This Article Abstract Full Text (PDF) All Versions of this Article: 145/5/2221    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, F. Articles by Hollenberg, A. N. Search for Related Content PubMed PubMed Citation Articles by Guo, F. Articles by Hollenberg, A. N.

Leptin Signaling Targets the Thyrotropin-Releasing Hormone Gene Promoter in Vivo

Thyroid Unit and Division of Endocrinology, Beth Israel Deaconess Medical Center and Harvard Medical School, Boston Massachusetts 02215

Address all correspondence and requests for reprints to: Anthony Hollenberg, M.D., Thyroid Unit, Division of Endocrinology, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston Massachusetts 02215. E-mail: thollenb{at}bidmc.harvard.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The regulation of TRH gene expression in the paraventricular nucleus of the hypothalamus (PVH) by leptin is critical for normal function of the thyroid axis in rodents and humans. The TRH neuron in the PVH expresses both leptin and melanocortin-4 receptors, suggesting that both signaling systems may regulate TRH gene expression in vivo. Indeed, the TRH promoter responds to both of these signaling pathways in cell culture through identified cis-acting elements, which include signal transducer and activator of transcription (STAT) 3 and cAMP-response element binding protein binding sites that mediate leptin and melanocortin responses, respectively. To determine whether leptin signaling can directly target the TRH promoter in vivo, we developed a chromatin immunoprecipitation assay to use on leptin-treated animals. After a single injection of leptin in fasting animals, we could detect a significant increase in TRH gene expression in the PVH that correlated well with the induction of phosphorylated-STAT3 in the hypothalamus. Furthermore, using a STAT3 antibody, we could immunoprecipitate the STAT-binding site containing regions of both the TRH promoter and the promoter of the suppressor of cytokine signaling-3 gene, another well-defined target of leptin action. In contrast, upstream regions of these promoters that lack STAT sites were not precipitated. Taken together these experiments demonstrate that STAT3 mediates transcriptional effects of leptin in vivo and that the TRH promoter is a likely direct site of leptin action. In addition, these experiments demonstrate that chromatin immunoprecipitation can be used to characterize leptin-signaling in vivo.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE REGULATION OF the thyroid axis by the adipocyte-derived polypeptide hormone leptin is a key adaptive process in mammals to food deprivation (1, 2). Leptin mediates its actions on the thyroid axis by regulating TRH gene expression in the paraventricular nucleus of the hypothalamus (PVH) such that an acute fast causes a rapid drop in TRH gene expression that can be prevented by the administration of leptin (2). Leptin acts to regulate TRH gene expression either directly through the long form of its receptor, ObRB, which is present on a subset of TRH neurons in the PVH, or indirectly through its actions on the arcuate nucleus (3, 4). Indeed, central administration of the proopiomelanocortin (POMC)-derived peptide MSH, prevents the fasting-induced suppression of TRH consistent with the known expression of its target, the melanocortin-4 receptor (MC4-R), on most TRH neurons (5). In addition, the arcuate nucleus produced peptides neuropeptide Y (NPY) and agouti-related peptide (AgRP), which are induced during fasting, are potent negative regulators of TRH gene expression in the PVH (6, 7). Thus, the TRH neuron is an important physiologic integrator of signals from nutritionally important pathways through the regulation of TRH gene expression.

The TRH promoter is poised to integrate signals from the pathways discussed. It contains a cAMP-response element binding protein site that interacts with cAMP-response element binding protein in vitro and responds to MSH signaling via the MC4-R (4, 8). Furthermore, leptin signaling via the ObRB can also target the TRH promoter in cell culture experiments (4). The ObRB is known to signal, in part, through the Janus kinase (JAK) 2-signal transducer and activator of transcription (STAT) 3 pathway in cell culture (9, 10, 11) and in vivo (12). Activation of this pathway by leptin leads to key changes in gene expression (13). Indeed, activation of STAT3 by leptin in cell lines transfected with the ObRB activates the TRH promoter via a canonical STAT-binding site that is conserved across species. Thus, the TRH gene provides an ideal target in which to study leptin signaling at the transcriptional level (4).

In addition to activating TRH gene expression, leptin-mediated activation of STAT3 correlates anatomically with the activation of other key nutritionally regulated genes including the suppressor of cytokine signaling (SOCS)-3 in multiple areas of the hypothalamus (14, 15), POMC in the arcuate nucleus (16, 17, 18, 19), and the cocaine and amphetamine-related transcript in both the arcuate and PVH (20, 21). However, because of the lack of hypothalamic cell lines that express the ObRB, there is little evidence supporting the exact role of STAT3 in the regulation of these target genes and TRH in vivo. To begin to address this issue, we designed a hypothalamic in vivo chromatin immunoprecipitation assay (ChIP) to directly assess the role of leptin signaling via STAT3. Using this assay we can demonstrate that STAT3 is recruited to the SOCS-3 and TRH promoters in a leptin-dependent fashion. Furthermore, using genetic models of obesity, we can confirm the relevance of leptin-mediated signaling pathways. Thus, using this ChIP assay, we can demonstrate that leptin transcriptionally regulates target genes via STAT3 in vivo.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animal care
Female FVB mice (36–49 d) were purchased from Charles River Laboratory (Wilmington, MA, and Kingston, NY). Female db/db mice, ob/ob mice together with control littermates on a C57/bl6 background were purchased from Jackson Laboratories (Bar Harbor, ME). The animals and procedures used were in accordance with the guidelines and approval of the Institutional Animal Care and Use Committees of the Beth Israel Deaconess Medical Center. Upon arrival, mice were fed a standard rodent chow for 1 wk to acclimate and were housed in groups of four. Room temperature was maintained between 22 and 23 C and a 12-h light, 12-h dark cycle was used. Mice were handled daily for 4–7 d before procedures. The day before experiments, mice were fasted overnight and then treated with recombinant leptin (purchased from Dr. E. Parlow, National Institute of Diabetes and Digestive and Kidney Diseases and the National Hormone and Pituitary Program, Torrance CA, 5 µg/g) or an equal volume of PBS by ip injection at the indicated time points. Animals were killed by CO₂ inhalation. Brains were then removed and the hypothalamus was quickly dissected and put into liquid nitrogen. They were stored at –80 C until used for analysis.

RT-PCR
FVB mice were either fed normally or fasted for 24 h, or fasted and then given leptin by ip injection. PVHs were then dissected as described 6 h after the administration of leptin. The PVH was dissected from sagittal sections of fresh brain (1-mm thickness from the midline of the brain). Coordinates for the dissection of the PVH in the section are as follows: anterior margin, anterior commisure; dorsal margin, border with thalamus; ventral margin, 1.5 mm ventral to the border with thalamus; posterior margin, white matter separating PVH and hypothalamus dorsomedial nucleus/ventromedial hypothalamic (nucleus). RNA was isolated by RNA STAT-60 and then extracted by chloroform and precipitated by addition of isopropanol, washed with ethanol, and solubilized in water. Equal amounts of RNA (500 ng) were then used for RT-PCR by using Advantage RT-PCR kit (Clontech, Palo Alto, CA). Primers used for PCR are available upon request. PCRs were performed for a number of different cycles to ensure linearity.

ChIP assay
ChIP assays were performed as described with the following modifications (22, 23). Each hypothalamus was weighed and incubated in1% formaldehyde (10 µl per µg tissue) at 37 C for 10 min. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M and incubating at 37 C for another 5 min. The tissue was then washed twice with ice-cold PBS and resuspended in sodium dodecyl sulfate lysis buffer. The chromatin was then sonicated to lengths of 200-1000 bp and the tissue homogenate was centrifuged at 14,000 rpm for 10 min at 4 C. At this point homogenate from individual hypothalami was pooled such that each sample represents material from two to three hypothalami. Then 200 µl of the sonicated cell pellet suspension was added to a fresh 2-ml microfuge tube with 1.8 ml of ChIP dilution buffer (Upstate Biotechnology, Lake Placid, NY). Fifty microliters of each sample were removed as the input control. Each 2-ml sample was precleared with 80 µl salmon sperm DNA/protein A agarose 50% slurry for 30 min at 4 C with rotation. Samples were immunoprecipitated with 2 µg anti-STAT3 antibody (SC-482, Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4 C with rotation. Each time a mock immunoprecipitation with no antibody was also performed. The next day 60 µl salmon sperm DNA/protein A agarose slurry was added and incubated for 1 h at 4 C to collect the precipitated complex. The pellet was washed for 5 min on a rotating platform with 1 ml of each buffer in the following order: low salt immune complex wash buffer; high salt immune complex wash buffer; LiCl immune complex wash buffer; 1 x Tris/EDTA buffer (two times). Elution was carried out in two separate 250-µl aliquots of elution buffer (1% sodium dodecyl sulfate, 0.1 M NaHCO₃). Then 20 µl of 5 M NaCl was added to the combined eluates. Protein-DNA cross-links were reversed by heating at 65 C for 4 h. The following day, 10 µl of 0.5 M EDTA, 20 µl 1 M Tris-HCl (pH 6.5), and 2 µl (10 mg/ml) proteinase K were added and the samples were incubated for 1 h at 45 C. DNA was collected after phenol/chloroform extraction. Pellets were resuspended in H₂O and subjected to PCR.

PCR primers were designed according to published sequences and the murine sequence in GenBank. Two sets of primers were designed for the SOCS-3 and TRH promoters: one set of primers was used to amplify the region that contains the STAT3 binding site, whereas the other set of primers was used to amplify an upstream region away from the STAT3 binding site. PCR was performed by annealing at 56 C for 30–35 cycles. All PCR products were initially verified by DNA sequencing. PCR primers are available upon request.

All experiments were repeated at least two times with control animals present in each experiment when ob/ob and db/db animals were used.

Western blot analysis
Hypothalami were placed in lysis buffer and homogenized. The resulting supernatant was resolved by SDS-PAGE, followed by transfer to a membrane. The nitrocellulose membrane was blocked with 10% nonfat dried milk in Towbin buffer [20 mm Tris-HCl (pH 7.4), 0.9% NaCl, and 0.05% Tween 20] for 1 h and then incubated with either STAT3 or phospho-STAT3 antibodies (Sigma Chemical Co., St. Louis, MO) in 5% milk in Towbin buffer at 4 C overnight. After washing, the membranes were incubated with antirabbit IgG in 2% milk in Towbin buffer for 1 h at room temperature. The targeted proteins were detected using enhanced chemiluminescence, as described by the manufacturer (Amersham International, Buckinghamshire, UK).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Leptin regulates TRH gene expression in the paraventricular nucleus
To examine the regulation of TRH gene expression in the PVH we isolated the PVH directly from groups of female FVB mice that were fed, fasted for 24 h or fasted but given leptin 6 h before being killed. As shown in Fig. 1, a 24-h fast causes a significant drop in TRH mRNA expression by more than 40% in the PVH. These data are consistent with in situ hybridization data demonstrating that TRH gene expression is regulated by leptin only in the PVH (24, 25). The administration of a single injection of leptin reverses this drop and rescues TRH gene expression within 6 h, consistent with a possible transcriptional effect. These data demonstrate that leptin is critical for the regulation of TRH expression in the PVH and can stimulate TRH mRNA expression rapidly, consistent with a transcriptional effect in vivo. However, this experiment does not answer the question of whether leptin directly targets the TRH neuron or activates TRH expression indirectly by enhancing POMC expression or MSH release from the arcuate nucleus.

View larger version (24K): [in this window] [in a new window]   FIG. 1. Leptin regulates TRH mRNA expression in the PVH. Groups of mice (n = 4) were fed, fasted, or fasted followed by a single injection of leptin. The PVH was isolated as described in Materials and Methods and total RNA isolated. RT-PCR was performed on equal amounts of input RNA with primers specific for murine TRH or cyclophilin. A, Ethidium bromide-stained gel displaying both TRH and cyclophilin PCR products from each individual animal. B, Data were quantified using NIH Image software, and TRH expression was corrected for cyclophilin expression. *, P < 0.05 fed vs. fasting; **, P < 0.05 fasting vs. fasting plus leptin. (student’s t test)

View larger version (28K): [in this window] [in a new window]   FIG. 2. STAT3 is phosphorylated for up to 6 h by leptin stimulation. A, Pairs of FVB mice that were fasted overnight were treated with leptin for the indicated time points. After isolation of the hypothalamus, extracts were prepared and equal amounts run on SDS-PAGE and immunoblotted with both STAT3 and P-STAT3 antibodies. B, Shown is the structure of the SOCS-3 and TRH promoters with the primers used for PCR in the ChIP assay. The STAT-binding sites are shown in black.

Given that STAT3 is rapidly phosphorylated by leptin in the hypothalamus and that the SOCS-3 gene is induced within 1 h in a variety of hypothalamic nuclei by leptin administration in animals (14), we chose to focus initially on whether the murine SOCS-3 promoter could recruit STAT-3 in vivo using ChIP. Previous studies performed by Auernhammer et al. (26) in the AtT-20 cell line had demonstrated that the murine SOCS-3 promoter contained two consensus STAT binding sites between –100 and +1 with the more proximal site between –72 and –64 playing a key role in mediating regulation of the promoter by STAT3 (Fig. 2B). Furthermore, this element was shown to bind STAT3 dimers well in EMSAs.

Fasted female FVB mice were treated with a single ip injection of leptin or PBS and their hypothalami were isolated. After precipitation of cross-linked DNA with a STAT3 antibody, PCR on regions of the SOCS-3 promoter was performed. Using this assay, it is clear that the proximal SOCS-3 promoter is precipitated after leptin treatment in the presence of STAT3 antibody only (compare lane 1 with lane 3, Fig. 3A). In the absence of a specific antibody the proximal STAT3 promoter is not immunoprecipitated (lanes 2 and 4). To ensure that the proximal murine promoter was being specifically precipitated, we also amplified, from the same immunoprecipitate, a region of the SOCS-3 promoter between –2665 and –2345 that is not involved in the STAT3 response in cell culture. Whereas the input chromatin showed strong amplification, there was no evidence that in the presence of leptin treatment and a STAT3 antibody that this region of the promoter was immunoprecipitated (lanes 5 and 7, Fig. 3B). Thus, the activation of STAT3 by leptin in the hypothalamus leads to the recruitment of STAT3 to the proximal SOCS-3 promoter.

View larger version (28K): [in this window] [in a new window]   FIG. 3. STAT3 is recruited to the proximal SOCS-3 promoter. A and B, Animals were treated with leptin or PBS for 30 min and ChIP was performed. The resulting immunoprecipitate was amplified with primers between –134 and +2 or 2665 to –2345 of the SOCS-3 promoter and run on an agarose gel. Before immunoprecipitation an aliquot of the sample was stored and then purified and represents input for each sample shown. C, ChIP was performed on animals treated with PBS or leptin for 30 min and 6 h. PCR was performed as described and the resulting products visualized on agarose gels.

The TRH promoter recruits STAT3 in vivo
To determine whether the STAT3 binding site present in the mTRH promoter between –146 and –137, which is necessary for leptin responsiveness in mammalian cell lines (Fig. 2B), can recruit STAT3 in vivo in response to leptin we again used ChIP. As shown in Fig. 4A (compare lane 1 to lane 3), in the presence of leptin and the STAT3 antibody, the proximal TRH promoter recruited STAT3 within 30 min of leptin stimulation. In contrast, an upstream region of the TRH promoter was unable to recruit STAT3 (compare lanes 5 and 7, Fig. 4B). Thus, like SOCS-3 the TRH promoter is directly targeted by leptin signaling in vivo. We also performed a time-course experiment and, similar to SOCS-3, the proximal TRH promoter was able to recruit STAT3 over a prolonged period, whereas the distal region was unable to recruit STAT3 (Fig. 4B). Thus, the TRH promoter is a direct target of leptin signaling in vivo.

View larger version (26K): [in this window] [in a new window]   FIG. 4. STAT3 is recruited to the TRH promoter. ChIP was performed on hypothalamic chromatin from mice treated with leptin for 30 min, and after immunoprecipitation the resulting DNA was subjected to PCR with oligonucleotides spanning the proximal promoter (A) or a distal region (B). C, ChIP was performed on animals treated with PBS or leptin for 30 min and 6 h. PCR was performed as described and the resulting products visualized on agarose gels.

View larger version (20K): [in this window] [in a new window]   FIG. 5. Phosphorylation of STAT3 in ob/ob and db/db mice. Groups of fasting ob/ob and db/db mice and control littermates were treated with either PBS or leptin for 30 min. After isolation of the hypothalamus, extracts were prepared and equal amounts were run on SDS-PAGE, transferred to nitrocellulose, and immunoblotted with either a pSTAT3 antibody or a STAT3 antibody.

View larger version (43K): [in this window] [in a new window]   FIG. 6. STAT3 recruitment in ob/ob and db/db mice to the SOCS-3 promoter. Groups of db/db (n = 3, A), littermates (n = 3, B), or ob/ob (n = 3, C) mice were treated with leptin or PBS for 30 min, and their hypothalami were isolated for ChIP assays using the STAT3 antibody. The left panels show the PCR results using the primers spanning the STAT3 binding site, whereas the right panels use the upstream region.

View larger version (48K): [in this window] [in a new window]   FIG. 7. STAT3 recruitment in ob/ob and db/db mice to the TRH promoter. The same immunoprecipitates from Fig. 6 were used with primers specific for the TRH promoter. A, db/db. B, C57bl/6 littermates. C, ob/ob. The left panels show the PCR results using the primers spanning the STAT3 binding site, whereas the right panels use the upstream region.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The regulation of the thyroid axis by leptin is of critical importance in rodents and also in man. Recent studies in humans demonstrate that controlled caloric restriction leading to weight loss results in a decline in serum thyroid hormone levels, which can be reversed by leptin (33), whereas acute fasting in humans leads to dysregulation of TSH secretion, which can also be reversed by leptin (34). Whereas leptin’s effects on the thyroid axis are mediated through its central effects on the TRH neuron, it remains unclear whether these effects are direct or indirect. Available genetic data support a direct role for leptin action that is independent of the melanocortin system. Both rodents and humans with leptin receptor mutations have central hypothyroidism (35). In contrast, murine MC4-R knockouts and human patients with MC4-R mutations have normal thyroid hormone levels (36, 37). However, substantial data also support an indirect role for leptin action via the arcuate nucleus. In addition to leptin receptors, the TRH neuron in the PVH expresses the MC4-R and NPY-1 and -5 receptor isoforms (4, 38, 39). Addition of MSH centrally to rodents can prevent the fasting-induced suppression of TRH in the PVH, whereas centrally administered AgRP or NPY can induce central hypothyroidism (5, 6, 7). Taken together, these data suggested that the POMC/cocaine and amphetamine-related transcript and AgRP/NPY neurons in the arcuate may also play an essential role in the regulation of PVH TRH gene expression. Given its structure, the TRH promoter likely integrates both the direct and indirect effects of leptin at the level of transcription.

Further complexity is also likely mediated by other signaling pathways that are altered by the nutritional state such as the glucocorticoid pathway, which is activated by fasting and may modulate leptin-signaling on many levels. Indeed, glucocorticoids inhibit TRH expression in the PVH (40), suggesting that they could also play a role in the suppression of TRH gene expression in the fasting state. Interestingly, glucocorticoids activate TRH gene expression in hypothalamic cultures so the mechanism by which they repress TRH expression in whole animals remains unclear (41, 42).

To begin to address the question of whether leptin can transcriptionally activate the TRH promoter directly and regulate the thyroid axis independently of arcuate nucleus inputs, we developed an in vivo ChIP assay using a STAT3 antibody to detect promoter binding sites after leptin stimulation. Indeed, a single leptin injection was able to phosphorylate STAT3 for a prolonged period of time in the hypothalamus. In addition, leptin was able to dynamically regulate TRH mRNA levels in the PVH within a time frame that is consistent with a transcriptional effect. Further studies will be required to determine when TRH gene expression first increases and when it reaches maximal levels. Previous studies on SOCS-3 gene expression demonstrate that it is maximally activated 1 h after leptin administration and returns to baseline by 6 h, suggesting that different transcriptional targets have different kinetics (15). The ChIP assay demonstrates that leptin stimulation leads to the recruitment of STAT3 to both the SOCS-3 and TRH promoters within 30 min of treatment. These data suggest that direct actions of leptin on the TRH neuron are critical for its transcriptional regulation. Furthermore, they prove that the STAT3 sites identified in vitro for both the TRH and SOCS-3 promoters are functional in vivo. The fidelity of the ChIP assay is further supported by the fact that upstream regions of the target promoters that lack STAT binding sites are also unable to recruit STAT3 in vivo.

The use of genetic models of obesity, which are defective in either leptin or its receptor, further support our hypothesis that leptin can directly target the TRH neuron in vivo. Whereas db/db mice show no evidence of leptin signaling, ob/ob, animals show both phosphorylation and recruitment of STAT3 to the TRH promoter. Previously other investigators had demonstrated decreased STAT3 mRNA and protein in ob/ob animals in specific nuclei of the hypothalamus (43). Whereas our data do not agree with this study, it is important to point out that we have measured only total hypothalamic STAT3 levels. Interestingly, levels of total STAT3 will need to be further investigated in db/db animals because our results suggest that total STAT3 levels are lower in this group of animals. A previous study by Madiehe et al. (44) demonstrated that leptin signaling did not affect total hypothalamic STAT3 protein levels, so more work is likely needed to address the likely complex regulation of STAT3 message and protein by leptin signaling. Together, our data indicate that leptin signaling is required for STAT3 induction in TRH neurons. Similar studies in MC4-R knockout animals will be necessary in the future to conclusively support the direct model.

Surprisingly, the POMC promoter was not precipitated above background levels using primers directed against its proximal promoter in the experiments shown (data not shown). This is despite data demonstrating that the POMC promoter can be activated by STAT3 via the proximal 91 bases of its promoter in heterologous cells (19). Our ChIP data suggest that this promoter may use alternate sites to respond to leptin in vivo or alternatively recruits STAT3 earlier or later than the time tested. We speculate that the former is correct and that the use of ChIP to identify the correct region of the promoter would have significant importance, given the possibility that mutations in the leptin-responsive regions of this promoter could result in the clinical expression of obesity. Further study of POMC and other leptin-responsive genes, such as AgRP, using the ChIP assay should lend considerable insight into how leptin signals to regulate target genes.

In summary, we have demonstrated the use of an in vivo ChIP assay, which can successfully identify STAT3 sites employed by leptin-responsive genes in vivo. Our data show that the TRH promoter is targeted by leptin in vivo and that leptin can likely regulate the thyroid axis directly without need for input from the arcuate nucleus. However, it is likely, given the structure of the TRH promoter and the inputs to the TRH neuron in the PVH, that arcuate inputs assist in the modulation of TRH gene expression. Further investigation of cross-talk between the leptin and melanocortin signaling systems on the TRH promoter should be possible with ChIP.

	Acknowledgments

 
We thank the laboratory of Christian Bjorbaek for helpful discussions, and the laboratory of Jang-Ho Cha for assistance with the ChIP assay.

	Footnotes

 
This work was supported by National Institutes of Health Grant DK57658; a grant from the Thyroid Research Advisory Council, Abbot Laboratories (to A.N.H.); and assistance from the Animal Metabolic Physiology Core of the Boston Area DERC P30 DK57521.

Abbreviations: AgRP, Agouti-related peptide; ChIP, chromatin immunoprecipitation assay; JAK, Janus kinase; MC4-R, melanocortin-4 receptor; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVH, paraventricular nucleus of the hypothalamus; SOCS-3, suppressor of cytokine signaling-3; STAT, signal transducer and activator of transcription.

Received September 30, 2003.

Accepted for publication January 26, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. 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]
2. 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]
3. Nillni EA, Vaslet C, Harris M, Hollenberg A, Bjorbak 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]
4. 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 107:1–11
5. Fekete C, Legradi G, Mihaly E, Huang Q-H, Tatro JB, Rand WM, Emerson CH, Lechan RM 2000 -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]
6. Fekete C, Kelly J, Mihaly E, Sarkar S, Rand WM, Legradi G, Emerson CH, Lechan RM 2001 Neuropeptide Y has a central inhibitory action on the hypothalamic-pituitary-thyroid axis. Endocrinology 142:2606–2613[Abstract/Free Full Text]
7. Fekete C, Sarkar S, Rand WM, Harney JW, Emerson CH, Bianco AC, Lechan RM 2002 Agouti-related protein (AGRP) has a central inhibitory action on the hypothalamic-pituitary-thyroid (HPT) axis: comparisons between the effect of AGRP and neuropeptide Y on energy homeostasis and the HPT axis. Endocrinology 143:3846–3853[Abstract/Free Full Text]
8. Hollenberg AN, Monden T, Flynn TR, Boers ME, Cohen O, Wondisford FE 1995 The human thyrotropin-releasing hormone gene is regulated by thyroid hormone through two distinct classes of negative thyroid hormone response elements. Mol Endocrinol 9:540–550[Abstract]
9. 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]
10. 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 [Erratum (1997) 272:12248][Free Full Text]
11. Bjorbak 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]
12. Bates SH, Stearns WH, Dundon TA, Schubert M, Tso AW, Wang Y, Banks AS, Lavery HJ, Haq AK, Maratos-Flier E, Neel BG, Schwartz MW, Myers Jr MG 2003 STAT3 signalling is required for leptin regulation of energy balance but not reproduction. Nature 421:856–859[CrossRef][Medline]
13. Bjorbaek C, Hollenberg AN 2002 Leptin and melanocortin signaling in the hypothalamus. Vitam Horm 65:281–311[Medline]
14. 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]
15. Bjorbaek C, El-Haschimi K, Frantz JD, Flier JS 1999 The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem 274:30059–30065[Abstract/Free Full Text]
16. Thornton JE, Cheung CC, Clifton DK, Steiner RA 1997 Regulation of hypothalamic proopiomelanocortin mRNA by leptin in ob/ob mice. Endocrinology 138:5063–5066[Abstract/Free Full Text]
17. Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV 1998 Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin [published erratum appears in Diabetes 1998;47:696]. Diabetes 47:294–297[Abstract]
18. Schwartz MW, Seeley RJ, Woods SC, Weigle DS, Campfield LA, Burn P, Baskin DG 1997 Leptin increases hypothalamic pro-opiomelanocortin mRNA expression in the rostral arcuate nucleus. Diabetes 46:2119–2123[Abstract]
19. 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]
20. Dhillon H, Ge Y, Minter RM, Prima V, Moldawer LL, Muzyczka N, Zolotukhin S, Kalra PS, Kalra SP 2000 Long-term differential modulation of genes encoding orexigenic and anorexigenic peptides by leptin delivered by rAAV vector in ob/ob mice. Relationship with body weight change. Regul Pept 92:97–105[CrossRef][Medline]
21. Elias CF, Lee CE, Kelly JF, Ahima RS, Kuhar M, Saper CB, Elmquist JK 2001 Characterization of CART neurons in the rat and human hypothalamus. J Comp Neurol 432:1–19[CrossRef][Medline]
22. Ho Y, Elefant F, Cooke N, Liebhaber S 2002 A defined locus control region determinant links chromatin domain acetylation with long-range gene activation. Mol Cell 9:291–302[CrossRef][Medline]
23. Shibusawa N, Hollenberg AN, Wondisford FE 2003 Thyroid hormone receptor DNA binding is required for both positive and negative gene regulation. J Biol Chem 278:732–738[Abstract/Free Full Text]
24. 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]
25. Abel ED, Ahima RS, Boers ME, Elmquist JK, Wondisford FE 2001 Critical role for thyroid hormone receptor ß2 in the regulation of paraventricular thyrotropin-releasing hormone neurons. J Clin Invest 107:1017–1023[Medline]
26. Auernhammer CJ, Bousquet C, Melmed S 1999 Autoregulation of pituitary corticotroph SOCS-3 expression: characterization of the murine SOCS-3 promoter. Proc Natl Acad Sci USA 96:6964–6969[Abstract/Free Full Text]
27. Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2000 Cofactor dynamics and sufficiency in estrogen-receptor regulated transcription. Cell 103:843–852[CrossRef][Medline]
28. Elias CF, Aschkenasi C, Lee C, Kelly J, Ahima RS, Bjorbaek C, Flier JS, Saper CB, Elmquist JK 1999 Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area. Neuron 23:775–786[CrossRef][Medline]
29. 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]
30. Mercer JG, Hoggard N, Williams LM, Lawrence CB, Hannah LT, Trayhurn P 1996 Localization of leptin receptor mRNA and the long form splice variant (Ob-Rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization. FEBS Lett 387:113–116[CrossRef][Medline]
31. 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]
32. Hubschle T, Thom E, Watson A, Roth J, Klaus S, Meyerhof W 2001 Leptin-induced nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei involved in body weight regulation. J Neurosci 21:2413–2424[Abstract/Free Full Text]
33. Rosenbaum M, Murphy EM, Heymsfield SB, Matthews DE, Leibel RL 2002 Low dose leptin administration reverses effects of sustained weight-reduction on energy expenditure and circulating concentrations of thyroid hormones. J Clin Endocrinol Metab 87:2391–2394[Abstract/Free Full Text]
34. Chan JL, Heist K, DePaoli AM, Veldhuis JD, Mantzoros CS 2003 The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men. J Clin Invest 111:1409–1421[CrossRef][Medline]
35. Clement K, Vaisse C, Lahlou N, Cabrol S, Pelloux V, Cassuto D, Gourmelen M, Dina C, Chambaz J, Lacorte JM, Basdevant A, Bougneres P, Lebouc Y, Froguel P, Guy-Grand B 1998 A mutation in the human leptin receptor gene causes obesity and pituitary dysfunction. Nature 392:398–401[CrossRef][Medline]
36. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, Gu W, Kesterson RA, Boston BA, Cone RD, Smith FJ, Campfield LA, Burn P, Lee F 1997 Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 88:131–141[CrossRef][Medline]
37. Farooqi IS, Keogh JM, Yeo GS, Lank EJ, Cheetham T, O’Rahilly S 2003 Clinical spectrum of obesity and mutations in the melanocortin 4 receptor gene. N Engl J Med 348:1085–1095[Abstract/Free Full Text]
38. 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]
39. Fekete C, Sarkar S, Rand WM, Harney JW, Emerson CH, Bianco AC, Beck-Sickinger A, Lechan RM 2002 Neuropeptide Y1 and Y5 receptors mediate the effects of neuropeptide Y on the hypothalamic-pituitary-thyroid axis. Endocrinology 143:4513–4519[Abstract/Free Full Text]
40. Kakucska I, Qi Y, Lechan RM 1995 Changes in adrenal status affect hypothalamic thyrotropin-releasing hormone gene expression in parallel with corticotropin-releasing hormone. Endocrinology 136:2795–2802[Abstract]
41. Luo LG, Bruhn T, Jackson IM 1995 Glucocorticoids stimulate thyrotropin-releasing hormone gene expression in cultured hypothalamic neurons. Endocrinology 136:4945–4950[Abstract]
42. Luo LG, Jackson IM 1998 Glucocorticoids stimulate TRH and c-fos/c-jun gene co-expression in cultured hypothalamic neurons. Brain Res 791:56–62[CrossRef][Medline]
43. Hakansson-Ovesjo ML, Collin M, Meister B 2000 Down-regulated STAT3 messenger ribonucleic acid and STAT3 protein in the hypothalamic arcuate nucleus of the obese leptin-deficient (ob/ob) mouse. Endocrinology 141:3946–3955[Abstract/Free Full Text]
44. Madiehe AM, Lin L, White C, Braymer HD, Bray GA, York DA 2001 Constitutive activation of STAT-3 and down-regulation of SOCS-3 expression induced by adrenalectomy. Am J Physiol Regul Integr Comp Physiol 281:R2048–R2058

This article has been cited by other articles:

				B. E. Levin Neurotrophism and energy homeostasis: perfect together Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R988 - R991. [Full Text] [PDF]

				D C Ferguson, Z Caffall, and M Hoenig Obesity increases free thyroxine proportionally to nonesterified fatty acid concentrations in adult neutered female cats J. Endocrinol., August 1, 2007; 194(2): 267 - 273. [Abstract] [Full Text] [PDF]

				E. Ortega, N. Pannacciulli, C. Bogardus, and J. Krakoff Plasma concentrations of free triiodothyronine predict weight change in euthyroid persons Am. J. Clinical Nutrition, February 1, 2007; 85(2): 440 - 445. [Abstract] [Full Text] [PDF]

				A Boelen, J Kwakkel, X G Vos, W M Wiersinga, and E Fliers Differential effects of leptin and refeeding on the fasting-induced decrease of pituitary type 2 deiodinase and thyroid hormone receptor {beta}2 mRNA expression in mice. J. Endocrinol., August 1, 2006; 190(2): 537 - 544. [Abstract] [Full Text] [PDF]

				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]

				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]

				J. E. McMinn, S.-M. Liu, H. Liu, I. Dragatsis, P. Dietrich, T. Ludwig, C. N. Boozer, and S. C. Chua Jr. Neuronal deletion of Lepr elicits diabesity in mice without affecting cold tolerance or fertility Am J Physiol Endocrinol Metab, September 1, 2005; 289(3): E403 - E411. [Abstract] [Full Text] [PDF]

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

				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]

				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]

				P. Joseph-Bravo Hypophysiotropic Thyrotropin-Releasing Hormone Neurons as Transducers of Energy Homeostasis Endocrinology, November 1, 2004; 145(11): 4813 - 4815. [Full Text] [PDF]

				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 Abstract Full Text (PDF) All Versions of this Article: 145/5/2221    most recent Author Manuscript (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Guo, F. Articles by Hollenberg, A. N. Search for Related Content PubMed PubMed Citation Articles by Guo, F. Articles by Hollenberg, A. N.