26
  55. Haskell-Luevano C, Cone RD, Monck EK, Wan YP 2001 Structure activity studies of the melanocortin-4 receptor by in vitro mutagenesis: identification of agouti-related protein (AGRP), melanocortin agonist and synthetic peptide antagonist interaction determinants. Biochemistry 10:6164–6179
  56. Nijenhuis WA, Oosterom J, Adan RA 2001 AgRP(83–132) acts as an inverse agonist on the human-melanocortin-4 receptor. Mol Endocrinol 15:164–171[Abstract/Free Full Text]
  57. Yang YK, Thompson DA, Dickinson CJ, Wilken J, Barsh GS, Kent SB, Gantz I 1999 Characterization of agouti-related protein binding to melanocortin receptors. Mol Endocrinol 13:148–155[Abstract/Free Full Text]
  58. Hahn TM, Breininger JF, Baskin DG, Schwartz MW 1998 Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons. Nat Neurosci 1:271–272[CrossRef][Medline]
  59. Chen P, Li C, Haskell-Luevano C, Cone RD, Smith MS 1999 Altered expression of agouti-related protein and its colocalization with neuropeptide Y in the arcuate nucleus of the hypothalamus during lactation. Endocrinology 140:2645–2650[Abstract/Free Full Text]
  60. Elias CF, Lee C, Kelly J, Aschkenasi C, Ahima RS, Couceyro PR, Kuhar MJ, Saper CB, Elmquist JK 1998 Leptin activates hypothalamic CART neurons projecting to the spinal cord. Neuron 21:1375–1385[CrossRef][Medline]
  61. Kim MS, Small CJ, Stanley SA, Morgan DG, Seal LJ, Kong WM, Edwards CM, Abusnana S, Sunter D, Ghatei MA, Bloom SR 2000 The central melanocortin system affects the hypothalamo-pituitary thyroid axis and may mediate the effect of leptin. J Clin Invest 105:1005–1011[Medline]
  62. Kim MS, Small CJ, Russell SH, Morgan DG, Abbott CR, al Ahmed SH, Hay DL, Ghatei MA, Smith DM, Bloom SR 2002 Effects of melanocortin receptor ligands on thyrotropin-releasing hormone release: evidence for the differential roles of melanocortin 3 and 4 receptors. J Neuroendocrinol 14:276–282[CrossRef][Medline]
  63. Powis JE, Bains JS, Ferguson AV 1998 Leptin depolarizes rat hypothalamic paraventricular nucleus neurons. Am J Physiol 274:R1468–R1472



This article has been cited by other articles:


Home page
 EndocrinologyHome page
L. Huo, K. M. Gamber, H. J. Grill, and C. Bjorbaek
Divergent Leptin Signaling in Proglucagon Neurons of the Nucleus of the Solitary Tract in Mice and Rats
Endocrinology, February 1, 2008; 149(2): 492 - 497.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
E. A. Nillni
Regulation of Prohormone Convertases in Hypothalamic Neurons: Implications for ProThyrotropin-Releasing Hormone and Proopiomelanocortin
Endocrinology, September 1, 2007; 148(9): 4191 - 4200.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
M. Perello, R. C. Stuart, and E. A. Nillni
The Role of Intracerebroventricular Administration of Leptin in the Stimulation of Prothyrotropin Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus
Endocrinology, July 1, 2006; 147(7): 3296 - 3306.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
L. Huo, H. J. Grill, and C. Bjorbaek
Divergent Regulation of Proopiomelanocortin Neurons by Leptin in the Nucleus of the Solitary Tract and in the Arcuate Hypothalamic Nucleus
Diabetes, March 1, 2006; 55(3): 567 - 573.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C. Fekete, P. S. Singru, E. Sanchez, S. Sarkar, M. A. Christoffolete, R. S. Riberio, W. M. Rand, C. H. Emerson, A. C. Bianco, and R. M. Lechan
Differential Effects of Central Leptin, Insulin, or Glucose Administration during Fasting on the Hypothalamic-Pituitary-Thyroid Axis and Feeding-Related Neurons in the Arcuate Nucleus
Endocrinology, January 1, 2006; 147(1): 520 - 529.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Endocrinol. Metab.Home page
C. D. Morrison, G. J. Morton, K. D. Niswender, R. W. Gelling, and M. W. Schwartz
Leptin inhibits hypothalamic Npy and Agrp gene expression via a mechanism that requires phosphatidylinositol 3-OH-kinase signaling
Am J Physiol Endocrinol Metab, December 1, 2005; 289(6): E1051 - E1057.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
R. Guillemin
Hypothalamic hormones a.k.a. hypothalamic releasing factors
J. Endocrinol., January 1, 2005; 184(1): 11 - 28.
[Abstract] [Full Text] [PDF]

Home page
 DiabetesHome page
S. H. Bates, T. A. Dundon, M. Seifert, M. Carlson, E. Maratos-Flier, and M. G. Myers Jr
LRb-STAT3 Signaling Is Required for the Neuroendocrine Regulation of Energy Expenditure by Leptin
Diabetes, December 1, 2004; 53(12): 3067 - 3073.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
G Tulipano, A V Vergoni, D Soldi, E E Muller, and D Cocchi
Characterization of the resistance to the anorectic and endocrine effects of leptin in obesity-prone and obesity-resistant rats fed a high-fat diet
J. Endocrinol., November 1, 2004; 183(2): 289 - 298.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
C. Fekete, D. L. Marks, S. Sarkar, C. H. Emerson, W. M. Rand, R. D. Cone, and R. M. Lechan
Effect of Agouti-Related Protein in Regulation of the Hypothalamic-Pituitary-Thyroid Axis in the Melanocortin 4 Receptor Knockout Mouse
Endocrinology, November 1, 2004; 145(11): 4816 - 4821.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/5/2516    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited