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Endocrinology Vol. 148, No. 5 2251-2263
Copyright © 2007 by The Endocrine Society

Leptin Suppresses Ghrelin-Induced Activation of Neuropeptide Y Neurons in the Arcuate Nucleus via Phosphatidylinositol 3-Kinase- and Phosphodiesterase 3-Mediated Pathway

Daisuke Kohno, Masanori Nakata, Fumihiko Maekawa, Ken Fujiwara, Yuko Maejima, Motoki Kuramochi, Takuya Shimazaki, Hideyuki Okano, Tatsushi Onaka and Toshihiko Yada

Divisions of Integrative Physiology (D.K., M.N., F.M., K.F., Y.M., M.K., T.Y.) and Neurophysiology (T.O.), Department of Physiology, Division of Histology and Cell Biology (K.F.), Department of Anatomy, and Department of Neuropsychiatry (M.K.), Jichi Medical University School of Medicine, Tochigi 329-0498, Japan; and Department of Physiology (T.S., H.O.), Keio University School of Medicine, Tokyo 160-8582, Japan

Address all correspondence and requests for reprints to: Dr. Toshihiko Yada, Department of Physiology, Division of Integrative Physiology, Jichi Medical University, School of Medicine, Tochigi 329-0498, Japan. E-mail: tyada{at}jichi.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Neuropeptide Y (NPY) neurons in the hypothalamic arcuate nucleus (ARC) play a central role in stimulation of feeding. They sense and integrate peripheral and central signals, including ghrelin and leptin. However, the mechanisms of interaction of these hormones in NPY neurons are largely unknown. This study explored the interaction and underlying signaling cross talk between ghrelin and leptin in NPY neurons. Cytosolic Ca2+ concentration ([Ca2+]i) in single neurons isolated from ARC of adult rats was measured by fura-2 microfluorometry. Ghrelin increased [Ca2+]i in 31% of ARC neurons. The [Ca2+]i increases were inhibited by blockers of phospholipase C, adenylate cyclase, and protein kinase A. Ghrelin-induced [Ca2+]i increases were suppressed by subsequent administration of leptin. Fifteen of 18 ghrelin-activated, leptin-suppressed neurons (83%) contained NPY. Leptin suppression of ghrelin responses was prevented by pretreatment with inhibitors of phosphatidylinositol 3-kinase and phosphodiesterase 3 (PDE3) but not MAPK. ATP-sensitive potassium channel inhibitors and activators did not prevent and mimic leptin suppression, respectively. Although leptin phosphorylated signal-transducer and activator of transcription 3 (STAT3) in NPY neurons, neither STAT3 inhibitor nor genetic STAT3 deletion altered leptin suppression of ghrelin responses. Furthermore, orexigenic effect of intracerebroventricular ghrelin in rats was counteracted by leptin in a PDE3-dependent manner. These findings indicate that ghrelin increases [Ca2+]i via mechanisms depending on phospholipase C and adenylate cyclase-PKA pathways in ARC NPY neurons and that leptin counteracts ghrelin responses via a phosphatidylinositol 3-kinase-PDE3 pathway. This interaction may play an important role in regulating ARC NPY neuron activity and, thereby, feeding.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ARCUATE NUCLEUS (ARC) in the hypothalamus senses and integrates a variety of peripheral and central signals involved in feeding and energy homeostasis (1, 2). Neuropeptide Y (NPY) neurons coexpressing agouti-related protein in ARC play a critical role in feeding; the expression and secretion of NPY in the hypothalamus are increased during fasting (3), intracerebroventricular (ICV) injection of NPY potently stimulates food intake (4), and abrogation of NPY neurons in adulthood causes anorexia (5, 6). The NPY neurons in ARC are the principal effector system for both the orexigenic effects of ghrelin and anorexigenic effects of leptin.

Ghrelin is abundantly produced in the stomach and, to a lesser extent, in the intestine and pancreas (7, 8, 9). In the brain, ghrelin is localized to neurons in either the ARC (7) or the internuclear space between the ARC, paraventricular, dorsomedial, ventromedial hypothalamic nuclei, and lateral hypothalamus (10). Plasma concentrations of ghrelin rise preprandially and fall postprandially (11). Both peripheral and ICV administrations of ghrelin increase food intake and body weight (12, 13). These observations suggested a possible physiological role for ghrelin in feeding. Ghrelin receptors are abundantly expressed in NPY neurons (14). The food intake stimulated by ghrelin and ghrelin receptor agonists is suppressed by ICV administration of NPY Y1 receptor antagonists (15, 16) and anti-NPY IgG (15) and in NPY knockout (KO) mice (17). Our previous study demonstrated that ghrelin directly activates ARC neurons, with NPY neurons as the major target (18). Ghrelin-induced food intake thus appears to be mediated primarily via NPY neurons, although vagus nerve-mediated pathway may also be involved (19).

Leptin, an adipocyte-derived hormone, is a key negative regulator of feeding and energy metabolism. Central injection of leptin in ob/ob mice reduces food intake and increases energy expenditure (20), and neuron-selective deletion of leptin receptors leads to obesity (21). Thus, leptin decreases food intake and body weight through its effects on the leptin receptors located in central neurons. Leptin receptors are abundantly distributed in ARC neurons, including both NPY and proopiomelanocortin (POMC) neurons (22). Restoring leptin signaling in ARC in leptin receptor-null mice decreases both food intake and body weight (23). Thus, the effects of leptin on ARC NPY neurons, as well as POMC neurons, play a critical role in the regulation of food intake and body weight.

Plasma levels of ghrelin and leptin are inversely correlated (24). Ghrelin stimulates and leptin inhibits food intake (15). At the neuronal level, ghrelin-induced activation of NPY neurons is suppressed by subsequent administration of leptin (18). Ghrelin and leptin thus reciprocally regulate NPY neurons, which may play a role in the physiological regulation of feeding. However, the precise nature of the interaction between ghrelin and leptin and the underlying signaling mechanisms in NPY neurons are largely unknown.

In this study, we isolated single neurons from ARC of rats aged 5–7 wk, at which time feeding function is matured, and monitored the activity of single ARC neurons by measuring cytosolic Ca2+ concentrations ([Ca2+]i) and investigated the intracellular signaling mechanisms for ghrelin, leptin, and their cross talk. We present evidence suggesting that ghrelin activates NPY neurons via both phospholipase C (PLC) and adenylate cyclase-cAMP-protein kinase A (PKA) pathways and that leptin counteracts ghrelin-induced activation via phosphatidylinositol 3-kinase (PI3K)-phosphodiesterase 3 (PDE3)-mediated attenuation of the adenylate cyclase-cAMP-PKA pathway in ARC NPY neurons.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Adult (5–7 wk old) male Sprague Dawley rats, wild-type C57BL/6 mice, signal transducer and activator of transcription (STAT) 3/nestin KO mice, and STAT3flox/– mice were maintained on a 12-h light, 12-h dark cycle and given conventional food and water ad libitum. The animal protocols for this study were approved by the Jichi Medical University Institute of Animal Care and Use Committee and accorded with the Japanese Physiological Society’s guidelines for animal care.

STAT3/nestin KO mice
Nestin-Cre mice expressing Cre recombinase under the control of the mouse nestin gene promoter (5.8 kb 5' fragment) and neural-specific enhancer (1.8 kb fragment of second intron) were mated with STAT3+/– to generate offspring carrying both the Cre gene and the heterozygous STAT3-null mutation (nestin-Cre STAT3+/–). These mice were further mated with STAT3flox/flox mice to generate neural-specific STAT3-KO mice (nestin-Cre STAT3flox/–; STAT3/nestin KO mice) (25).

Preparation of single neurons from ARC
The ARC was excised from the brains of rats or mice aged 5–7 wk (see Fig. 3Go). Single neurons were then prepared according to procedures reported previously (18) with slight modifications. Briefly, rats were anesthetized with urethane (ethyl carbamate; 1 g/kg, ip) and decapitated, and their brains were removed. Brain slices containing the entire ARC were prepared, and the entire ARC was excised from the left and right sides. The dissected tissues were washed with 10 mM HEPES-buffered Krebs-Ringer bicarbonate buffer (HKRB) containing 10 mM glucose. They were then incubated in HKRB supplemented with 20 U/ml papain (Sigma Chemical Co., St. Louis, MO), 0.015 mg/ml deoxyribonuclease, 0.75 mg/ml BSA, and 1 mM cysteine for 16 min at 36 C in a shaking water bath, followed by gentle mechanical trituration for 5–10 min. After trituration, the cell suspension was centrifuged at 100 x g for 5 min. The pellet was resuspended in HKRB and distributed onto coverslips. The cells were kept at 30 C in moisture-saturated dishes for 30 min and then 25 C for up to 10 h.


Figure 3
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  		FIG. 3. Leptin suppressed ghrelin-induced [Ca2+]i increases via a PI3K-mediated mechanism. A and B, Preincubation with PI3K inhibitors LY294002 at 50 µM (A) or wortmannin at 100 nM (B) for 1 h abolished leptin-induced suppression of ghrelin-induced [Ca2+]i increases. C, Preincubation with U0126 at 10 µM for 30 min did not alter leptin-induced suppression of ghrelin responses. D, Percentage of neurons in which ghrelin-induced [Ca2+]i increases were suppressed by leptin. Effects of leptin were quantified by reduction of amplitude of [Ca2+]i response to ghrelin, expressed as percentages (E) or by AUC (F). Suppressions of [Ca2+]i by leptin, thus expressed as amplitude and AUC, were significantly attenuated after treatment with LY294002 (LY29) and wortmannin (Wort) but not U0126. Cont, Control. *, P < 0.05; **, P < 0.01. Glucose concentration was 10 mM in A–F. G–J, Leptin suppressed ghrelin-induced [Ca2+]i increases via a PI3K-mediated mechanism under low-glucose condition. G, Under low (2.5 mM) glucose condition, ghrelin at 10–10 M increased [Ca2+]i in ARC neurons and leptin at 10–12 M suppressed ghrelin-induced [Ca2+]i increases. H, Preincubation with PI3K inhibitor LY294002 at 50 µM for 1 h abolished leptin-induced suppression of ghrelin-induced [Ca2+]i increases under low-glucose condition. I, Percentage of neurons in which ghrelin-induced [Ca2+]i increases were suppressed by leptin. J, Reduction of amplitudes of ghrelin-induced [Ca2+]i responses by leptin, as expressed by percent inhibition, was significantly attenuated after treatment with LY294002. *, P < 0.05.

 
Measurements of [Ca2+]i in single ARC neurons
At 2–10 h after cell preparation, [Ca2+]i was measured by ratiometric fura-2 microfluorometry in combination with digital imaging, as previously reported (18, 26). Briefly, after incubation with 2 µM fura-2/AM for 30 min at room temperature, the cells were mounted in a chamber and superfused with HKRB at 1 ml/min at 33 C. Fluorescence images due to excitation at 340 and 380 nm were detected every 8.0 sec with an intensified charge-coupled device camera, and the ratio image was produced by an Argus-50 system (Hamamatsu Photonics Co., Hamamatsu, Japan). Ratio values were converted to [Ca2+]i values according to calibration curves (27). Data were obtained from single cells identified as neurons by procedures reported previously (18); briefly, they have a relatively large diameter (≥10 µm), their cell bodies are clear and round on phase-contrast microscopy, and they responded to glutamate administration with rapid increases in [Ca2+]i. Cells with astrocyte-like flat morphology were excluded. Data were obtained from cells that met these criteria for neurons.

Criteria for [Ca2+]i responses and expression of results
Ghrelin and leptin were administered in the superfusion solution. Amplitudes of [Ca2+]i increases in response to agents were calculated by subtracting prestimulatory basal [Ca2+]i levels from peak [Ca2+]i levels. When increases in [Ca2+]i took place within 5 min after addition of agents and their amplitudes were 150 nM or larger, they were considered responses. In the studies of ghrelin effects shown in Fig. 1Go, when the amplitude of [Ca2+]i responses to the second ghrelin administration with drug treatment was 40% or smaller than that of control [Ca2+]i responses to the first ghrelin administration without drugs, inhibition was judged to have occurred. In Figs. 2–6GoGoGoGoGo, the suppression by leptin was judged by the following criteria: the peak amplitude of ghrelin-induced [Ca2+]i increase was decreased to a level of 40% or smaller, the reduction lasted for 7 min or longer, and the recovery of [Ca2+]i increase was observed after washing out leptin. Area under the curve (AUC) was calculated by integrating and averaging amplitudes of [Ca2+]i changes during 5 min after the ghrelin administration (Fig. 1Go) or leptin administration (Fig. 3Go).


Figure 1
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  		FIG. 1. Inhibitors of adenylate cyclase, cAMP, and PLC suppressed ghrelin-induced [Ca2+]i increases in ARC neurons. A, Administration of ghrelin twice caused repeated [Ca2+]i increases in a neuron isolated from ARC, which was subsequently proven to contain NPY. Scale bar, 10 µm. B–E, Preincubation with the adenylate cyclase inhibitor SQ22536 (B), the competitive cAMP antagonist Rp-8Br-cAMP (C), and the PLC inhibitor U73122 (D) suppressed ghrelin-induced [Ca2+]i increases, whereas U73343, an analog of U73122 without inhibitory effect on PLC, did not alter ghrelin-induced [Ca2+]i increase (E). F–H, Percentage of neurons with increases in [Ca2+]i in response to ghrelin (F), the [Ca2+]i increase assessed by amplitude (G) and AUC (H) were significantly decreased after treatment with SQ22536 (SQ), Rp-8Br-cAMP (Rp8Br), and U73122 (U731), compared with the control (Cont) without drugs or with U73343 (U733). The numbers above each point indicate the number of neurons that responded over the number of neurons examined (F). The number above each bar indicates the number of neurons examined (G and H). *, P < 0.05; **, P < 0.01; #, P < 0.01. Glucose concentration was 10 mM in all experiments.

 

Figure 2
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  		FIG. 2. Leptin suppressed ghrelin-induced [Ca2+]i increases in NPY neurons. A, Ghrelin at 10–10 M increased [Ca2+]i in ARC neurons. B, Leptin at 10–12 M suppressed ghrelin-induced [Ca2+]i increases (left panel) in a neuron that was proven to be NPY immunoreactive (right panel). Scale bar, 10 µm. C, Percentage of leptin-suppressed neurons among neurons that responded to ghrelin with [Ca2+]i increases. D, Percentage of NPY-immunoreactive neurons among neurons in which ghrelin-induced [Ca2+]i increases were suppressed by leptin.

 

Figure 4
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  		FIG. 4. Leptin suppressed ghrelin-induced [Ca2+]i increases via a PDE3-mediated mechanism. A, Preincubation with the PDE3 inhibitor milrinone (10 µM) for 30 min abolished suppression by leptin of ghrelin-induced [Ca2+]i increases in ARC neurons. B, After 30 min pretreatment with and in the presence of the PDE3 inhibitor cilostamide (1 µM), leptin suppression of ghrelin-induced [Ca2+]i increases was blunted. C, In the presence of 100 nM glibenclamide, a KATP channel inhibitor, leptin suppressed ghrelin-induced [Ca2+]i increases. D, Pinacidil (100 nM), a KATP channel activator, did not suppress ghrelin-induced [Ca2+]i increases. E and F, Percentage of neurons in which ghrelin-induced [Ca2+]i increases were suppressed by leptin (E) and percent reduction of amplitude of ghrelin-induced [Ca2+]i increases by leptin (F) were significantly attenuated after treatments with milrinone (Milr) and cilostamide (Cilo) but not glibenclamide (Gilb) and tolbutamide (Tolb), a KATP channel inhibitor. *, P < 0.05. G and H, Leptin counteracted ghrelin-stimulated food intake and this effect of leptin was abolished by a PDE3 inhibitor. G, Effects of single ICV injections of saline (n = 11), 150 pmol ghrelin (n = 13), and 125 pmol leptin (n = 7) and coinjection of ghrelin and leptin (n = 16) on overnight (14 h) cumulative food intake in 8-wk-old rats. Ghrelin-induced enhancement of food intake was suppressed by coinjection of leptin. *, P < 0.05; **,P < 0.01 vs. saline-treated group. H, Cilostamide (2 µg) was coadministered with ghrelin (n = 8) or ghrelin and leptin (n = 10). Treatment with cilostamide abolished leptin suppression of ghrelin-stimulated food intake.

 

Figure 5
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  		FIG. 5. A–C, Leptin induced phosphorylation of STAT3 in NPY neurons. Rats (n = 4) were given a single ICV injection of leptin (20 µg/rat) or saline and killed 20 min later. A, In leptin-injected rats, NPY mRNA and pSTAT3 were detected in ARC by in situ hybridization of NPY mRNA with digoxigenin-labeled riboprobe (blue) and by immunohistochemistry using anti-pSTAT3 (Tyr795) antibody (brown). Scale bar, 50 µm B, Magnification of the area boxed in A. Black arrowheads indicate NPY mRNA-expressing neurons, white arrowheads indicate pSTAT3-immunoreactive neurons, and red arrowheads indicate NPY mRNA and pSTAT3-coexpressing neurons. C, In saline-injected rats, little pSTAT3 immunoreactivity was observed. 3V, Third ventricle. D–F, STAT3 inhibitor peptide failed to alter leptin effect on rat ARC neurons. Preincubation with STAT3 inhibitor peptide at 500 µM for 30 min did not alter the leptin-induced suppression of ghrelin-induced [Ca2+]i increases (D). Percentage of neurons in which ghrelin-induced [Ca2+]i increases were suppressed by leptin (E). Cont, Control; Inhib, inhibitor. Reduction of amplitudes of ghrelin-induced [Ca2+]i increases by leptin is expressed by percent inhibition (F). No significant difference was observed in the absence and presence of STAT3 inhibitor peptide. G–J, Leptin equally inhibited ARC neurons from wild-type and STAT3/nestin KO mice. Ghrelin increased [Ca2+]i in ARC neurons from wild-type mice (G) and STAT3/nestin KO mice (H), and these [Ca2+]i increases were suppressed by leptin in a similar manner. Neither percentage of neurons in which ghrelin-induced [Ca2+]i increases were suppressed by leptin (I) nor reduction of amplitudes of ghrelin-induced [Ca2+]i responses by leptin, as expressed by percent inhibition (J), was significantly different between STAT3/nestin KO, STAT3flox/–, and wild-type mice.

 

Figure 6
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  		FIG. 6. Leptin suppression of adenylate cyclase activator-induced [Ca2+]i increases and its blockade by PI3K inhibitor. A and B, Forskolin, an adenylate cyclase activator, at 10 µM increased [Ca2+]i in ARC neurons (A), and this [Ca2+]i increase was suppressed by 10–10 M leptin (B). C–F, Leptin suppression of forskolin-induced [Ca2+]i increases were blocked by preincubation with LY294002 (LY29; 50 µM, 1 h) and milrinone (Milr; 10 µM, 30 min) (C and D). Percentage of neurons in which forskolin-induced [Ca2+]i increases were suppressed by leptin (E) and percent reduction of amplitude of forskolin-induced [Ca2+]i increases by leptin (F) were significantly attenuated after treatments with LY294002 and milrinone. Cont, Control. *, P < 0.05.

 
Values are presented as the mean ± SEM (n = number of neurons). Each experiment was based on at least seven neurons prepared from at least four rats. A total of 535 neurons was examined. Student’s paired or unpaired t test was used to evaluate differences, with values of P < 0.05 considered significant.

Immunocytochemical identification of NPY neurons
After [Ca2+]i measurement, the cells were fixed with 4% paraformaldehyde overnight. They were pretreated with 3% H2O2 in methanol for 1 h and blocked in 10% normal goat serum and in 0.1 M PBS for 1 h at room temperature. Cells were incubated overnight at 4 C with primary antiserum to NPY (DiaSorin, Stillwater, MN) diluted 1:10,000 in PBS containing 1.5% normal goat serum. The antiserum were then rinsed and incubated with biotinylated secondary antibody raised against rabbit IgG (Vector Laboratories Inc., Burlingame, CA; diluted 400-fold) for 1 h at room temperature. The secondary antibody was then rinsed, and the sections were labeled with avidin-peroxidase complex (ABC kit; Vector) for 1 h and color developed with 3,3'-diaminobenzidine. Control experiments were carried out by omitting the primary antiserum.

Correlation of [Ca2+]i and immunocytochemical findings was performed using procedures reported previously (18). In brief, at the end of [Ca2+]i imaging, we took photographs of all cells in the microscopic field subjected to [Ca2+]i measurements. Based on these photographs, the cells in which [Ca2+]i was recorded were correlated with the corresponding immunocytochemical results.

ICV injection
Rats and mice were anesthetized with Avertin (tribromoethanol; 200 mg/kg, ip) and placed in a stereotaxic frame. A stainless steel guide cannula (26-gauge) was inserted into the brain with the tip in the third ventricle (rats) or lateral ventricle (rats, mice), and secured to the skull with screws and dental cement. The cannula tip was located 1.4 mm caudal to bregma and 9 mm below the skull for rat third ventricle, 0.8 mm caudal and 1.6 mm right to the bregma and 3.5 mm below the skull for rat lateral ventricle, and 0.22 mm caudal and 1 mm right to bregma and 1.7 mm below the skull for mouse ventricle. The position was verified with methylene blue injected through the cannula after experiments. Seven to 10 d after surgery, hormones and/or agents were injected through a 30-gauge injection needle.

Immunohistochemistry and in situ hybridization after ICV injection of leptin
Leptin (20 µg per 20 µl/rat, 2 µg per 2 µl/mouse) or saline (rats) was injected into third ventricle. Twenty minutes (rats) and 30 min (mice) after ICV administration of leptin or saline, rats and mice were anesthetized with urethane and were perfused transcardially with ice-cold 4% paraformaldehyde for 20 min. The brains were removed and postfixed in the same fixative for 1 h and in paraformaldehyde solution containing 15% sucrose overnight at 4 C. They were then transferred to 30% sucrose solution in 0.1 M phosphate buffer for 2 d. The brains were frozen on dry ice and kept at –80 C until sectioning. Coronal sections (40 µm) were cut using a freezing microtome and collected at 160-µm intervals.

According to the free-floating in situ hybridization method described previously (28), sections were hybridized with digoxigenin-labeled sense or antisense RNA probes synthesized from rat NPY template cDNA (positions 1–503, GenBank no. M15880). Visualization of NPY mRNA was performed with alkaline phosphatase-conjugated antidigoxigenin antibody using 4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate.

Immunohistochemistry was then performed. The sections were rinsed in PBS for 30 min and pretreated with 3% H2O2 in PBS for 40 min. They were rinsed again in PBS and immersed in 0.3% glycine PBS for 25 min. After a further rinse, sections were placed in 0.03% sodium dodecyl sulfate for 25 min. All sections were rinsed again; placed in 4% normal serum, 0.4% Trition X-100, and 1% BSA for 30 min; and then incubated with a rabbit polyclonal primary antibody to phosphorylated STAT3 at Tyr705 (1:500; Cell Signaling Technology, Beverly, MA) overnight at 4 C in 1% normal serum, 0.4% Triton X-100, and 1% BSA. The sections were then treated with Envision system labeled polymer-horseradish peroxidase antirabbit (DakoCytomation, Glostrup, Denmark) for 1 h and color developed with 3,3'-diaminobenzidine.

Measurements of food intake after ICV injection of ghrelin, leptin, and cilostamide
Ghrelin (150 pmol) and/or leptin (125 pmol) diluted in 3.5 µl saline were ICV injected to 8-wk-old rats at the onset of darkness, and overnight (14 h) cumulative food intake was measured. In a study, cilostamide (2 µg per 3 µl) was coinjected with ghrelin and/or leptin in 3.5 µl saline.

Solutions and chemicals
Measurements were carried out in HKRB solution composed of 129 mM NaCl, 5.0 mM NaHCO3, 4.7 mM KCl, 1.2 mM KH2PO4, 1.8 mM CaCl2, 1.2 mM MgSO4, and 10 mM HEPES (pH 7.4). fura-2/AM was obtained from Dojin Chemical (Kumamoto, Japan). Ghrelin was obtained from Peptide Institute, Inc. (Osaka, Japan), and leptin was from R&D Systems (Minneapolis, MN). Forskolin, and LY294002 were from Wako Chemicals (Osaka, Japan), milrinone and STAT3 inhibitor peptide were from EMD Biosciences, Inc. (Calbiochem; San Diego, CA), PD98059 was from Alexis Platform (Montréal, Canada), U0126 was from Cell Signaling Technology, and all other chemicals were from Sigma. All agents were dissolved in distilled water or dimethylsulfoxide as stock solutions, and they were diluted in the superfusion solution (HKRB) at 1:1000 or less. Pharmacological agents were administered at the concentrations that specifically influence the particular molecule or pathway based on the literatures. In control experiments, 0.1% dimethylsulfoxide administered alone had no effect on [Ca2+]i in ARC neurons.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Ghrelin increased [Ca2+]i in single ARC neurons, and the [Ca2+]i increases were suppressed by inhibitors of PLC, adenylate cyclase, and cAMP
Administration of ghrelin to the perfusate increased [Ca2+]i in 117 of 383 single neurons (31%) isolated from ARC, confirming the findings of our previous study (18). The [Ca2+]i increases usually took place in an oscillatory manner. Of 32 ghrelin-responding neurons examined, 25 (78%) were shown to contain NPY by subsequent immunocytochemistry using anti-NPY antibody (Fig. 1AGo). When 22 ghrelin-responding neurons were subjected to the second challenge with ghrelin, 20 neurons (>90%) responded to it with [Ca2+]i increases, and the patterns of [Ca2+]i responses to the first and second ghrelin administration were similar (Fig. 1Go, A and F). Therefore, to examine the effects of drugs, the second ghrelin administration was performed in the presence of or after pretreatment with drugs. Of 12 ARC neurons that responded to ghrelin with increases in [Ca2+]i (ghrelin-responding neurons), preincubation with 100 µM SQ22536, an adenylate cyclase inhibitor, for 20 min blunted [Ca2+]i responses to the second ghrelin administration in six cells, showing a reduced incidence of ghrelin response to 50% (six of 12 cells) (Fig. 1Go, B and F). SQ22536 is a substituted adenine derivative that forms a dead-end complex with activated adenylate cyclase by occupying the P site of the enzyme (29, 30). In the presence of a competitive cAMP antagonist (31), 8-bromo-cAMP Rp-isomer (Rp-8Br-cAMP) (10 µM), ghrelin increased [Ca2+]i in only nine of 21 (33%) ghrelin-responding ARC neurons (Fig. 1Go, C and F).

After preincubation with 1 µM U73122, a PLC inhibitor (32), for 20 min, ghrelin increased [Ca2+]i in only three of 14 ghrelin-responding neurons (21%), whereas with preincubation with 1 µM U73343, a related compound not capable of inhibiting PLC, ghrelin increased [Ca2+]i in 13 of 18 ghrelin-responding neurons (Fig. 1Go, D–F). The amplitude of [Ca2+]i increase was significantly (P < 0.05) decreased by treatments with SQ22536 (179.6 ± 48.6 nM, n = 12), Rp-8Br-cAMP (205.2 ± 47.9 nM, n = 21), and U73122(147.1 ± 35.9 nM, n = 14), compared with control (349.1 ± 31.9 nM, n = 22) and U73343 treatment (351.0 ± 59.6 nM, n = 18) (Fig. 1GGo). AUC was also significantly decreased by treatments with SQ22536 (79.5 ± 15.9 nM, n = 12), Rp-8Br-cAMP (80.1 ± 17.3 nM, n = 21), and U73122 (76.6 ± 14.4 nM, n = 14), compared with control (152.1 ± 31.2 nM, n = 22) and U73343 treatment (158.3 ± 26.0 nM, n = 18) (Fig. 1HGo). Thus, quantification of [Ca2+]i changes by amplitude and that by AUC yielded similar results.

Leptin suppressed ghrelin-induced [Ca2+]i increases in NPY neurons
Administration of ghrelin induced sustained increase in [Ca2+]i in ARC neurons (Fig. 2AGo). The [Ca2+]i increase was suppressed by coadministration of leptin (Fig. 2BGo). The effect of leptin on [Ca2+]i in ARC neurons was concentration dependent over the range of 10–16 to 10–12 M (Fig. 2CGo). The neuron from which the recording in Fig. 2BGo was taken was subsequently stained positively with anti-NPY antiserum (Fig. 2BGo, right panel). Fifteen of 18 ghrelin-activated and leptin-inhibited neurons (83%) were found to be NPY-immunoreactive neurons (Fig. 2DGo). These findings indicated that the large majority of ghrelin-activated, leptin-inhibited neurons in ARC are NPY neurons. Because it is reported that glucose level is lower in the brain than the blood (33), we examined the effect of leptin in the presence of lower glucose concentration (2.5 mM). Under the low glucose condition, leptin suppressed ghrelin-induced [Ca2+]i increases in ARC neurons in a manner similar to that observed with 10 mM glucose (Fig. 3GGo).

Effects of PI3K and MAPK inhibitors on the ability of leptin to suppress ghrelin activation of ARC neurons
To explore the intracellular signal transduction, we first examined the involvement of PI3K, a signaling molecule related to leptin-induced anorexia (34), in the leptin suppression of ghrelin responses. Preincubation for 1 h with the PI3K inhibitors, LY294002 at 50 µM and wortmannin at 100 nM (35), abolished the leptin suppression of ghrelin-induced [Ca2+]i increases (Fig. 3Go, A and B). Similar results were observed in the corresponding experiments under conditions with lower glucose concentration of 2.5 mM in the superfusate (Fig. 3HGo). We then examined the involvement of MAPK, a key molecule in another signaling pathway for leptin (36). Preincubation with U0126, a MAPK inhibitor (35), at 10 µM for 30 min did not significantly alter the suppression by leptin of ghrelin-induced [Ca2+]i increases (Fig. 3CGo). Leptin exerted the suppressive effect in nine of 20 ghrelin-responding neurons in the control group without inhibitors (45%), two of 12 with LY294002 treatment (17%), one of 11 with wortmannin treatment (9%), and five of 15 with U0126 treatment (33%) (Fig. 3DGo). Effects of leptin were quantified by leptin-induced reduction of amplitude of [Ca2+]i responses to ghrelin, as expressed by the percentage. The ability of leptin to suppress ghrelin-induced [Ca2+]i increases, expressed by amplitude, was significantly attenuated after treatment with LY294002 (21.5 ± 9.1%, n = 12) and wortmannin (13.8 ± 6.0%, n = 11), compared with the control (48.1 ± 8.9%, n = 20), whereas it was not significantly altered by treatment with U0126 (34.2 ± 9.6%, n = 15) (Fig. 3EGo). Suppressive effect of leptin, as expressed by AUC, was also significantly attenuated aftertreatment with LY294002 (9.2 ± 6.3 nM, n = 12) and wortmannin (17.4 ± 6.5 nM, n = 11), compared with the control (49.6 ± 14.5 nM, n = 20), whereas it was not significantly altered by treatment with U0126 (45.6 ± 16.5 nM, n = 15) (Fig. 3FGo).

PDE3, but not ATP-sensitive potassium (KATP) channel, is involved in the ability of leptin to suppress ghrelin activation of ARC neurons and food intake
Both PDE3, a subtype of cAMP catabolic enzyme, and KATP channels are considered downstream effectors of PI3K (37, 38, 39, 40). Preincubation for 30 min with 10 µM milrinone, a PDE3 inhibitor (41), abolished leptin suppression of ghrelin-induced [Ca2+]i increases in ARC neurons (Fig. 4AGo). Similarly, after 30 min pretreatment with and in the presence of 1 µM cilostamide, another PDE3 inhibitor (41), also blunted suppression by leptin (Fig. 4BGo). On the other hand, the KATP channel inhibitors (42), glibenclamide at 100 nM (Fig. 4CGo) and tolbutamide at 300 µM (data not shown), did not significantly influence leptin suppression of ghrelin-induced [Ca2+]i increases. The KATP channel activators (42), pinacidil at 100 µM (Fig. 4DGo) and diazoxide at 200 µM for 20 min (data not shown), did not mimic leptin’s suppression of ghrelin-induced [Ca2+]i increases. Leptin exerted suppressive effects in nine of 20 ghrelin-responding neurons in the control without drugs (45%), four of 18 with milrinone (22%), three of 15 with cilostamide (20%), three of 10 with glibenclamide (30%), and six of 19 with tolbutamide (32%) (Fig. 4EGo). Suppression by leptin of ghrelin-induced [Ca2+]i increases, as expressed by amplitude, was significantly attenuated after treatment with milrinone (22.8 ± 8.6%, n = 18) and cilostamide (21.5 ± 7.2%, n = 15), compared with the control (48.1 ± 8.9%, n = 20), whereas it was not significantly altered by treatment with glibenclamide (31.7 ± 10.4%, n = 11) or tolbutamide (36.6 ± 7.4%, n = 18) (Fig. 4FGo).

Based on the in vitro data indicating critical role of PDE3 in the regulation of NPY neurons, we examined the involvement of PDE3 in the in vivo feeding regulation. ICV injection of ghrelin at the onset of darkness increased food intake, whereas that of leptin inhibited food intake (Fig. 4GGo). The ghrelin-induced stimulation of food intake was counteracted by simultaneous ICV leptin injection (Fig. 4GGo). This leptin’s action to counteract ghrelin was abolished by ICV coadministration of cilostamide, a PDE3 inhibitor (Fig. 4HGo).

Involvement of STAT3 in leptin signaling in NPY neurons
Possible involvement of STAT3 in the counteraction by leptin of ghrelin activation of ARC neurons was investigated. We first examined whether leptin induces phosphorylation (p) of STAT3 in ARC NPY neurons. Immunohistochemistry and in situ hybridization in combination revealed that ICV leptin injection induced pSTAT3 (brown) in a substantial fraction of neurons in ARC, some of which also expressed NPY-mRNA (blue) (Fig. 5Go, A and B). Of 2219 ARC neurons that expressed NPY-mRNA (from three rats; one of four slices for each rat was examined), 601 neurons (27%) also exhibited pSTAT3 immunoreactivity. In control experiments, ICV vehicle injection did not induce pSTAT3 (Fig. 5CGo). Thus, STAT3 is present and activated by leptin in a substantial fraction of ARC NPY neurons. Next, the possible involvement of STAT3 in the effect of leptin on [Ca2+]i was examined using the STAT3 inhibitor peptide and neuron-specific STAT3-KO (STAT3/nestin KO) mice. Preincubation for 30 min with the STAT3 inhibitor peptide (500 µM), an agent that inhibits STAT3 activity by blocking its SH2 domains (43), did not alter leptin suppression of ghrelin responses (Fig. 5Go, D–F). Leptin suppressed nine of 20 ghrelin-responding neurons in the control (45%) and seven of 17 ghrelin-responding neurons treated with STAT3 inhibitor peptide (41%) (Fig. 5EGo). The suppression by leptin of ghrelin-induced [Ca2+]i increases, expressed by amplitude, was not significantly altered by treatment with STAT3 inhibitor peptide (39.0 ± 9.2%, n = 17), compared with the control (48.1 ± 8.9%, n = 20) (Fig. 5GGo).

In wild-type mice, approximately half of the neurons that expressed NPY mRNA were immunoreactive to pSTAT3 in response to leptin (supplemental Fig. A, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org), whereas the leptin-induced pSTAT3 immunoreactivity was observed in few NPY mRNA expressing neurons in STAT3/nestin KO mice (supplemental Fig. B). Leptin suppressed ghrelin-induced [Ca2+]i increases in ARC neurons isolated from STAT3/nestin KO mice; the pattern (Fig. 5Go, G and H), incidence (Fig. 5IGo), and magnitude (Fig. 5JGo) of suppression were indistinguishable from those observed in the neurons from STAT3flox/– mice and wild-type mice. Leptin-induced suppression occurred in five of 14 ghrelin-responding neurons in wild-type mice (36%), five of 17 in STAT3flox/– mice (29%), and seven of 22 in STAT3/nestin KO mice (32%) (Fig. 5IGo). The suppression by leptin of ghrelin-induced [Ca2+]i increases, expressed by amplitude, was not significantly altered in STAT3/nestin KO mice (25.3 ± 7.9%, n = 22), compared with wild-type mice (26.0 ± 10.0%, n = 14) and STAT3flox/– mice (29.9 ± 8.7%, n = 17) (Fig. 5JGo).

Leptin suppressed forskolin-induced [Ca2+]i increases
Because our findings suggested that the adenylate cyclase-PKA pathway is involved in ghrelin activation of ARC neurons, we examined whether [Ca2+]i increases induced by the adenylate cyclase-PKA pathway are suppressed by leptin. Administration of forskolin, an adenylyl cyclase activator, at 10 µM induced long-lasting [Ca2+]i increases in 32 of 62 ARC neurons (51%) (Fig. 6AGo), nine of which (28%) exhibited suppression by leptin (Fig. 6BGo). After preincubation with 50 µM LY294002 for 1 h and 10 µM milrinone for 30 min, the suppressive effect of leptin was either abolished or markedly reduced (Fig. 6Go, C and D): leptin suppressed forskolin responses in only three of 25 cells (12%) and three of 23 cells (13%), respectively (Fig. 6EGo). The percent inhibition of amplitude of forskolin responses by leptin was significantly attenuated after treatment with LY294002 (9.9 ± 4.2%, n = 24) and milrinone (9.2 ± 4.8%, n = 23), compared with the control (26.5 ± 6.4%, n = 32) (Fig. 6FGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study presents findings indicative of novel signaling mechanisms for ghrelin, leptin, and their interaction in the ARC NPY neurons of adult rats. More than 80% of ghrelin-activated ARC neurons were proven to be NPY neurons, confirming the findings of a previous study (18). Because this study investigated ghrelin-activated ARC neurons, data were obtained primarily from NPY neurons. Ghrelin increases [Ca2+]i via mechanisms dependent on both the adenylate cyclase-cAMP-PKA and PLC pathways. Leptin counteracts ghrelin-induced [Ca2+]i increases via PI3K- and PDE3-mediated pathway, whereas MAPK, STAT3, and KATP channels appear not to play a significant role, if any (Fig. 7Go).


Figure 7
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  		FIG. 7. Proposed model for signaling cross talk between ghrelin and leptin in ARC NPY neurons. Ghrelin increases [Ca2+]i via mechanisms dependent on both adenylate cyclase-cAMP-PKA and PLC pathways. Leptin suppresses ghrelin-induced [Ca2+]i increases via PI3K- and PDE3-mediated pathway. This interaction may play a role in regulation of the activity of NPY neurons and, thereby, feeding. G, GTP binding protein.

 
Based on the reports that plasma leptin level is around 10–9 M and that 1:1000 of the plasma leptin enters the brain through blood-brain barrier (BBB) (44), the estimated leptin level in the brain is around 10–12 M. It was documented that leptin concentration measured in cerebrospinal fluid was 1.6 x 10–11 M (45). The leptin concentration in ARC might be somewhat higher because BBB in this area is thought to be relatively poor (46). Hence, 10–12 and 10–10 M, the leptin concentrations used in the present study, are in the physiological range. In contrast, physiological concentrations of ghrelin in ARC are difficult to estimate for the following reasons. Transport of ghrelin through BBB appears to be complicated including the species difference and remains to be further clarified (47, 48). Ghrelin neurons are reported to be located in ARC and/or adjacent area, providing an additional source for ghrelin delivery to ARC. The concentration of ghrelin used in this experiment (10–10 M) is close to the preprandial plasma ghrelin level (11). Together the concentrations of leptin and ghrelin used in this study appear to be in an appropriate range if not precisely accurate, and the results obtained would at least partly reflect the physiological effects of ghrelin and leptin.

We previously showed that ghrelin activates NPY neurons in a PKA-dependent manner (18). The present study further demonstrated that an adenylate cyclase inhibitor and a cAMP competitive inhibitor counteracted ghrelin-induced [Ca2+]i increases. Unexpectedly, a PLC inhibitor also blocked ghrelin-induced [Ca2+]i increases. There are two possible explanations for these results: first, both adenylate cyclase and PLC pathways are activated by ghrelin; second, one of these pathways is activated by ghrelin, whereas the basal activity of the other assists ghrelin’s signaling to increase [Ca2+]i.

Regarding the first possibility, studies on primary cultured pituitary cells and somatotropinomas indicated that ghrelin and ghrelin receptor agonist stimulate cAMP production (49, 50, 51), and a study in GH secretagogue receptor-expressing oocytes indicated that ghrelin receptors are coupled to the G protein {alpha}11-subunit that activates PLC but not Gq, G16, Go, G13, Gi1, or Gi3 (52). Certain types of adenylate cyclases, including type II, could be activated by the combination of G protein ß{gamma}-subunits (types II, IV, and VII) (53). mRNA for adenylate cyclase type II is reportedly expressed in ARC (54). If ghrelin receptor is coupled with G protein {alpha}11-subunit and its ß{gamma}-subunits activate adenylate cyclase type II in NPY neurons, ghrelin could activate both PLC and adenylate cyclase in NPY neurons. However, the specific type(s) of G-proteins coupled with ghrelin receptor in NPY neurons remain to be clarified. Regarding the second possibility that ghrelin activates one signaling pathway whereas basal activity of the other signaling pathway assists it, basal PLC activity could play a role. PLC regulates the concentration of its substrate, PIP2, phosphatidylinositol 4,5-bisphosphate, which is implicated in the regulation of ion channel activities (55). If this is the case in ARC NPY neurons, the basal activity of PLC could regulate the ion channel activity, membrane potential, and excitability, thereby supporting ghrelin signaling to increase [Ca2+]i. Alternatively, basal activity of adenylate cyclase could assist ghrelin signaling to lead to [Ca2+]i increases. In support of this notion, oscillation of cAMP level is capable of inducing rapid on-off Ca2+ responses in pancreatic ß-cells (56). It was shown that ghrelin inhibits [Ca2+]i via pertussis toxin-sensitive G protein in islet ß-cells (9), showing that ghrelin has tissue-specific signaling pathway. Thus, ghrelin can elicit different signaling pathways in a tissue-specific manner, which could underlie the opposing central and peripheral actions of ghrelin. Further studies are required to thoroughly elucidate the signaling pathways for ghrelin in ARC NPY neurons, including possible involvement of PKC.

Leptin inhibited ghrelin-induced [Ca2+]i increases in approximately 45% of ghrelin-responding neurons. The remaining half of ghrelin-responding neurons were apparently leptin insensitive; however, this particular subpopulation was not the subject of the present study. More than 80% of ghrelin-responding, leptin-inhibited neurons were NPY neurons. This study demonstrated that PI3K primarily mediates leptin suppression of ghrelin-induced [Ca2+]i increases. It has been reported that feeding at the onset of the dark cycle in rodents is suppressed by administration of leptin and that this effect of leptin is prevented by ICV infusion of PI3K inhibitors (34). This effect of leptin could be due at least partly to suppression of effects of ghrelin because plasma ghrelin concentrations rise substantially during fasting and preprandially (11). Leptin might thus exert an anorexigenic effect in part via PI3K-mediated counteraction of the ghrelin effect in NPY neurons in certain metabolic conditions and/or during certain periods of the day.

We demonstrated that PDE3, a cAMP catabolic enzyme, serves as a key signal downstream of leptin. It has previously been reported that leptin activates PDE3 in a PI3K-dependent manner in some peripheral tissues (37, 57). Furthermore, we found that leptin suppressed ghrelin-stimulated food intake and that this effect of leptin was abolished by ICV administration of a PDE3 inhibitor. This result is in accord with the previous report that a PDE3 inhibitor blocked the leptin-induced suppression of dark phase food intake (58). Therefore, activation of PI3K-PDE3 and consequent down-regulation of the cAMP pathway in the hypothalamus, particularly in ARC NPY neurons, may serve as a mechanism by which leptin counteracts the effects of ghrelin (Fig. 7Go). Chronic central infusion of leptin for 2 d suppresses food intake; increases PI3K, PDE3B, and pSTAT3 activities; and decreases cAMP levels in the hypothalamus (59). By contrast, chronic infusion for 16 d induces leptin resistance manifested by reduced anorexia and down-regulation of the PI3K-PDE3B pathway (59). Therefore, the unbalanced interaction between ghrelin and leptin due to the dysregulated PI3K-PDE3 pathway in NPY neuron could be related to leptin resistance causing elevated feeding.

ICV administration of leptin stimulated STAT3 phosphorylation in NPY neurons. However, neither pharmacological blockade nor genetic deletion of STAT3 affected the leptin suppression of ghrelin-induced [Ca2+]i increases. These results suggest that the rapid action of leptin on the cellular activities, such as [Ca2+]i and membrane potentials, is exerted via the PI3K pathway and does not require STAT3. Similarly, it has been reported that the PI3K signaling in POMC neuron is related to rapid regulation of cellular activities (60) and that the activation of PI3K can occur independently of STAT3 signaling (61). The STAT3-mediated route of the leptin signaling could regulate feeding (62) via more chronic effects linked to regulation of gene transcription, neurogenesis (63), and/or neurite growth (64) in NPY, POMC, and possibly other feeding regulatory neurons.

This study demonstrated that ghrelin activates the ARC NPY neurons of adult rats via mechanisms, depending on both adenylate cyclase-cAMP-PKA and PLC pathways. Leptin suppresses the ghrelin effect by activating PI3K-PDE3 pathway, which may counteract the adenylate cyclase-cAMP-PKA system implicated in the effects of ghrelin. The interaction between orexigenic ghrelin and anorexigenic leptin via this specific signaling cross talk could serve as a mechanism for the temporal and optimal regulation of ARC NPY neurons and possibly feeding.


   Footnotes
 
This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science, that on Priority Areas (15081101), a grant from the 21st Century Center of Excellence program, the Science Research Promotion Fund from the Promotion and Mutual Aid Corporation for Private Schools of Japan, an Insulin Research Award from Novo Nordisk Pharma Ltd., and a grant from Japan Diabetes Foundation (to T.Y.).

Disclosure Statement: The authors have nothing to disclose.

First Published Online February 15, 2007

Abbreviations: ARC, Arcuate nucleus; AUC, area under the curve; BBB, blood-brain barrier; [Ca2+]i, cytosolic Ca2+ concentration; HKRB, HEPES-buffered Krebs-Ringer bicarbonate buffer; ICV, intracerebroventricular; KATP, ATP-sensitive potassium; KO, knockout; NPY, neuropeptide Y; p, phosphorylation; PDE3, phosphodiesterase 3; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PLC, phospholipase C; POMC, proopiomelanocortin; Rp-8Br-cAMP, 8-bromo-cAMP Rp-isomer; STAT, signal transducer and activator of transcription.

Received September 11, 2006.

Accepted for publication February 5, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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