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Endocrinology, doi:10.1210/en.2007-0822
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Endocrinology Vol. 148, No. 12 6073-6082
Copyright © 2007 by The Endocrine Society

Leptin Sensitivity in the Developing Rat Hypothalamus

A.-S. Carlo, M. Pyrski, C. Loudes, A. Faivre-Baumann, J. Epelbaum, L. M. Williams and W. Meyerhof

Department of Molecular Genetics (A.-S.C., M.P., W.M.), German Institute of Human Nutrition Potsdam-Rehbruecke, 14558 Nuthetal, Germany; Institut National de la Santé et de la Recherche Médicale Unité 549 (A.-S.C., C.L., A.F.-B., J.E.), 75014 Paris, France; and Metabolic Health Group (A.-S.C., L.M.W.), Rowett Research Institute, Aberdeen AB21 9SB, Scotland, United Kingdom

Address all correspondence and requests for reprints to: Lynda Williams, Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, United Kingdom. E-mail: lmw{at}rri.sari.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In adults, the adipocyte-derived hormone, leptin, regulates food intake and body weight principally via the hypothalamic arcuate nucleus (ARC). During early postnatal development, leptin functions to promote the outgrowth of neuronal projections from the ARC, whereas a selective insensitivity to the effects of leptin on food intake appears to exist. To investigate the mechanisms underlying the inability of leptin to regulate food intake during early development, leptin signaling was analyzed both in vitro using primary cultures of rat embryonic ARC neurones and in vivo by challenging early postnatal rats with leptin. In neuronal cultures, despite the presence of key components of the leptin signaling pathway, no detectable activation of either signal transducer and activator of transcription 3 or the MAPK pathways by leptin was detected. However, leptin down-regulated mRNA levels of proopiomelanocortin and neuropeptide Y and decreased somatostatin secretion. Leptin challenge in vivo at postnatal d (P) 7, P14, P21, and P28 revealed that, in contrast to adult and P28 rats, mRNA levels of neuropeptide Y, proopiomelanocortin, agouti-related peptide and cocaine- and amphetamine-regulated transcript were largely unaffected at P7, P14, and P21. Furthermore, leptin stimulation increased the suppressor of cytokine signaling-3 mRNA levels at P14, P21, and P28 in several hypothalamic nuclei but not at P7, indicating that selective leptin insensitivity in the hypothalamus is coupled to developmental shifts in leptin receptor signaling. Thus, the present study defines the onset of leptin sensitivity in the regulation of energy homeostasis in the developing hypothalamus.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
LEPTIN IS SYNTHESIZED by adipocytes in proportion to the level of stored triglycerides (1) and is a key hormone in the regulation of food intake and energy expenditure (2). Leptin acts via distinct hypothalamic regions, including the ventromedial (VMH) and dorsomedial (DMH) nuclei, and the lateral hypothalamus in addition to the arcuate nucleus (ARC), all of which express the long, signaling isoform of the leptin receptor (Ob-Rb) (3). The ARC lies close to the third ventricle and median eminence; thus, neurones in this region have direct access to leptin in both the cerebrospinal fluid and the blood circulation (4). ARC neurones project to other energy balance centers in the hypothalamus, including the VMH, DMH, lateral hypothalamus, and paraventricular nucleus (PVN). Damage to the ARC, for example by monosodium glutamate treatment, severely affects the hypothalamic network involved in the regulation of energy balance and results in hyperphagia and obesity (5). Studies have demonstrated that the ARC plays a key role in the development of leptin resistance in obesity (6). Leptin resistance after diet-induced obesity has been observed in humans and rodents and is the result of the inability of the hypothalamus to respond to increasing leptin levels (7, 8, 9, 10).

Leptin, acting via Ob-Rb (11), induces phosphorylation of signal transducer and activator of transcription 3 (STAT3), which dimerizes and translocates to the nucleus in which it functions as a transcription factor (12, 13) for target genes that mediate the effects of leptin (14). Leptin resistance has been shown to emerge early during a high-fat diet and in one study was accompanied by the lack of STAT3 phosphorylation in the ARC (10). Consistent with this, neuronal STAT3 knockout mice exhibit obesity and leptin resistance (15, 16). Other signaling cascades activated by leptin include the MAPK (17) pathway, phosphatidylinositol 3-kinase (PI3K) (18) and 5'-AMP-activated protein kinase (AMPK) (19).

Among the different neurones present in the ARC, two distinct populations, both expressing Ob-Rb, represent the major effectors of energy balance regulation (20, 21). Neuropeptide Y (NPY) and agouti-related protein (AgRP) are orexigenic peptides, which are coexpressed within one population (22). The second population expresses the anorexigenic proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) (23, 24). NPY/AgRP neurones are inhibited by leptin (22, 25, 26, 27, 28), whereas leptin stimulates POMC/CART neurones (29, 30, 31).

In contrast to the high leptin concentrations seen during the early neonatal period, rodents maximize food intake to support growth and differentiation at this time (32, 33). In keeping with this apparent discrepancy between high leptin levels and high food intake, leptin administration fails to influence feeding or metabolic rates in animals less than 2 wk old (34, 35, 36). Moreover, leptin-deficient ob/ob mice exhibit only slightly higher body weight than normal littermates during the first 2 wk of age (34). Similarly, hyperphagia in Zucker fatty (fa/fa) rats, which lack functional Ob-Rb, does not emerge until between postnatal d 9 and 12 (37). Taken together, these results suggest that endogenous leptin plays a minor role, if any, in the regulation of energy balance in early postnatal life. However, the presence of Ob-Rb mRNA in the mouse ARC as early as embryonic d (E) 18.5 strongly suggests that leptin does play a role at this stage of development (38). In line with this concept, leptin challenge in primary hypothalamic embryonic neuronal cultures has been shown to promote pro-TRH biosynthesis (39) and potentiate the effect of insulin on GnRH secretion (40). Leptin has also been shown to exert neurotrophic effects promoting the formation of hypothalamic pathways in the neonate (41, 42).

Thus, during early postnatal life, hypothalamic neurones appear selectively insensitive to the regulatory effects of leptin on energy balance. To investigate this insensitivity to leptin, two independent models were studied. First, primary neuronal cultures derived from the ARC at E18 were challenged with leptin. Second, leptin-responsiveness was analyzed in the hypothalamus of rats during early postnatal development. In the present study, we show that hypothalamic neurones are partially insensitive to leptin before 2 wk of age and that they acquire the ability to fully respond to leptin around weaning time.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Rats were purchased from Charles River Laboratories (Sulzfeld, Germany) or bred in house (Max Rubner Laboratory, German Institute of Human Nutrition). Animals were housed in individual cages under standard conditions with a 12-h light, 12-h dark cycle (lights on at 0600 h) at 22 C with ad libitum access to water and standard rat chow (Altromin; Altromin GmbH, Lage, Germany). Animal experimentation complied with the ethical guidelines for the care and use of laboratory animals of the Ministry of Agriculture for Nutrition and Forestry (State Brandenburg, Germany, approval 32/48-3560-0/3 and 32-44457 + 35).

Primary ARC neuronal culture
Timed-pregnant Sprague Dawley CD rats on d 18 of gestation were anesthetized using ketamine [100 mg/kg body weight (BW)] (Albrecht, Aulendorf, Germany) and xylazine (10 mg/kg BW) (Rompun; Bayer Vital, Leverkusen, Germany). Primary neuronal cultures were prepared from dissociated ARC as described earlier (43). Briefly, tissue fragments were pooled and cells mechanically dissociated in PBS containing 10% fetal calf serum, 0.6% glucose, and antibiotics. Cells were seeded at the equivalent of three dissected ARC (~680,000 cells) onto 35-mm cell culture dishes (Corning, Wiesbaden, Germany) or, for immunocytochemistry, on glass coverslips (VWR International, Darmstadt, Germany) both coated with gelatin/poly-L-lysine/fetal calf serum. Cells were maintained for up to 21 d in serum-free, DMEM/Ham’s F-12 medium (D8437; Sigma-Aldrich, Chemie, Germany) supplemented with N-2 formulation (Invitrogen, Karlsruhe, Germany), 100 nM corticosterone, 1 pM 17ß-estradiol, 1 µg/ml arachidonic acid, 0.5 µg/ml docosahexaenoic acid, 10 nM triiodo-L-thyronine, and 0.5 mM L-glutamine at 37 C and 7% CO2. To inhibit glial cell proliferation, 1 µM cytosine arabinoside (Invitrogen) was added from 5 d in vitro (DIV 5) onward.

Leptin challenge and tissue collection
Male Wistar rats aged 7, 14, 21, and 28 d received a single ip injection of either 2 µg/g BW leptin (R & D Systems, Wiesbaden-Nordenstadt, Germany) or pyrogen-free saline (PFS) (Sigma, Munich, Germany). Pups were then returned to their mother and killed 2 h later. Treatments were carried out in the early light phase. Pups from small litters were excluded from the study to avoid body weight gain due to postnatal overfeeding. Trunk blood was collected and serum frozen at –80 C. Brains were rapidly collected and frozen in isopentane chilled over dry ice and stored at –80 C. Twenty-micrometer transverse sections were cut on a Leica CM1900 cryostat (Leica Microsystems AG, Wetzlar, Germany). Sections were thaw mounted onto poly-L-lysine-coated slides for in situ hybridization or onto 0.5% gelatin-coated slides for [125I]leptin binding.

NPY, somatostatin, and leptin RIA
To evaluate the effects of leptin on neuropeptide secretion, primary ARC cultures at DIV 11 were stimulated for 15 min at 37 C with PBS or 20 nM leptin (R & D Systems) in Leeman’s buffer containing 3 mM KCl. After stimulation, medium was collected and centrifuged and the supernatant aspirated and mixed 1:10 (vol/vol) with 2 N acetic acid. Samples were lyophilized and kept at –80 C. The somatostatin RIA was carried out as previously described (44). Leptin and NPY RIAs were carried out according to the manufacturer’s recommendations (Linco Research, Inc., St. Charles, MO, and Phoenix, Karlsruhe, Germany, respectively).

RT-PCR
Total RNA from cultured ARC neurones was isolated using TRIzol reagent (Invitrogen). One microgram total RNA was used for cDNA synthesis (50 µl total volume) using 10 µM oligo (dT)-primers, 40 U RNase A (MBI Fermentas, St. Leon-Rot, Germany), in the presence or absence (–RT) of 200 U Superscript RT II reverse transcriptase (RNase H–) (Invitrogen) for 60 min at 42 C. Buffer conditions were as described by the manufacturer (Invitrogen). After heat inactivation (15 min, 70 C) cDNA was diluted with 30 µl sterile water and stored at –80 C. cDNA was amplified using gene-specific primers (Table 1Go). For detection of Ob-Rb, each 12.5-µl reaction contained 1x Taq buffer (Eppendorf, Hamburg, Germany), 0.8 mM deoxynucleotide triphosphate mix, 1 µM Ob-Rb primer, 0.16 µM ß-actin primer, 1 µl cDNA, and 0.5 U Taq DNA polymerase (Eppendorf). PCR cycling conditions consisted of an initial denaturation at 96 C for 4 min followed by 30–42 cycles at 94 C for 45 sec, 60 C for 1 min, and 72 C for 1.5 min. ß-Actin primers were added to the reaction at cycle 11 to detect both genes simultaneously in the exponential amplification phase, as defined in initial experiments (not shown). For semiquantification, aliquots were removed every second cycle and run on 1% agarose gels. At each time point, the OD of the target gene was normalized to that of ß-actin. For RT-PCR of AgRP, CART, GH secretagogue receptor (GHS-R), NPY, and POMC mRNAs, the PCR was doubled and 0.4 µM of each primer was used (Table 1Go). PCR cycling conditions consisted of an initial denaturation at 96 C (94 C for GHS-R) for 4 min, followed by 35 cycles at 94 C for 45 sec (96 C for 30 sec for GHS-R), 62 C (64 C for AgRP and 60 C for GHS-R) for 1 min, and 72 C for 30 sec (1 min for GHS-R) and a final extension of 10 min at 72 C. For all amplifications, –RT control reactions that omitted reverse transcriptase either during cDNA synthesis or template DNA were performed.


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  		TABLE 1. List of the gene-specific primers, size of the amplicon, accession number, annealing temperature, and elongation time

 
Real-time PCR
To measure the effect of leptin challenge on neuropeptide gene expression, cultured ARC neurones at DIV 12 were exposed to 20 nM leptin or PFS in supplemented DMEM/Ham’s F-12 medium for 1, 2, 4, and 8 h. After total RNA extraction and cDNA synthesis, real-time PCR was carried out using the SYBR green PCR master mix (Stratagene, Amsterdam, The Netherlands). Twenty-five microliters PCR consisted of 1 µl of cDNA, 1x SYBR green PCR master mix, 30 nM internal reference dye, and 0.4 µM of primer (Table 1Go). PCR conditions corresponded to the manufacturer’s instructions, and each template was run in triplicate. DNA templates were replaced by water for controls. The specificity of the PCR products was examined by melting curve analysis using an ICycler real-time PCR machine (Bio-Rad, Muenchen, Germany) and by agarose gel electrophoresis. Real-time PCR values were normalized to neurone-specific enolase and ß-actin at the corresponding time points. Data were analyzed using ICycler software (Bio-Rad).

Western blot analyses
For biochemical analyses of STAT3 and MAPK activation, ARC neurones were treated with PFS, 20 nM leptin, or 100 ng/ml ciliary neurotrophic factor (CNTF) (Peprotech, London, UK) in supplemented DMEM/Ham’s F-12 medium at 37 C. After stimulation, cells were washed with ice-cold PBS and lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 0.2 mM EDTA, 0.2 mM EGTA, 280 mM NaCl, 10% glycerol, 1 mM dithiothreitol, 1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium-orthovanadate, 1 mM sodium-fluoride, and 10 mM sodium-pyrophosphate. After centrifugation at 12,000 x g for 30 min at 4 C, the protein content of the supernatant was determined by Bradford assay (Bio-Rad Laboratories). Equal amounts of cellular protein extracts were subjected to SDS-PAGE on 8–12.5% gels, and Western blotting was carried out according to standard procedures. After blocking, membranes were incubated overnight at 4 C in polyclonal rabbit antisera directed against mouse STAT3 (sc-482; Santa Cruz Biotechnology, Inc., Heidelberg, Germany), human phospho Tyr705-STAT3 (sc-7993R; Santa Cruz), or human phospho Thr202/Tyr204-ERK1/2 (no. 9101; Cell Signaling, Frankfurt, Germany), all at a dilution of 1:1000. Bound antibody was visualized by one of two detection methods. Blots were either incubated with alkaline phosphatase-conjugated sheep antirabbit secondary antibody at a dilution of 1:10,000 to 1:20,000 (A-8702; Sigma) followed by standard colorimetry or with horseradish peroxidase-conjugated antirabbit secondary antibody at a dilution of 1:10,000 (Amersham Biosciences GmbH, Freiburg, Germany) followed by enhanced chemiluminescence detection according to the manufacturer’s recommendations (Amersham Biosciences). ERK1/2 activation was quantified by optical densitometry using ImageJ software (http://rsb.info.nih.gov/ij/). Because the level of STAT3 was shown to remain stable during leptin challenge, the OD of the STAT3 band was used as internal control to verify equal protein loading on the gel used to determine changes in ERK1/2 phosphorylation.

Immunocytochemistry
Cultured neurones were washed in PBS, fixed for 20–30 min in 4% paraformaldehyde at room temperature, and washed in PBS before incubation in blocking buffer (4% horse serum, 0.1% Triton X-100 in PBS) for 1 h. Neurones were incubated overnight at 4 C in primary rabbit antiserum directed against rat CART (H-003-62; Phoenix), rat NPY (T-4070; Peninsula Laboratories Inc., Weil am Rhein, Germany), or {alpha}-MSH (AB-5087; Chemicon International, Temecula, CA) at dilutions of 1:400, 1:200, and 1:400, respectively, in blocking buffer. Bound antibody was detected by incubation with fluorescence-conjugated goat antirabbit secondary antibody, Alexa 488 (A-11034; Molecular Probes, Karlsruhe, Germany) at a concentration of 1:400 for CART and 1:1000 for NPY or with fluorescence-conjugated donkey antisheep secondary antibody Alexa 488 (A-11015; Molecular Probes) for 1 h. Coverslips were mounted (Dako Cytomation, Dako, Carpinteria, CA) on microscope slides (Superfrost; Roth, Karlsruhe, Germany). Fluorescence staining was visualized by confocal microscopy (Leica TCS SP2 laser scan; Leica Microsystems AG, Wetzlar, Germany).

[125I]leptin binding
Cryosections were brought to room temperature before washing in low-pH, high-salt (pH 2, 0.5 M NaCl) HEPES buffer at 4 C for 6 min to dissociate bound leptin from the receptor. Sections were then washed in 100 mM HEPES buffer (pH 7.4) for 2 x 2 min and then incubated with 1 nM [125I]leptin (PerkinElmer Life Sciences, Buckinghamshire, UK) with the specific activity adjusted to approximately 250,000 cpm/pmol with unlabeled leptin (R & D Systems) in HEPES buffer for 2 h at room temperature. Control sections were incubated with the [125I]leptin plus 10–6 M unlabeled leptin. Sections were then washed 4 x 5 min in HEPES buffer at 4 C and briefly rinsed in distilled water to remove salts before being air dried and apposed to Kodak X-AR OMAT film (Sigma) together with [125I] microscales (Amersham) for 6 wk.

In situ hybridization
Generation of riboprobes complementary to fragments of NPY, AgRP, POMC, CART, and suppressor of cytokine signaling (SOCS)-3 and hybridization procedure were as described earlier (45, 46). In brief, sections were fixed, acetylated (for SOCS-3 only), and hybridized overnight at 58 C with [35S]-labeled riboprobes at 104 cpm/µl. Nonhybridized RNA was digested with RNase A (Sigma) at 37 C for 30 min. Sections were then desalted through a series of washes in saline-sodium citrate to a final stringency of 0.1x saline-sodium citrate at 60 C for 30 min and finally dehydrated in ethanol. Air-dried slides were apposed to Hyperfilm ß-max (Amersham Biosciences) together with autoradiographic [14C] microscale standards (Amersham Biosciences) for 1 wk (SOCS-3, CART), 2 wk (AgRP, NPY), or 6 wk (POMC) at room temperature.

Quantification
For in situ hybridization, integrated ODs of hypothalamic areas were measured using the Image Pro-plus system (Media Cybernetics, Wokingham, UK) and converted to nanocuries per gram tissue using a [14C] microscale standard curves. For [125I] leptin binding, mean ODs of choroid plexus were converted to nanocuries per milligram tissue using [125I] microscale standard curve.

Statistics
All results were analyzed by one- or two-way ANOVA using Statistica software (StatSoft, Hamburg, Germany). Post hoc differences were compared using Student-Newman-Keuls multiple range test or Dunnett’s test for analysis of neuropeptide expression and the activation of MAPK. The results of leptin challenge in postnatal rats were analyzed by Student’s t test. Whenever leptin and PFS treatment at any given age were found not to be statistically different (P > 0.2), the values were grouped and used to test for differences between ages by Student-Newman-Keuls multiple range test. Significance was set as P < 0.05. All values are presented as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Primary cultures of ARC neurones express peptides and receptors important in energy balance
RT-PCR revealed amplicons of the expected size for all peptides and receptors tested: 251 bp for NPY, 222 bp for AgRP, 362 bp for POMC, 645 bp for GHS-R, and 738 bp for Ob-Rb. Replicate samples for GHS-R demonstrate the reproducibility of the results. Increasing cycles for Ob-Rb and ß-actin show that the density of the bands increase in parallel (Fig. 1AGo and Table 1Go). For CART, two amplicons of 526 and 565 bp were observed, corresponding to the known alternatively spliced forms of CART mRNA (47). The expression of these genes was confirmed at different DIVs and in several cultures (n ≥ 4). The presence of the gene products, NPY, CART, and {alpha}-MSH was confirmed by immunocytochemistry (Fig. 1Go, B–F). Semiquantitative PCR showed the level of expression of Ob-Rb mRNA throughout the culture period as remaining relatively stable from DIV 9 through DIV 17 (Table 2Go). RT-PCR analyses of Ob-Rb mRNA carried out on dissected ARC tissue at E18, day of birth [postnatal day (P) 0] and P7 revealed that Ob-Rb mRNA levels increased from E18 to P0 by a factor of 1.2 and then remained constant until P7 (Table 2Go). However, there was no detectable [125I]leptin binding above background levels in cultured ARC neurones (data not shown).


Figure 1
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  		FIG. 1. Gene expression in cultured neurones. A, Gel electrophoreses of RT-PCR products from ARC neuronal cultures at DIV 9, DIV 14 for AgRP, and DIV 15 for GHS-R for replicates 1, 2, and 3. The number of PCR cycles is indicated above the gel showing Ob-Rb and ß-actin. The double band observed for CART corresponds to the long and short forms of CART. No products derived from control reactions omitting reverse transcriptase (–RT) or template DNA (–T). B–F, Immunocytochemistry for NPY, {alpha}-MSH, and CART in cultured ARC neurones at DIV 11 and 14. NPY immunoreactivity is present in the neuritic processes (B; arrowheads; scale bars, 28 µm) and the cytosol (C; scale bar, 14 µm). D, Not all neurones contain NPY. Scale bars, 14 µm. E, CART expression is evident in the soma and processes of ARC neurones. Scale bar, 20 µm. F, {alpha}-MSH was detected in the soma and dendrites of cultures neurones. Scale bar, 20 µm. No staining was observed if primary antibody was omitted (not shown).

 

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  		TABLE 2. Semiquantification of Ob-Rb mRNA levels at different developmental stages

 
Leptin does not phosphorylate STAT3 and MAPK in cultured ARC neurones
STAT3 activation in ARC neurones in culture measured 30 min after leptin challenge using Western blotting and a STAT3-specific antibody revealed a 92-kDa band corresponding to STAT3 (Fig. 2AGo). Nonstimulated cells showed a weak band corresponding to phosphorylated STAT3 levels (48). Leptin challenge of ARC neurones failed to increase the level of phospho-STAT3 after 30 min. However, challenge of ARC neurones with CNTF, which also couples to the Janus kinase (JAK)-2/STAT3 signaling pathway (49) resulted in a significant induction of STAT3 phosphorylation after 30 min (Fig. 2AGo). These results were reproducible in cultures of different ages (n > 10) and for leptin concentrations between 10 and 100 nM (not shown).


Figure 2
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  		FIG. 2. A, Western blot analysis of STAT3 and p-STAT3 in ARC neurones at DIV 10 stimulated with PFS, 20 nM leptin (L), and 100 ng/ml CNTF (C) for 30 min. Leptin treatment failed to increase STAT3 phosphorylation, but CNTF challenge results in an increase in the density of the pSTAT3 band. Total cellular STAT3 was constant under each experimental condition. B, Cultured ARC neurones at DIV 10 were treated with PFS, 20 nM leptin (L), or 100 ng/ml CNTF (C) for 10, 20, and 30 min. Western blotting revealed the phosphorylated forms of the MAPKs, ERK1, and ERK2. STAT3 was used as internal control to verify equal gel loading. C and D, The bands were analyzed by densitometry. Arbitrary units were normalized to control levels and expressed as ratios. Leptin was unable to activate MAPK in ARC neuronal cultures; however, CNTF promoted ERK1 and ERK2 phosphorylation. **, P < 0.01.

 
Leptin is also known to activate the MAPK signaling pathway via a STAT3-independent mechanism (17, 50). Western blotting of control neurones showed two specific bands of 42 and 44 kDa, corresponding to low basal levels of phosphorylated ERK1 and 2 (Fig. 2BGo). Leptin stimulation had no significant effect on the level of phosphorylation of either ERK1 or ERK2 (Fig. 2Go, B and C). In contrast, CNTF challenge tended to increase ERK1 phosphorylation (P = 0.09 at 20 and 30 min) and induced a strong activation of ERK2 with a maximum at 30 min (Fig. 2Go, B and D). Thus, cultured ARC cells possess a functional MAPK system, which can be activated by CNTF but not leptin.

Leptin modulates the level of neuropeptide release and gene expression in cultured ARC neurones
RIA of ARC neurone culture media at DIV 11 treated with PFS or 20 nM leptin showed a decrease of around 30% in somatostatin release in response to leptin challenge. Leptin challenge decreased NPY release by around 19%, although this effect was not significant (Fig. 3AGo).


Figure 3
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  		FIG. 3. A, Effect of leptin challenge (4 h) on somatostatin and NPY secretion in cultured neurones at DIV 11 (n = 4 culture dishes/treatment). Results are expressed as percentage of control value (PFS = 100%). B and C, Time course of leptin challenge on ARC neurones. Results are expressed in percentage of the PFS control (100%). Significant differences between treatment periods are indicated: *, P < 0.05 and **, P < 0.01.

 
The effect of leptin on NPY and POMC mRNA expression levels was examined by real-time PCR at DIV 12 over an 8-h time course. The level of expression of NPY mRNA appeared to be reduced in a time-dependent manner (Fig. 3BGo). There was a leptin-induced reduction of POMC mRNA levels, which reached statistical significance after 4 h (Fig. 3CGo).

Activation of leptin signaling in postnatal hypothalamic neurones
To further determine the onset of activation of leptin signaling in the developing hypothalamus, postnatal rats at P7, P14, P21, and P28 d were acutely challenged with PFS or leptin. To confirm successful ip injection, leptin concentration in terminal serum was assayed by RIA. In all PFS-treated animals, serum leptin concentrations were less than 1 ng/ml, whereas leptin administration increased serum leptin concentrations to above 50 ng/ml. SOCS-3 is a negative feedback regulator of the intracellular response to leptin, and as such SOCS-3 gene expression is strongly induced by leptin (51). Basal SOCS-3 mRNA expression was detected in the ARC (Fig. 4Go, A and B) and the CA-1–3 region of the hippocampus (not shown) at all developmental stages tested. Administration of leptin-induced SOCS-3 mRNA expression in the ARC, VMH, DMH, and ventral premammillary nucleus (PMV) from P14 onward (Fig. 4Go, C and D) (PMV not shown).


Figure 4
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  		FIG. 4. A–D, Localization of SOCS-3 mRNA in the brain of P7 (A and B) and P14 (C and D) rats using in situ hybridization. D, Leptin challenge induces SOCS-3 mRNA expression in the ARC, VMH, and DMH nuclei at P14. E–G, Quantification of SOCS-3 mRNA induction in the ARC (E), VMH (F), and DMH (G). The hybridization signal is expressed as a percentage of P7 control values in the ARC (100%). Significant differences between treatments are: *, P < 0.05 and **, P < 0.01. ND, No detectable signal. Animal numbers are shown in brackets. Scale bar, 2 mm (A and B) and 1 mm (C and D).

 
Effect of leptin challenge on [125I]leptin binding in the postnatal brain
Specific [125I]leptin binding was found over the choroid plexus of the lateral and dorsal third ventricles and in the leptomeninges, the piriform cortex, and more diffusely in the thalamus. No [125I]leptin binding was detected in hypothalamic areas (Fig. 5AGo) as previously reported (52). Analysis of basal [125I]leptin binding density over the choroid plexus revealed that it is lowest at P7 and increases between 1 and 2 wk of age. Leptin challenge caused a decrease in [125I]leptin binding in the choroid plexus at P14 onward but not at P7 (Fig. 5Go, C and D).


Figure 5
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  		FIG. 5. [125I] leptin binding sites in early postnatal rat brains. A, Representative section showing specific [125I] leptin binding in the lateral ventricle (LV), dorsal third ventricle (D3V), and thalamus (T) at P28. B, Nonspecific [125I]leptin binding in presence of 10–6 M nonlabeled rat leptin. C and D, Changes in leptin binding 2 h after single ip leptin challenge. Results are expressed as percentage of P7 control values (100%). Significant differences between treatments and ages are indicated: *, P < 0.05, **, P < 0.01, and ***, P < 0.001. a, b, c, P14 controls, P21 controls, and P28 controls, respectively. Numbers of animals analyzed are shown in brackets. Scale bar, 2 mm

 
Neuropeptide gene expression in the postnatal hypothalamus after leptin challenge
In situ hybridization studies revealed that neuropeptide mRNA levels varied in an age-dependent manner with a peak of expression occurring around P21 for all peptides measured apart from NPY, which peaked at P14 (Fig. 6Go).


Figure 6
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  		FIG. 6. Quantification of NPY (A and B; scale bar, 1 mm), AgRP (C), POMC (D), and CART (E and F) mRNA expression in the rat hypothalamus 2 h after a single leptin ip challenge. Results are expressed as percentage of P7 control values (100%). a, b, c, P14, P21, and P28 rats, respectively. Significant differences are indicated: *, P < 0.05, **, P < 0.01, and ***, P < 0.001. ND, Not detected.

 
Leptin administration significantly increased NPY mRNA levels in the DMH at d 21 but significantly decreased NPY gene expression at P28, with no NPY mRNA signal being detectable after leptin challenge (Fig. 6Go, A and B). Leptin challenge reduced AgRP mRNA levels in the ARC at P28. This reduction did not reach statistical significance because a single animal exhibited AgRP mRNA levels approximately 3-fold higher than the other animals in the group (Fig. 6CGo). Leptin increased POMC mRNA expression at P14 (Fig. 6DGo). Leptin challenge induced a decrease in CART in the ARC and PVN at P7, whereas it increased CART at P28 in the ARC and PVN (Fig. 6Go, E and G).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Using a combination of RT-PCR and immunocytochemistry, the present study demonstrates the viability of using cultured ARC neurones to study cellular events involved in leptin signaling. Cultured ARC neurones express not only Ob-Rb but also leptin target genes including the orexigenic peptides NPY and AgRP and the anorexigenic POMC and CART. Previous studies have shown that in vivo the phenotype of ARC neurones is identifiable during late embryonic development with NPY mRNA detectable at E18 (53). Similarly, CART mRNA expression is first seen at E18 in the rat brain, and AgRP mRNA expression in the ARC is found at birth (54, 55). The presence of NPY and POMC has also been previously identified in E18 ARC neurones in culture (56). The present study also demonstrates the presence GHS-R gene expression in ARC neurones in culture. Thus, taken together our data demonstrate that important energy balance-related neuropeptides and receptors are present in ARC neurones and that their presence is maintained throughout the culture period used in the present study.

Signal transduction mediated by Ob-Rb is predominantly via the JAK2/STAT3 pathway, which is required for the effects of leptin on energy balance (14). Ob-Rb also signals via the MAPK pathway. Using Western blotting techniques, the presence of these signaling pathways was demonstrated in cultured ARC neurones. In the adult animal, leptin challenge results in rapid STAT3 phosphorylation (57). However, in cultured ARC neurones, there was no detectable increase in STAT3 phosphorylation in response to leptin challenge. Neither was there any discernible leptin-induced phosphorylation of MAPK. The leptin concentrations used in the present study (10–100 nM) have been previously shown to be effective in inducing leptin mediated effects in primary hypothalamic neuronal cultures (39, 58). Stimulation with CNTF, an IL-6 type cytokine that activates leptin-like pathways (49), resulted in a rapid increase in phosphorylated STAT3 and MAPK. These findings demonstrate a functional JAK2/STAT3 and MAPK signaling system in cultured ARC cells, which, however, appears to be nonresponsive to leptin.

The quantification of Ob-Rb mRNA levels in cultured ARC neurones between DIV 9 and 17 indicates that no changes in gene expression occur throughout the culture period, with values ranging around the 0.85 value, comparable with a value of 0.87 found in dissected E18 ARC. However, in the dissected ARC tissue, the level of expression of Ob-Rb increases between E18 and birth (P0) over approximately 3 d. Thus, the Ob-Rb gene expression level in cultured ARC neurones fails to show the expected increase seen in the intact animal. However, the reduced level of Ob-Rb mRNA expression is unlikely to be responsible for the impairment of leptin-induced STAT3 and MAPK signaling pathways in cultured ARC neurones. The observation that leptin regulates somatostatin secretion in cultured ARC neurones provides evidence that Ob-Rb protein is present and capable of eliciting leptin-stimulated events. Also, in a previous study, leptin has been shown to regulate pro-TRH biosynthesis in hypothalamic neuronal cultures from E17 rats, although in this case Ob-Rb mRNA levels were found to be approximately 4 times lower than in the adult (39). However, the increase in Ob-Rb gene expression, which occurs in the intact animal but not the cultured neurones, indicates that the signal for this increase is not present in the cultured neurones.

As previously reported, there was no detectable [125I]leptin binding to hypothalamic nuclei using in vitro autoradiography (52). The absence of leptin binding in the hypothalamus has also been described in the neonatal and adult brain (59, 60, 61, 62), which may be explained by the fact that most of the leptin receptor in neurones of the hypothalamus is found in the Golgi apparatus rather than at the cell surface (63, 64, 65). Also only low levels of Ob-Rb expression are detected at the cell surface of transiently transfected HeLa cells (66). Thus, the majority of Ob-R mRNA may not be translated into receptor protein destined for the cell surface.

The quantification of the level of gene expression for AgRP, CART, and POMC in the developing ARC showed a peak of expression for these neuropeptides at P21 corresponding to the beginning of the weaning period. NPY gene expression peaked at P14. These findings confirm other studies, which showed that NPY mRNA levels in the ARC peak at around P15 (67) and that NPY content in the hypothalamus rapidly increases between P4 and P20 (68). In mice, the level of AgRP and POMC gene expression in the ARC has been shown to peak of expression at P21 and P22, respectively (35, 55). CART mRNA expression has also been shown to increase between P7 and P14 to reach its maximum at P21 (35). Similarly, the level of Ob-Rb in the hypothalamus also increases in the first 3 wk postnatally (69). Taken together, these results indicate that the neuropeptide system involved in the regulation of energy balance develops between 2 and 3 wk of age.

Leptin signaling is under the negative feedback control of SOCS-3 (51, 70, 71). Up-regulation of SOCS-3 gene expression may be one of the mechanisms underlying the development of leptin resistance (72). In the present study, consistent with studies in adult rats, SOCS-3 mRNA was strongly induced 2 h after a single leptin challenge in the ARC, DMH, PMV, and VMH (51). This induction occurred in P14, P21, and P28 rats, whereas no change in SOCS-3 expression was observed at P7, even after prolonged exposure of the film to maximize signal detection. Because the transcriptional up-regulation of SOCS-3 gene expression is mediated by the STATs (73, 74, 75), this finding substantiates the lack of response of the STAT3 pathways to leptin challenge in primary ARC neuronal cultures. These results demonstrate that basal SOCS-3 mRNA levels are low in the developing hypothalamus and that leptin is able to induce SOCS-3 mRNA expression in the hypothalamus only after P7. One possible explanation for the lack of induction of SOCS-3 gene expression may be the low levels of expression of Ob-Rb at early postnatal ages and hence the ability of leptin to elicit a measurable response (69). The fact that Ob-Rb gene expression did not increase in cultured ARC neurones intimates that the presence of circulating factors in the postnatal animal may be responsible for this increase. The most obvious of these would be the increase in leptin seen in the second week of postnatal life (32). It has been demonstrated that any changes in the timing or the magnitude of this postnatal leptin surge by manipulation of maternal nutrition can have long lasting repercussions on energy balance and metabolic health in the offspring (76).

To confirm and extend these observations, the effects of leptin challenge on the gene expression of hypothalamic neuropeptides was quantified in ARC neurones in culture and during early postnatal development. In contrast to the lack of response of the STAT3 and MAPK pathways to leptin challenge in cultured neurones, the level of NPY and POMC gene expression was regulated by leptin. Leptin significantly down-regulated NPY mRNA, consistent with the response seen in the adult animal (26) and also consistent with that found in immortalized embryonic hypothalamic neurones (77). However, leptin decreased POMC mRNA levels. This is in contrast to the adult animal in which leptin increases POMC gene expression (30, 31, 78). The leptin-induced increase in POMC gene expression seen in the adult has been shown to be phosphorylated STAT3 dependent (14, 78). The inability of leptin to activate STAT3 in embryonic ARC neurones may underlie this unexpected result. In contrast to POMC, the regulation of NPY gene expression by leptin is via a STAT3-independent mechanism (14). Together these findings may explain the leptin-induced reduction of both NPY and POMC mRNA levels in the absence of STAT3 phosphorylation.

In contrast to the results obtained in ARC cultures, no significant differences were observed in neuropeptide mRNA levels in animals challenged with leptin at P14 and P21 apart from AgRP mRNA, which showed a tendency to be decreased in response to leptin challenge at P21, and POMC gene expression, which was up-regulated at P14. In a previous study, daily leptin injection from P10 to P17 did not alter NPY, AgRP, CART, or POMC gene expression in neonatal mice (35). In a separate study, NPY mRNA levels in the ARC were found to be unchanged 2 h after acute leptin challenge at P10 (36). However, leptin challenge in pups previously chronically treated with leptin (P3-P9) was found to decrease NPY mRNA levels in the caudal region of the ARC at P10 (36). In the present study, leptin challenge at P21 did not affect the level of NPY mRNA in the ARC but increased gene expression in the DMH. Region-specific changes in NPY expression have been previously identified (79) in which maternal deprivation increases NPY mRNA levels in the ARC and reduces those in the DMH. CART mRNA levels were found to be down-regulated 2 h after leptin challenge in the ARC and PVN at P7, in contrast to the known effect of leptin in adult animals (31). Leptin challenge had no effect on the gene expression of any other neuropeptide at P7.

In conclusion, these findings demonstrate the inability of leptin to stimulate the JAK/STAT and MAPK signaling cascades in embryonic ARC neurones and the JAK/STAT pathway in postnatal hypothalamus before P7. These results taken together with the low basal levels of expression of neuropeptides before P21 and leptin-induced changes in POMC and CART gene expression in opposition to those seen in the adult animal explain the inability of leptin to regulate food intake in early postnatal animals. These data also indicate that the effects of leptin on neuronal growth in the developing ARC are not mediated via the JAK/STAT or MAPK pathways.


   Acknowledgments
 
We thank J. Duncan, I. Jäger, E. Thom, E. Steinmeyer, and A. Morris for excellent technical assistance and P. Bain for assistance with graphics.


   Footnotes
 
A.-S.C. was a recipient of a Research Training Grant at Obesechool funded by European Community framework V Program: HPMT-CT-2001-00410 and NuGo Exchange Grant DS05-007.

Disclosure Summary: The authors have nothing to disclose.

First Published Online September 13, 2007

Abbreviations: AgRP, Agouti-related protein; ARC, arcuate nucleus; BW, body weight; CART, cocaine- and amphetamine-regulated transcript; CNTF, ciliary neurotrophic factor; DIV, days in vitro; DMH, dorsomedial nucleus; E, embryonic day; GHS-R, GH secretagogue receptor; JAK, Janus kinase; NPY, neuropeptide Y; Ob-Rb, long, signaling isoform of the leptin receptor; P, postnatal day; PFS, pyrogen-free saline; PMV, ventral premammillary nucleus; POMC, proopiomelanocortin; PVN, paraventricular nucleus; SOCS-3, suppressor of cytokine signaling-3; STAT3, signal transducer and activator of transcription 3; VMH, ventromedial nucleus.

Received June 19, 2007.

Accepted for publication September 4, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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