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Endocrinology, doi:10.1210/en.2005-1533
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Endocrinology Vol. 147, No. 7 3296-3306
Copyright © 2006 by The Endocrine Society

The Role of Intracerebroventricular Administration of Leptin in the Stimulation of Prothyrotropin Releasing Hormone Neurons in the Hypothalamic Paraventricular Nucleus

Mario Perello, Ronald C. Stuart and Eduardo A. Nillni

Division of Endocrinology (M.P., R.C.S., E.A.N.), Department of Medicine, Brown Medical School/Rhode Island Hospital, Providence, Rhode Island 02903; and Department of Molecular Biology, Cell Biology and Biochemistry (E.A.N.), Brown University, Providence, Rhode Island 02903

Address all correspondence and requests for reprints to: Dr. Eduardo A. Nillni, Division of Endocrinology, Brown Medical School/Rhode Island Hospital, 55 Claverick Street, 4th Floor, Room 430, Providence, Rhode Island 02903. E-mail: Eduardo_Nillni{at}Brown.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We previously have shown that leptin regulates proTRH in the paraventricular nucleus (PVN) of the hypothalamus through two pathways. The first one acts directly on proTRH neurons, and the second one (indirect) acts through the melanocortin system (arcuate nucleus). However, it is unknown whether the direct or the indirect pathways of leptin action on proTRH neurons occurs on separated or on the same subsets of neurons within the PVN region. We used immunostaining for the phosphorylated signal transducer and activator of transcription 3 to localize direct leptin signaling, and the phosphorylated cAMP response element binding protein to localize indirect signaling on proTRH neurons in animals intracerebroventricularly injected with leptin. With this approach we were able to identify two subsets of neuronal populations responsive to leptin, which are distributed in different regions within the PVN. ProTRH neurons directly responsive to leptin were located mainly in the medial and posterior part of the PVN, and they were not primarily related to the hypothalamic pituitary thyroid axis. Whereas, proTRH neurons indirectly responsive (through {alpha}-MSH) to leptin were located mainly in the anterior, medial, and periventricular part of the PVN, and related to the hypothalamic pituitary thyroid axis. In addition, {alpha}-MSH showed to affect the processing of proTRH and up-regulated the prohormone convertase 1/3. In this study, we show evidence supporting the hypothesis that in the PVN there are subpopulations of proTRH neurons responding to leptin, which is dependent upon the way leptin reaches its primary target(s) in the hypothalamus. These findings are critical to a better understanding of leptin-mediated actions in energy expenditure.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A GREAT DEAL of effort has been invested in the last few years to understand the precise neural circuits through which leptin affects appetite and neuroendocrine function (1, 2, 3). Among its many actions, leptin acts directly on specific regions of the hypothalamus and activates the hypothalamic pituitary thyroid (HPT) axis, a key regulator of energy expenditure (4). During fasting, when circulating levels of leptin are low, the biosynthesis of proTRH (26 kDa), a precursor to TRH, decreases in the hypothalamic paraventricular nucleus (PVN), resulting in low plasma levels of TSH and thyroid hormones, and exogenous administration of leptin treatment reverses these effects (4, 5, 6). This indicates that low leptin signaling is a mechanism to conserve energy by decreasing thyroid hormone-dependent thermogenesis. The up-regulatory effect of leptin on proTRH is associated with an increase in the biosynthesis of two members of the prohormone convertase family (PC1/3 and PC2), which are necessary for the processing of proTRH to TRH (7, 8, 9, 10).

From published work done in our laboratory and others, there is compelling evidence supporting both the direct and indirect action of leptin on proTRH neurons (11, 12, 13, 14, 15). For example, leptin binds to its receptor (ObRb) present in proTRH neurons and activates a receptor-associated kinase, which phosphorylates signal transducer and activator of transcription 3 (P-STAT3) (16). This transcription factor increases the preproTRH gene transcription in proTRH neurons (17). Therefore, nuclear immunostaining for P-STAT3 represents a specific and sensible marker to functionally map direct leptin signaling (18). Evidence supporting the indirect pathway was demonstrated by the action of leptin on the arcuate nucleus (ARC) of the hypothalamus, a nuclei that produces neuropeptide Y and Agouti-related protein (AgRP) peptides with an inhibitory effect on proTRH biosynthesis and TRH release, and {alpha}-MSH derived from proopiomelanocortin, with a stimulatory effect on proTRH (19, 20, 21, 22, 23, 24, 25, 26). The cAMP response element binding protein (CREB) is critical in the indirect leptin action on proTRH neurons. {alpha}-MSH binds to the Gs protein-coupled melanocortin 4 receptor (MC4R), which signals by increasing cAMP levels and, then, phosphorylation of CREB (P-CREB) (27). Subsequently, P-CREB activates the preproTRH gene (11). AgRP acts as a competitive antagonist or inverse agonist on the melanocortin receptors (28, 29), and blocks {alpha}-MSH-induced effects on TRH release (30). The neuropeptide Y action on proTRH neurons is mediated by Y1 and Y5 receptors (24), which couple to Gi protein and, when activated, decrease cAMP levels (31). Therefore, nuclear immunostaining for P-CREB can be used as a specific marker to functionally label the indirectly leptin-activated proTRH neurons.

In the present study, we systematically examined leptin-activated proTRH neurons throughout the PVN of the rat when the leptin inputs were either directly or indirectly (throughout the ARC) produced. We found clear evidence that the PVN contains different leptin-responsive populations of proTRH neurons with distinct anatomic location. Additional analysis demonstrated that proTRH neurons stimulated by the indirect, {alpha}-MSH, pathway were mostly involved in the regulation of the HPT axis. The indirect pathway also affected the processing of proTRH in a coordinated fashion causing an increase in the production of the PC1/3 enzyme.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
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). Angiotensin II and SHU9119 were obtained from Sigma (St. Louis, MO). The rabbit polyclonal phospho-STAT3 (Tyr705) and mouse monoclonal phospho-CREB (Ser133) antibodies were obtained from Cell Signaling (Beverly, MA). Dr. Nabil Seidah donated the antibodies against PC1/3 and PC2 (IRCM, Montreal, Canada). Rabbit polyclonal anti-TRH, anti-{alpha}-MSH, and anti-preproTRH178–199 (pFE22) antibodies were developed in our laboratory. Biotinylated goat antirabbit was from Jackson ImmunoResearch Laboratories (West Grove, PA). Normal donkey serum and normal goat serum were obtained from Invitrogen Life Technologies (Grand Island, NY). Fluorescent goat antirabbit immunoglobulin conjugate (Alexa fluor 594) and fluorescent goat antimouse immunoglobulin conjugate (Alexa fluor 488) were obtained from Molecular Probes (Eugene, OR). Avidin biotin complex vectastain and fluorescence mounting solution were from Vector Laboratories (Burlingame, CA) and diaminobenzidine-developing solution was obtained from Roche (Basel, Germany). Male Sprague Dawley rats, 6–7 wk of age, were obtained from Charles River Laboratories, Inc. (Wilmington, MA). The Institutional Animal Care and Use Committee of Rhode Island Hospital/Brown University approved the experimental protocols and euthanasia procedures.

Surgeries, treatments, and samples
Rats were anesthetized with Ketamin 50 mg/kg-Domitor 0.25 mg/kg (ip) and then were stereotaxically implanted with an intracerebroventricular (ICV) 21-gauge stainless steel guide cannula (Plastic One, Roanoke, VA). The placement coordinates for the lateral hypothalamic ventricle, obtained from the Paxinos and Watson atlas, were as follows: anteroposterior –0.8 mm; lateral –1.2 mm, and ventral –3.6 mm. Cannulas were fixed to the skull surface with two screws and dental acrylic cement and then occluded with a dummy cannula. After surgery, animals were caged individually and were allowed to recover for at least 7 d. Correct placement of the cannulas was verified by the measurement of water intake in response to the ICV injection of angiotensin II (40 ng/rat). The acceptance criteria were for those animals that consumed more than 5 ml of water in the 15 min after treatment. The rats were acclimated to being handled by the removal of the dummy cannula and then connection to an empty cannula connector daily for at least 4 d before treatment to reduce stress. Animals fasted for 48 h starting 1 wk after surgery were given 6 µl artificial cerebral spinal fluid (aCSF) (140 mM NaCl, 3.35 mM KCl, 1.15 mM MgCl2, 1.26 mM CaCl2, 1.2 mM Na2HPO4, and 0.3 mM NaH2PO4, pH 7.4) alone or containing SHU9119 (1 µg/rat) via the ICV cannula. After 30 min, rats from each group were injected with 6 µl of aCSF alone or containing leptin (3.5 µg/rat) via the same cannula. The resultant animals groups were: 1) aCSF, 2) SHU9119, 3) leptin, and 4) SHU9119 + leptin treatments. All ICV injections were made in freely moving animals through a 30-gauge needle that extend 0.5 mm below the guide cannula connected by polyethylene tubing to a 25-µl Hamilton syringe. Injections were made over 3 min time course by a microprocessor-controlled infusion pump (Bioanalytical Systems, West Lafayette, IN), and the needle was then left in place for 30 sec to prevent back flow of the injected solution.

Animals were killed by decapitation at 1.5, 3, or 6 h after leptin treatment, each contained five to 10 animals per treatment per time point. The blood was immediately collected for TSH, T3, and T4 analysis. The PVN and median eminence (ME) were rapidly removed from the hypothalamus by surgical dissection and subjected to an acid peptide extraction cocktail with 2 N acetic acid freshly supplemented with a protease inhibitor cocktail (AEBSF, pepstatin A, E64, bestatin, leupeptin, and aprotin) (Sigma) as previously described (32). Cell extracts were then heated at 95 C for 15 min and sonicated, and cell disruption was performed by 15 strokes using Dounce homoginizer. After cell disruption, the samples were centrifuged at 15,000 rpm at 4 C for 30 min. Finally, supernatants were collected, and protein concentrations were determined by the Bradford assay (Coomassie Protein Assay Reagent; Pierce, Rockford, IL). The supernatant was used to measure peptides by specific RIA methods, or further purified by fractionation on a SDS-PAGE followed by gel slicing, elution, and RIA analysis. For Western blot analysis, PVN and ME were immediately immersed in extraction buffer (50 mM Tris/Cl, pH 7.2; 150 mM NaCl; 1% Triton X-100; 0.5% sodium deoxycolate; 0.1% SDS) freshly supplemented with the same protease inhibitor cocktail and homogenized with glass microhomogenizers (Wheaton, Milvale, NJ). The samples were then centrifuged at 15,000 rpm at 4 C for 30 min. Supernatants were collected and protein concentrations were determined by the Bradford assay.

Because ip anesthesia could alter hypothalamic P-CREB levels, animals used for immunohistochemistry (IHC) experiments were implanted with a jugular cannula to ensure rapid and stress-free anesthesia at the end of the experiment as previously described (26). One week after the implants of the ICV cannula, rats were anesthetized using Ketamin 50 mg/kg and Domitor 0.25 mg/kg (ip) and catheters consisting of medical grade polyethylene tubing were positioned within the right jugular vein. At the end of implantation, the tubing was filled with heparinized sterile saline solution (500 UI/ml). The distal ends of these catheters were exteriorized at an interscapular location and sealed with a sterile stainless steel plug. Animals were allowed to recover from the jugular vein cannulation for 2 d before initiation of fasting. All injections were performed between 0900 and 1030 h. Rats, fasted for 48 h, were injected via ICV cannulas with leptin (3.5 µg/rat) or the vehicle aCSF (n = 4 in each group). After 30 min, animals were deeply anesthetized with sodium pentobarbital (50 mg/kg) via the jugular catheter, and the heart was exposed and systemically perfused with heparinized saline for 5 min via the left ventricle, followed by 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 45 min. The brain was then carefully removed, postfixed for 2 h in 4% paraformaldehyde in buffered solution, and, finally, cryoprotected in 20% sucrose solution. Brains were frozen on dry ice and cut in 25-µm-thick coronal sections on a sliding cryostat, collected in four series, and stored in 0.02% sodium azide containing PBS at 4 C until further use.

Double IHC
For double P-STAT/proTRH IHC, the procedure was performed as described (12). 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. Sections were then blocked for 1 h with 3% normal goat serum in PBS/0.25% Triton X-100/0.2% sodium azide. The anti-P-STAT3 antibody was added to blocking solution (1:1500) and incubated overnight at 4 C. The next day, sections were washed, incubated with biotinylated secondary goat antirabbit antibody for 2 h (1:1000), and then treated with avidin biotin complex solution for 1 h. Finally, the signal was developed with diaminobenzidine solution, resulting in a brown precipitate. Consecutively, IHC for proTRH was performed by incubating the tissues overnight at 4 C with the primary antibody (anti-proTRH, 1:5000) in blocking solution. The next day, sections were washed and incubated with a fluorescent secondary goat antirabbit Alexa 488 (green) antibody for 1 h. After several washes, the floating sections were mounted on glass slides containing fluorescence mounting solution with 4',6'-diamidino-2-phenylindole (DAPI). The nuclear DAPI was used as an easy way to identify the nucleus. Because both antibodies were made in rabbit, control staining experiments were run in parallel, but omitting the first anti-P-STAT3 antibody to make sure that the anti-proTRH antibody stained the same cellular pattern. Results were visualized using either fluorescence (proTRH) or bright-field light (P-STAT3) sources. Fluorescent images (12 bit) and DAB images (24 bit) were acquired with a Nikon E800 microscope (Nikon Inc., Melville, NY) and a Spot II digital camera (Diagnostic Instruments, Sterling Heights, MI). Using ImageJ [National Institutes of Health (NIH), Springfield, VA] and Adobe Photoshop (Adobe, San Jose, CA), fluorescence and bright-field photographs were combined using RGB channels to visualize double-labeled cells.

For P-CREB/proTRH IHC double fluorescent staining was developed. That was accomplished by pretreating tissue with 0.3% glycine for 10 min and 0.03% SDS for 20 min. Sections were then blocked for 1 h with 3% normal donkey serum in PBS/0.25% Triton X-100/0.2% sodium azide. A combination of the mouse anti-P-CREB (1:500) and rabbit anti-proTRH (1:5000) antibodies in blocking solution was added to the incubation reaction for 72 h at 4 C. The next day, sections were washed and incubated with a combination of donkey antimouse Alexa 488 (green) and donkey antirabbit Alexa 594 (red) secondary antibodies for 2 h (1:1000). After several washes, the floating sections were mounted on glass slides and covered in fluorescence mounting solution with DAPI. Confocal images were acquired with a Nikon PCM 2000 (Nikon Inc., Melville, NY) using the Argon (488 nm) and the green Helium-Neon (543 nm) laser. Serial optical sections were performed with Simple 32 C-imaging computer software (Compix Inc., Tualilatin, OR). Z series sections were collected at 0.7 µm with x40 PlanApo lens or x10 Plan Apo lens. A scan zoom of x1 was used in the acquisition of images, and then processed and reconstructed in NIH Image shareware. Adobe Photoshop was then used for the assembly of figures.

Quantitative analysis of P-CREB and P-STAT-labeled nuclei and proTRH immunoreactive perikarya in the PVN
Double-labeled sections containing P-STAT/proTRH or P-CREB/proTRH were used for quantitative analysis. ProTRH immunostaining was confined to the perikarya and dendrites allowing one to see the nucleus containing either brown or fluorescent label for P-STAT or P-CREB, respectively. The percentage of proTRH neurons containing a labeled nucleus was determined from three distinct levels of the PVN (anterior, medial, and posterior) from four animals in each condition. The levels of the PVN were identified based on the rat brain atlas of Paxinos and Watson and by the examination of the characteristic distribution of proTRH neurons (26). All nuclei with positive staining were counted in the proTRH neurons of the PVN with visible DAPI-positive nuclei on each side of the third ventricle. That relationship was expressed as a percentage, which represents positive proTRH neurons in each section of the PVN compared with the total number of DAPI-positive proTRH neurons observed.

SDS-polyacrylamide gel fractionation of ProTRH peptides
In this experiment, 80 µg of PVN-extracted protein of the different animal groups were used. Adequate volumes of each supernatant were evaporated using an ultra cold speed vacuum system and then dissolved in sample buffer (0.0625 M Tris, pH 6.8/1% SDS/15% glycerol/0.05% bromophenol blue), boiled for 5 min, and loaded onto a discontinuous tricine-PAGE system (1.5 mm thick). A stacking gel was made to 3% crosslinking (acrylamide/bis-solution), and the separating gel was made to 6% crosslinking (acrylamide/bis-solution). The low range Prestained Protein Standards (Boston Bioproducts, Boston, MA) were used as molecular mass markers. After electrophoresis, gels were cut into 2-mm slices in a gel slicer (Hoeffer Scientific Instruments, San Francisco, CA) and peptides were extracted from the gel slices by incubating the slices in 0.5 ml 2 N acetic acid for 18 h at 4 C. These fractions were then evaporated using a refrigerated ultra cold speed vacuum system (ATR, Laurel, MD). The dried samples were reconstituted in 0.5 ml RIA buffer and then assayed by RIA for the pFE22 peptide.

Static incubation of hypothalamic explants
Animals fasted for 48 h were killed by decapitation, and the brain was immediately removed. The brain was placed in a brain matrix (Kent Scientific Corp., Torrington, CT) with the ventral surface on top, and cut using the optic chiasm and rostral edge of mamillary bodies as rostral and caudal limits, respectively. Hypothalamic sulci were used as lateral limits and a 3 mm-thick slice was taken parallel to the base of the hypothalamus. The hypothalamic explants were maintained in a static incubation system using aCSF gassed with 95% O2 and 5% CO2. After an initial 45-min equilibration period, the hypothalami were incubated for 2 h in 1 ml of the following treatments: 1) aCSF; 2) leptin 100 nM; 3) SHU9119 100 nM; 4) leptin 100 nM plus SHU9119 100 nM. Finally, the viability of the tissue was verified by 45 min of exposure to 56 mM KCl solution. At the end of the last two periods, the aCSF was collected and evaporated using a refrigerated speed vacuum system, reconstituted in 0.5 ml RIA buffer, and measured for TRH by RIA methods. The hypothalamic explants that did not respond to 56 mM KCl were excluded in the data analysis.

RIA analysis
Plasma TSH levels in rat were determined using a highly sensitive double antibody method, developed by A. F. Parlow, director of the National Hormone and Pituitary Program, (Harbor-University of California Los Angeles Medical Center, Torrance, CA) as described previously (4). Plasma T3 and T4 levels were measured using RIA kits from MP Biomedicals Diagnostic Division (Orangeburg, NY). The sensitivity of the T3 and T4 assays were 25 ng/dl and 1.2 µg/dl, and the intra and interassay variability were approximately 5–7 and 10–11%, respectively. The RIA assay used to measure TRH was developed in our laboratory using a specific TRH antiserum that recognizes only mature TRH neuropeptide. To study proTRH processing, we used the C-terminal antibody, anti- preproTRH178–199 (pFE22), which recognizes preproTRH160–255 (10 kDa), preproTRH160–199 (5.6 kDa), preproTRH178–199 (2.8 kDa), and preproTRH186–199 (1.7 kDa). The assay used for pFE22-related peptides was developed in our laboratory using custom made peptides and primary antibodies. The RIA assay to measure {alpha}-MSH was developed in our laboratory using a specific {alpha}-MSH antiserum as previously described (33). The tracers were iodinated using the Chloramine T oxidation-reduction method followed by HPLC purification. The sensitivity of the three RIA systems developed in our laboratory for TRH, pFE22, and {alpha}-MSH were 2.0, 40.0, and 15.0 pg/tube, respectively. The intra and interassay variability were 5–6 and 9–12%.

Western blot analysis of PC1/3 and PC2
In this experiment, we used 25 µg of PVN-extracted protein of the different animal groups. The adequate volumes of each supernatant were evaporated using an ultra cold speed vacuum system and then dissolved in sample buffer, boiled for 5 min before SDS-PAGE. The samples were fractionated with 8% glycine-SDS-PAGE gels. The Precision Plus Protein standards (dual color) were used as molecular mass markers (Bio-Rad Laboratories, Richmond, CA). After the electrophoresis, proteins were electroblotted onto PDVF membranes (Millipore Laboratories) for immunodetection, and blocked with 5% milk in TBS (50 mM Tris and 150 mM NaCl, pH 7.4). The membranes were probed overnight at 4 C with a 1:3000 dilution of anti-PC1/3 or anti-PC2 antiserum in 0.5% milk in TTBS (TBS containing 0.1% Tween 20) as previously described (10). An alkaline phosphatase-linked goat antirabbit immunoglobulin secondary antibody (1:2000) was then used, and immunoreactive bands were visualized by the Immunostar Assay, as described by the manufacturer (Bio-Rad Laboratories).

Statistical analysis
The results are presented as the mean ± SEM. Statistical significance was determined by ANOVA followed by post hoc Newman-Keuls test. Differences were considered to be significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ICV administration of leptin activates P-CREB and P-STAT3 in distinct populations of proTRH neurons within the parvocellular division of the PVN
To test the hypothesis that both pathways of leptin action on proTRH neurons could operate on different subsets of neurons within the PVN, we mapped the anatomic distribution of proTRH neurons using phosphorylation of STAT3 as an indicator of the direct pathway, and phosphorylation of CREB as an indicator of the indirect pathway of leptin regulation. Figure 1Go depicts a set of representative images from the anterior, medial, and posterior parts of the PVN containing proTRH neurons from animals perfused with aCSF or leptin. The brown nuclear staining represents P-STAT3 and the green cytoplasmic color represents proTRH. The merged images depicted in the right panels for each condition show the colocalization of the P-STAT3 with proTRH. See also in the bottom panels, high-magnification microphotographs indicating the specific colocalization. Consistent with our earlier report (12), a dense population of cells with nuclear P-STAT3 staining was seen in the PVN from leptin-treated rats and very few P-STAT3-positive-cells were found from aCSF-treated animals. Untreated animals (aCSF) showed that less than 1% of the proTRH cells were positive for P-STAT3. In contrast, the quantitative analysis (more than 32 fields per condition) done in leptin-treated animals showed that 4% (±1%), 28% (±3%), and 37% (±5%) of proTRH neurons were positive for P-STAT3 in the anterior, medial, and posterior parts of the PVN neurons, respectively (n = 4, P < 0.05; Fig. 1Go, graph panel). These data suggest that proTRH neurons directly responsive to leptin are mostly concentrated in the posterior and medial parts of the parvocellular subdivision of the PVN.


Figure 1
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  		FIG. 1. ProTRH neurons responding directly to leptin are concentrated in the caudal and medial parts of the parvocellular subdivision of the PVN. Fasted rats were given a single ICV injection of leptin (220 pmols/rat) or vehicle (aCSF) and killed 30 min later. Coronal brain sections were subjected to double IHC using anti-PSTAT3 (brown staining) and anti-pro-TRH (green fluorescent staining) antiserum. The upper set of panels depicts in low magnification the anterior, medial, and posterior parts of the PVN from animals treated with leptin and aCSF, and the visualization of P-STAT3 and proTRH. Colocalization of proTRH with P-STAT3 is seen in the merged panels, and the bottom panels show a detailed colocalization with a higher magnification. Arrowheads point to TRH-labeled cells, lines to single-labeled P-STAT3 cells, and arrows to dual-labeled cells. Scale bars, 50 µm (upper panels), 20 µm (lower panel). The bar graph represents the statistical analysis in percentages of proTRH neurons positive for P-STAT3 taken from eight PVNs in each condition and treatment. A total of 32 fields per section and condition were analyzed to construct the percentage analysis data. *, P < 0.005 vs. aCSF.

 
To identify activated proTRH neurons through the indirect pathway, we employed a double-IHC method for P-CREB using a green fluorescent secondary antibody to visualize mouse monoclonal anti-P-CREB antibody, and a red fluorescent secondary antibody to visualize rabbit polyclonal anti-proTRH antibody. P-CREB labeling was mostly seen as nuclear staining, but in some cases there was a display of a weak cytoplasmic distribution. Figure 2Go depicts a set of representative images from the anterior, medial, and posterior parts of the PVN containing proTRH neurons in aCSF or leptin-treated animals. Unstressed fasted rats treated with aCSF showed, as expected, minimal P-CREB staining in the parvocellular subdivision of the PVN. The staining was limited to the magnocellular subdivision, according to previous reports (6, 26). A massive increase of neurons with nuclear P-CREB staining was seen 30 min after leptin administration, particularly in the medial and anterior part of the PVN. The bottom panel of Fig. 2Go shows a higher magnification of the typical proTRH neurons that were positives for P-CREB (see arrows). Quantitative analysis of more than 32 fields (see bar graph in Fig. 1Go) indicated that 3% (±3%) of the proTRH cells were positive for P-CREB in aCSF-treated animals without any preferential distribution. On the contrary, quantitative analysis in four leptin-treated animals indicated a significant increase of P-CREB labeling in proTRH neurons (P < 0.05). The anterior, medial, and posterior parts of the PVN had 92% (±7%), 66% (±8%), and 18% (±5%) of proTRH cells also positive for P-CREB, respectively. The proTRH+/P-CREB+ neurons were predominantly located in the periventricular subdivision of the PVN. It is important to note that even although the distribution of proTRH+/P-STAT3+ and proTRH+/P-CREB+ throughout the PVN were different there was some overlapping in staining, particularly in the middle portion of the PVN, suggesting the possibility that a group of proTRH neurons could share both ObRb and MC4R. For both conditions, proTRH+/P-STAT3+ and proTRH+/P-CREB+ no adjustments in the percentages of colocalization were necessary because the means of 40 nuclei diameter counted between proTRH+/P-CREB– and proTRH+/P-CREB+ as well as for proTRH+/P-STAT3– and proTRH+/P-STAT3+ neurons was statistically very similar.


Figure 2
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  		FIG. 2. ProTRH neurons responding indirectly to leptin are concentrated in the anterior and medial parts of the parvocellular subdivision of the PVN. Fasted rats were given a single ICV injection of leptin (220 pmols/rat) or vehicle (aCSF) and killed 30 min later. Coronal brain sections were obtained and subjected to double IHC using anti-P-CREB (green fluorescence staining) and anti-proTRH (red fluorescence staining) antiserum. The upper set of panels depicts in low magnification the anterior, medial, and posterior part of the PVN from animals treated with leptin and aCSF, and the visualization of P-CREB and proTRH. Colocalization of proTRH with P-CREB is seen in the merged panels, and the bottom panels show a detailed colocalization in a higher magnification. Arrowheads point to TRH-labeled cells, lines point to single-labeled P-CREB cells, and arrows point to dual-labeled cells. Scale bars, 50 µm (upper panels), 20 µm (lower panel). The bar graph represents the statistical analysis in percentages of proTRH neurons positive for P-CREB taken from eight PVNs in each condition and treatment. A total of 32 fields per section and condition were analyzed to construct the percentage analysis data. *, P < 0.005 vs. aCSF.

 
Leptin increases TRH biosynthesis through the direct and indirect pathways
To further determine the role of the two pathways of leptin action on the regulation of TRH biosynthesis, we killed animals after 1.5, 3, and 6 h after central leptin administration, with and without blocking the melanocortin system, and analyzed the physiological end points of the HPT axis. As shown in Fig. 3AGo, leptin increases {alpha}-MSH levels in the ARC at 3 and 6 h, independently of the SHU9119 treatment. The maximum increase for the TRH peptide was observed at 3 h after leptin treatment (Fig. 3CGo), which went from 1.18 ± 0.08 pg/µg protein in the control to 2.22 ± 0.09 pg/µg protein [P < 0.05, vs. control (CTR)]. The pretreatment of animals with the melanocortin antagonist SHU9119 only partially blocked the leptin-induced synthesis of TRH (1.65 ± 0.09 pg/µg protein, P < 0.05 vs. CTR) suggesting that both pathways might be involved in the biosynthesis of TRH. In the PVN, after 6 h after leptin treatment (Fig. 3CGo), the TRH levels were approximately 75% (2.07 ± 0.08 pg/µg protein) greater than in the untreated controls, but this effect was completely reversed by the administration of SHU9119 (1.26 ± 0.09 pg/µg protein). The TRH content in the ME decreased from 2.03 ± 0.11 pg/µg protein to 1.45 ± 0.06 pg/µg protein after leptin treatment (Fig. 3BGo), likely due to an increase in the neurosecretion of TRH to the portal hypophysiotropic system. As a consequence of the neurosecretion of TRH, an increase in plasma TSH levels was observed, which ranged from 0.8 ± 0.1 to 1.9 ± 0.2 ng/ml with a maximum at 3 h posttreatment (Fig. 3DGo). The pretreatment of animals with the melanocortin antagonist completely blocked the leptin-induced decrease in TRH in the ME, as well as the increase in plasma TSH levels. This finding suggests that hypophysiotropic proTRH neurons are regulated mostly through the action of the melanocortin system. Plasma values of T3 and T4 were unchanged during all these treatments (data not shown).


Figure 3
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  		FIG. 3. Leptin increases TRH biosynthesis through the direct and indirect pathways. The panels shown represent comparative values of specific RIAs (see Materials and Methods) for {alpha}-MSH in the ARC (A), TRH in ME (B), TRH in the PVN (C), and serum TSH (D) from five to 10 animals used in each condition. Values are the mean ± SEM. ANOVA was followed by a multiple comparison using a Newman-Keuls test. *, P < 0.05 vs. time zero. a, P < 0.05 vs. leptin-treated animal at the same time.

 
The melanocortin system mediates the leptin-induced secretion of TRH from hypothalamic explants
Because our in vivo results suggest that leptin increases the neurosecretion of TRH from the ME through the melanocortin system, we used hypothalamic explants to corroborate this finding. The results depicted in Fig. 4Go showed that the release of TRH from hypothalamic explants increased from 1.9 ± 0.2 to 3.4 ± 0.3 pg/explant (P < 0.05) after 2 h of leptin treatment. Although the treatment of tissue with SHU9119 alone did not change the TRH release (2.1 ± 0.2 pg/explant), the melanocortin antagonist completely blocked the TRH release induced by leptin (2.3 ± 0.1 pg/explant).


Figure 4
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  		FIG. 4. Secretion of TRH induced by leptin from hypothalamic explants is dependent of melanocortin system. Secretion of TRH from hypothalamic explants treated for 2 h with leptin (100 nM), SHU9119 (100 nM), or leptin (100 nM) plus SHU9119 (100 nM) was measured with RIA. Hypothalamic explants were removed from rats fasted for 48 h. Values are the mean ± SEM. ANOVA was followed by a multiple comparison using a Newman-Keuls test. * P < 0.05, vs. CTR animal.

 
The melanocortin system partially mediates the leptin-induced increase in synthesis and posttranslational processing of proTRH
To determine whether the biosynthesis of proTRH is dependent on the direct or the indirect action of leptin on proTRH neurons, we examined the production profile of C-terminal proTRH-derived peptides in different experimental groups. We chose PVN samples taken after 3 h after leptin treatment because, at this time point, both pathways appear to be operating. We used an antiserum raised against the preproTRH178–199 (pFE22) sequence that recognizes 10, 5.6, 2.6, and 1.7 kDa proTRH-derived peptides (34, 35) (Fig. 5AGo). The first striking data obtained from these experiments was the fact that, in leptin-treated animals, the C-terminal intermediate preproTRH160–255 (10 kDa) form was more rapidly processed (decreased ~17% compared with untreated controls) to its smaller peptides by causing an accumulation of approximately 19% for the proTRH160–199 (5.6 kDa) and proTRH178–199 (2.6 kDa) respectively (Fig. 5BGo). The decrease of the 10-kDa and increase of the 2.6-kDa peptides respectively were statistically significant. The proTRH186–199 (1.7 kDa) was unchanged. Administration of the melanocortin antagonist partially reversed the effects of leptin in processing (Fig. 5CGo). When we calculated cumulatively the total amount of C-terminal peptides synthesized under leptin treatment as compared with untreated control, we found an overall increase in peptide biosynthesis from 3.3 ± 0.2 to 4.6 ± 0.3 fmol/µg of total protein analyzed (P < 0.05), and this action was partially mediated by the melanocortin system. Pretreatment of animals with SHU9119 partially reversed the effect of leptin by showing a decrease of total cumulative peptides from 4.6 ± 0.3 to 3.8 ± 0.2 fmol/µg of total protein.


Figure 5
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  		FIG. 5. The increase in biosynthesis and posttranslational processing of proTRH induced by leptin is partially dependent on the melanocortin system. A, Depiction of rat proTRH and the TRH-related peptides recognizes by anti-preproTRH178–199 (pFE22) antiserum. B, A representative analysis of an electrophoretic separation of PVN samples extracted from CTR, SHU9119-treated, leptin-treated, and SHU9119- plus leptin-treated animals. Tricine SDS-PAGE followed by gel slicing performed the separation, acid extraction of gel slices, and RIA assay against pFE22 peptide. Molecular masses of the identified peaks are indicated based on the migration pattern of molecular weight standards. This result represents a typical profile of three independent experiments. C, Distribution of pro-TRH-derived peptides expressed as the average of picomoles percentage for each proTRH-derived peptide of three independent experiments. *, P < 0.005 vs. CTR.

 
The melanocortin system partially mediates the leptin-induced increase of PCs levels in the PVN
We recently showed that, as part of the multiple effects that leptin has in the hypothalamus, it also regulates PC1/3 and PC2 enzymes in the PVN, which are key for the maturation of all proneuropeptides (4). Therefore, we wanted determine whether blocking the melanocortin system, in leptin stimulated animals, has an impact on the PCs. Extracted proteins from the PVN in animals subjected to SHU9119, leptin, or SHU9119 plus leptin treatments, and controls were analyzed by a Western blot system using antibodies against PC1/3 (87 kDa) and PC2 (75 and 68 kDa) as described earlier (4). The results depicted in the Fig. 6AGo show that the leptin-treated rats had an increase of approximately 50% in the PC1/3 levels in the PVN. Conversely, the pretreatment with SHU9119 antagonist reduced the leptin effect in PC1/3 by approximately 20%. Although the mature form of PC2 (68 kDa) showed a similar tendency to increase during leptin treatment, it was not as significant (Fig. 6BGo) as seen for PC1/3.


Figure 6
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  		FIG. 6. Leptin increases the level of PC1/3 in the PVN through the direct and indirect pathways. Typical Western blots for PC1/3 (A) and PC2 (B) from the PVN of animals treated with aCSF (CTR), SHU9119, leptin, or leptin plus SHU9119. The upper panels in A and B depict a single typical Western blot while the lower panels show the average integrated optical density of six Western blots. NIH Image software was used to obtain integrated optical densities. The molecular mass for PC1/3 and PC2 are indicated in A and B. A total of six animals per condition were used in this study. ANOVA was followed by a multiple comparison using a Newman-Keuls test. * P < 0.05, vs. CTR animal; a, P < 0.05 vs. leptin-treated animal.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The PVN house a complex neuronal system, where a variety of neuroendocrine, behavioral, and autonomic functions are integrated and regulated (36). Because it is one of the most important brain centers involved in the regulation of the energy expenditure, the understanding of its function is critical in the development of new therapies for energy balance abnormalities, such as obesity. In this study, we have demonstrated for the first time that there are two subpopulations of proTRH neurons responsive directly to leptin or {alpha}-MSH. ProTRH neurons located in the medial and posterior part of the PVN showed to be directly responsive to leptin, and they were not primarily related to the HPT axis. The second set of proTRH neurons, which are located in the anterior, medial, and periventricular part of the PVN were indirectly responsive to leptin (via the melanocortin system), and most of these neurons were implicated in the activation of the HPT axis.

Although the PVN is not considered to be one of the most functionally active hypothalamic targets for leptin, we confirmed that, consistent with our previous studies (11, 12, 37), there are leptin-responsive proTRH neurons, which are mostly concentrated in the caudal region of the PVN. The percentage of proTRH neurons positive for P-STAT3 is smaller than our earlier estimates, but this may be due to differences in the experimental protocols used. Although leptin-treated animals showed a strong staining for P-STAT3 in the ependymal cells and in periventricular neurons of the parvocellular subdivision of the PVN, not too many of them were proTRH neurons. This point is particularly important because these neurons are believed to send their projections to the ME and neurosecrete TRH to the portal vessels for later action in the pituitary gland (38). The P-CREB staining increased particularly in the anterior, medial, and periventricular part of the parvocellular subdivision of the PVN 30 min after leptin administration. This neuronal distribution of leptin activated neurons via {alpha}-MSH is consistent with the profile of P-CREB staining observed by us (data not show) and others (26) after the ICV administration of {alpha}-MSH.

The {alpha}-MSH-containing axons mostly innervate proTRH neurons in the anterior, ventral, and periventricular parvocellular subdivisions of the PVN, whereas fewer proTRH neurons are contacted in the medial and dorsal parvocellular subdivision (39). Also, the MC4R has been found in the medial and periventricular subdivision of the PVN (11, 40), and it was shown that only 30% of proTRH neurons in the medial parvocellular subdivision of the PVN were innervated by axons containing {alpha}-MSH (39). These proTRH neurons showed an increase in preproTRH mRNA after {alpha}-MSH administration (39). Although effective in restoring preproTRH mRNA levels in the PVN to normal levels in fasting rats, {alpha}-MSH alone was not able to fully replicate the effects of leptin administration in restoring the HPT axis to the normal condition. Fasted animals treated with {alpha}-MSH restored only 50% of thyroid hormone levels compared with the fed values. In support of these observations, we found that, in the posterior part of the PVN, approximately 40% of the proTRH neurons were P-STAT3 positive, and only approximately 20% were P-CREB positive. Although the percentage of P-CREB-positive cells could be different at different time points, it must be noted that in the anterior and the medial regions the P-CREB+/proTRH+ colocalization values are similar to the maximum levels found by other authors (26). Different from the observed STAT3 signaling, we found that 90% of the proTRH neurons located in the anterior part of the PVN responded to leptin via the indirect pathway through the activation of P-CREB, whereas, in this region, only approximately 4% of them were P-STAT3 positive. Altogether, these results confirmed our hypothesis that both pathways of leptin action in the PVN operate on partially different subsets of neurons within this nucleus. It is currently unknown whether there is a subset of proTRH neurons in the PVN sharing both the ObRb and the MC4R. Identifying P-STAT3 and P-CREB signaling in the same proTRH neurons will require more challenging triple colocalization methods. Consistent with the view of different subsets of proTRH neurons responding to different stimuli, it was recently proposed that a functional and anatomical specialization of proTRH neurons in the PVN in response to cold stress and suckling exists (41).

The data also showed that TRH levels in the ME decreased while plasma TSH levels increased after 1.5 h after leptin treatment (Fig. 3Go). This suggests that leptin stimulates the neurosecretion of TRH from axon terminals located in the ME, which might also provoke the release of other neuropeptides and neurotransmitters synthesized in the same neuron (42). This rapid leptin-induced TRH secretion appears to be independent of new protein biosynthesis, consistent with our previous studies using primary neuronal cultures (37). Although plasma TSH levels increase after ICV leptin treatment, we did not find significant changes in plasma thyroid hormone levels at the time points analyzed. Treatment with SHU9119 compound completely reversed the leptin-induced TRH release both in vivo and in vitro (Figs. 3Go and 4Go). These results are in agreement with the studies of Kim et al. (30) showing that the endogenous antagonist of the melanocortin receptors, AgRP, completely blocks the leptin-induced secretion of TRH from hypothalamic slices. These data, together with our IHC results, strongly suggest that the hypophysiotropic proTRH neurons, located in the periventricular and medial subdivision of the PVN respond to leptin mainly via the melanocortin system.

Carboxyl-terminal intermediate forms and end products of proTRH processing including TRH, increased in the PVN at 3 h after leptin treatment, likely due to a faster rate of proTRH biosynthesis, which takes approximately 2 h (43). Identical results were found for the amino-terminal products of proTRH in animals treated with leptin (4). The fact that addition of the SHU9119 compound to the leptin treatment of animals did not inhibit proTRH biosynthesis completely provides the strongest evidence supporting an activating of proTRH directly by leptin, independent of the melanocortin system. However, this dual response was abolished at 6 h of treatment when only the melanocortin system remained active. The activation of proTRH neurons, independent of the HPT axis, could be related to leptin actions on the sympathetic nervous system or on the food intake (3). TRH is an important activator of the sympathetic nervous system (35). It could mediate other sympathetic leptin-induced effects, which can be blocked with melanocortin antagonists, such as the increase of uncoupling protein 1 in the brown adipose tissue (44). It has also been shown that diencephalic TRH mediates the leptin-induced pressor effects (45). It is unknown whether proTRH neurons located in the PVN send direct inputs to the sympathetic nervous system. Interestingly, the melanocortin system mediates the anorectic leptin actions (46); therefore, this subtype of proTRH neurons could act as a second order neuron in the satiety pathway, because central administration of TRH inhibits food intake without affecting the HPT axis (47). The results also showed evidence indicating that leptin up-regulation of the PCs was partially dependent on the activation of the melanocortin system. The PC1/3 promoter contains two CREB response elements, which can be transactivated by CREB-1 (48). This is the first data showing that {alpha}-MSH increases the biosynthesis of proTRH in the PVN in a coordinated fashion with increased biosynthesis for PC1/3, which is consistent with leptin up-regulation of the PC1/3 promoter via P-STAT3 (4).

It is not yet known what is the specific role(s) of proTRH neurons when activated directly by leptin. We speculate that leptin could also mediate an increase in thermogenesis via nonhypophysiotropic pathway. For example, it was previously shown that the administration of TRH into the preoptic area, dorsomedial nucleus, anterior hypothalamus, and particularly the ventromedial hypothalamus increases energy expenditure via the TRH receptor-1 (49). Also, recent studies have showed that leptin has a thermogenic effect (3) through the sympathetic nervous system, independent of the melanocortin pathway (50, 51). Figure 7Go depicts the proposed model of leptin action in the hypothalamus to activate positively proTRH neurons. In summary, we have provided the first clear evidence for the existence of two distinct subgroups of proTRH neurons responsive to leptin and {alpha}-MSH independent from each other. This point has special interest because leptin-resistance associated to obesity seems to be specifically located in the ARC (52). In the same line of thought, it is also possible that resistance to obesity might be attributed in part to a more active thyroid axis or sympathetic activity from those proTRH neurons that were not subjected to leptin resistance. Therefore, if the PVN is still leptin-responsive in obesity, the direct leptin signaling on this hypothalamic nucleus could be a potential target for new strategies in the treatment of obesity disorders.


Figure 7
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  		FIG. 7. Proposed model of leptin action on proTRH neurons through the direct and indirect pathways. This current model proposes that, in the PVN, two subgroups of proTRH neurons exists. They are responsive to P-STAT3 and P-CREB signaling through the stimulation of leptin acting directly on proTRH neurons carrying the ObRb receptor, and on {alpha}-MSH neurons that in turn release {alpha}-MSH and stimulate proTRH through the MC4R. A third unidentified yet group of proTRH neurons is proposed to be potentially present in the PVN carrying both ObRb and MC4R. It is also proposed that most of the proTRH neurons carrying the MC4R might be involved in the regulation of the HPT axis. Other neurons stimulated by the melanocortin pathway might be involved in sympathetic activity or food intake regulation. ProTRH neurons activated directly by leptin might be involved in thermogenesis through sympathetic regulation.

 


   Acknowledgments
 
We thank Virginia Hovanesian of Rhode Island Hospital who assisted us in the acquisition and presentation of the images used in this study.


   Footnotes
 
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases/National Institutes of Health Grant R01 DK58148 and National Institute of Neurological Disorders and Stroke/National Institutes of Health Grant R01 NS045231 to E.A.N.

First Published Online April 20, 2006

Abbreviations: aCSF, Artificial cerebral spinal fluid; AgRP, Agouti-related protein; ARC, arcuate nucleus; CREB, phosphorylated cAMP response element binding protein; CTR, control; DAPI, 4',6'-diamidino-2-phenylindole; HPT, hypothalamic pituitary thyroid; ICV, intracerebroventricular; IHC, immunohistochemistry; MC4R, melanocortin 4 receptor; ME, median eminence; P-STAT3, phosphorylated signal transducer and activator of transcription 3; PC, prohormone convertase; P-CREB, phosphorylated cAMP response element binding protein; PVN, paraventricular nucleus.

Received December 2, 2005.

Accepted for publication April 10, 2006.


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