Interplay Between Galanin and Leptin in the Hypothalamic ... - CiteSeerX

Interplay Between Galanin and Leptin in the Hypothalamic Control of Feeding via Corticotropin-Releasing Hormone and Neuropeptide Y Gabriela E. Bergonzelli,1 Franc¸ois P. Pralong,1 Micheline Glauser,1 Claudia Cavadas,2,3 Eric Grouzmann,2 and Rolf C. Gaillard1

Over long periods, feeding and metabolism are tightly regulated at the central level. The total amount of nutrients ingested is thought to result from a delicate balance between orexigenic and anorexigenic factors expressed and secreted by specialized hypothalamic neuronal populations. We have developed a system of perifused hypothalamic neurons to characterize the relationships existing between the orexigenic peptide galanin and two other physiological modulators of feeding: neuropeptide Y (NPY) and corticotropin-releasing hormone (CRH). We demonstrated that galanin stimulates CRH and NPY secretion from hypothalamic neurons in a dose-dependent manner. Exposure to leptin for 24 h before galanin stimulation decreased NPY secretion by 30%, leaving the responsiveness of CRH neurons intact. These results suggest that CRH and NPY neurons participate to the intrahypothalamic signaling pathway of galanin, an observation that can explain the lower potency of galanin to stimulate food intake in vivo compared with NPY. The differential effects exerted by leptin on CRH and NPY suggest that there exists a subset of NPY neurons that are exquisitely sensitive to marked variations in leptin levels, and that the CRH neurons are less responsive to increases in leptin concentrations. Diabetes 50:2666 –2672, 2001

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ecent evidence suggests that the central nervous system plays a key role in controlling food intake and metabolic adaptations (1). A delicate interplay seems to exist between the activities of highly specialized neuronal subpopulations involved in the regulation of feeding and energy storage, or of satiety and energy expenditure. Overall, these regulations, operating From the 1Division of Endocrinology, University Hospital, Lausanne, Switzerland; the 2Division of Hypertension, University Hospital, Lausanne, Switzerland; and the 3Laboratory of Pharmacology, Faculty of Pharmacy, Coimbra, Portugal. Address correspondence and reprint requests to Franc¸ois Pralong, Division of Endocrinology, CHUV BH-19-707, CH-1011 Lausanne, Switzerland. E-mail: [email protected]. G.E.B. is currently affiliated with the Nutrition Department, Nestle Research Center, Lausanne, Switzerland. G.E.B. and F.P.P. contributed equally to this work. Received for publication 4 July 2001 and accepted in revised form 18 September 2001. araC, cytosine ␤-D-arabinofuranoside; AUC, area under the secretory curve; CRH, corticotropin-releasing hormone; GDH, glyceraldehyde phosphate dehydrogenase; GFAP, glial fibrillary acid protein; IRMA, immunoradiometric assay; MAP2, mitogen-activated protein 2; MCH, melanin-concentrating hormone; NPY, neuropeptide Y; POMC, pro-opio-melanocortin; PVN, paraventricular nucleus; RT-PCR, reverse transcriptase–polymerase chain reaction. 2666

at the level of the central nervous system, achieve the tight control of energy balance observed in mammals (2). The neurons involved are mainly located within the hypothalamus, where they serve as central integrators of peripheral metabolic signals. One important function of leptin (3) seems to be informing these neurons about levels of energy stored in the body (4). Changes in circulating leptin levels can therefore trigger an appropriate response of the central nervous system to modulate food intake and metabolism according to the total amount of body fat (5) as well as the variations of energy balance (6). However, the relations between the different hypothalamic neuronal subpopulations seem to be extremely complex (1). The systems involved are redundant, and thus they are most difficult to individualize in vivo. Neuropeptide Y (NPY) and galanin can both elicit strong feeding behavior when injected centrally (7–10) and are expressed in hypothalamic areas associated with feeding and metabolic regulations (10 –12). NPY is one of the most abundant peptides of the hypothalamus (11) and remains among the most potent orexigenic factors (13). In addition, it has also been implicated in the control of neuroendocrine adaptations to fasting or unfavorable metabolic conditions (14,15). Like NPY, galanin plays a physiologically important role in the regulation of neuroendocrine functions affected by metabolic changes, such as reproduction and growth (16). The hypothalamic corticotrophin-releasing hormone (CRH) system was initially recognized for its central role as the primary regulator of the stress response (17). In addition, CRH neurons located in the paraventricular nucleus of the hypothalamus are involved in the regulation of energy balance (18), independent of the stress-regulated production of adrenal glucocorticoids. Like NPY and galanin, CRH and its closely related peptide urocortin (19) can alter feeding behavior when administered centrally. However, CRH and urocortin are potent inhibitors of food intake, while also stimulating metabolism and energy expenditure (20,21). In addition to sharing a role in the control of feeding, NPY, galanin, and CRH are closely related anatomically. Galanin is present in the hypothalamic paraventricular nucleus, and some galanin-positive neurons also express CRH (10,22). Furthermore, these cells receive a dense innervation from NPY-ergic neurons located in the hypothalamic arcuate nucleus (23). Therefore, it can be hypothesized that these neurotransmitters DIABETES, VOL. 50, DECEMBER 2001

G.E. BERGONZELLI AND ASSOCIATES

are functionally related in their modulation of feeding and metabolism. We used cultures of fetal rat hypothalamic neurons in dynamic perifusion experiments to demonstrate that galanin can directly stimulate hypothalamic CRH and NPY secretion. Leptin was found to downregulate this galaninstimulated NPY secretion, whereas CRH release was not affected. Our results provide a novel mechanism of action for galanin, implicating CRH as well as NPY neurons downstream of its feeding effects. Furthermore, the differential effect of leptin on NPY and CRH secretion confirms in vivo observations suggesting that NPY neurons represent an important target of leptin’s effects (24 –26), but that CRH neurons do not respond to elevations of leptin levels above normal physiological values (27). Overall, these results demonstrate that the use of primary neurons in culture can be a useful adjunct to in vivo studies of hypothalamic function. RESEARCH DESIGN AND METHODS Isolation of embryonic hypothalamic neurons. Hypothalamuses were obtained from E18 or E19 rat fetuses as follows: whole brains were rapidly removed from the skull, and the hypothalamus was extracted with ophthalmic scissors as previously described (28). Two coronal cuts (anteriorly at the level of the optic chiasma and posteriorly at the level of the mammillary bodies) were followed by two parasagittal cuts along the hypothalamic sulci and a final dorsal cut at depths of ⬃2 mm from the ventral surface of the tissue block. Enzymatic digestion of the tissue leads to poor survival of the neurons in vitro. Therefore, dispersion was performed mechanically in phosphatebuffered saline (PBS) buffer (without calcium or magnesium; Gibco) supplemented with 0.06% glucose (Fluka) and 100 units/100 ␮g/ml penicillin/ streptomycin (Seromed). Freshly excised whole hypothalamuses were gently passed several times through Pasteur pipettes flamed in the middle of the procedure to decrease the diameter of their opening. Nondispersed tissue was allowed to settle for 5 min, and supernatant was transferred to a clean tube. The remaining pellet was resuspended in 4 ml PBS buffer, and mechanical dispersion was repeated. Supernatants from the first and second dispersion were mixed, centrifuged for 5 min at 100g, and the cellular pellet was gently resuspended in 5 ml neurobasal medium (Gibco) with 0.04% B27 supplement (Gibco) containing 500 ␮mol/l glutamine and 25 ␮mol/l glutamate (Sigma). This whole process, which was always performed by the same person, is extremely delicate because cellular yield and cell death both increase with the strength of pipetting. Viability of hypothalamic neurons is low compared with other neuronal cell types (30 – 40% viable cells immediately after the dispersion and before plating, as assessed by Trypan blue exclusion), with a yield of ⬃2 ⫻ 105 live cells/hypothalamus. Cultures were then performed as previously described (29,30): cells were seeded at a density of 500 live cells/mm2 in 60-mm diameter Petri dishes (Falcon) or in 6- or 12-well plates (Costar) containing round coverslips (Assistent) coated with 5 ␮g/ml poly-D-lysine (Sigma). After 48 h, 2 ␮mol/l cytosine ␤-D-arabinofuranoside (araC; Sigma) was added to prevent proliferation of nonneuronal cells. At 48 h after araC addition, half of the medium was replaced by neurobasal medium with 0.04% B27 supplement containing 500 ␮mol/l glutamine but without glutamate to avoid toxicity. Cells were maintained in culture for up to 6 weeks by renewing half of the medium every 4th day. Immunocytochemistry. Immunocytochemistry was performed at room temperature using an avidin biotinylated horseradish peroxidase macromolecular complex kit (ABC Elite; Vector, Burlingame, CA). Cells were fixed for 40 min in 4% paraformaldehyde (Merck) and rinsed three times in PBS and one time in TNT (0.05 mol/l Tris [Sigma], pH 7.4, 0.6% NaCl, and 0.3% Triton X-100 [Merck] with 10% goat serum [Gibco]). After the quenching of endogenous peroxidase activity, nonspecific binding was blocked by a 1-h incubation in TNT. Incubations with the primary antibodies were performed overnight, and then cells were washed and further incubated for 1 h with anti-mouse or anti-rabbit biotinylated secondary antibodies (Jackson Immunoresearch Laboratories, West Grove, PA). After three washes in 0.1 mol/l Tris-HCl, pH 7.7, cells were incubated in the ABC reagent, washed again, and finally incubated in DAB peroxidase substrate solution (50 mg/100 ml 3-3⬘ diaminobenzidine [Sigma] in 0.1 mol/l Tris-HCl and 0.018% H2O2, pH 7.7). Coverslips were counterstained with hematoxylin (Vector). Glial cells were identified with a human monoclonal anti-glial fibrillary acid DIABETES, VOL. 50, DECEMBER 2001

protein (GFAP) antibody (Dako) at a final concentration of 2.5 ␮g/ml. Neurons were identified using a rat monoclonal antibody raised against the microtubule-associated protein mitogen-activated protein 2 (MAP2; a gift of Dr. Beat Riederer) (31) at a final dilution of 1:75. CRH-positive neurons were identified using a polyclonal antibody (a-hCRH; a gift of Dr. Robert Benoit) at a final dilution of 1:5,000, whereas NPY-positive neurons were identified using a monoclonal antibody at a final dilution of 1:1,000 (32). Negative controls were obtained by incubating the primary antiserum with the corresponding hormone before staining. Northern blot. Total RNA was prepared with the TriPure isolation reagent containing 1 ␮l of 20 ␮g/␮l glycogen (Boehringer Mannheim). Northern blot analysis of 10 ␮g total RNA was performed as previously described (33). Relative changes in mRNA levels were determined by hybridization to specific (␣32)dCTP-labeled cDNA probes. Exposure time of all membranes to X-ray films (Amersham) were chosen to optimize the signals in a linear range. Densitometric analysis of mRNA signals on autoradiograms was performed with a Molecular Dynamics scanner (Sunnyvale, CA). To correct for variations in the amount of RNA loaded, all membranes were rehybridized with a probe for the ubiquitously expressed gene glyceraldehyde phosphate dehydrogenase (GDH). The ratio of the specific and the corresponding GDH signal was determined. DNA probes. The probes used were: the 770-bp BamHI fragment of the rat CRH cDNA (kindly supplied by Dr. L. Bain, University of Michigan) (34); a ProNPY-specific riboprobe (courtesy of Dr J.-A Haefliger, Lausanne, Switzerland); the 1.1-kb HindIII-EcoRI fragment of rat GDH cDNA (35); a 600-bp PstI fragment of the rat pro-opio-melanocortin (POMC) exon III (kindly provided by Dr. I. de Keyzer, University Rene´ Descartes); and a 410-bp fragment of rat orexin cDNA and a 503-bp fragment of the rat melanin-concentrating hormone (MCH) cDNA obtained by reverse transcriptase–polymerase chain reaction (RT-PCR) from total rat hypothalamic RNA (see below). RT-PCR. For all reactions, 1 ␮g total RNA was reverse-transcribed using oligo d(T)16 and amplified by PCR (Gene Amp RNA PCR kit; PE Biosystems) in a Perkin Elmer Gene Amp PCR System 9600 thermocycler. The intracellular region of the long isoform of the leptin receptor OB-Rb was amplified as previously described (36) using the following oligomers: 5⬘-TATCTATTAT TTAGGGGTCACC-3⬘ (sense) and 5⬘-ACCCACAACTATAATCTATTACAC-3⬘ (antisense). The amplification of the 88 – 497 fragment of orexin cDNA was performed at an annealing temperature of 50°C, using 5⬘-GGAATTCGTCG ACCATGAACCTTCCTTCTACAAAGG-3⬘ (SalI tail) as sense and 5⬘-GAATT CAAGCTTGATAGAAGACGGGTTCAGACTCTGG-3⬘ (HindIII tail) as antisense primers. The amplification of the 36 –538 fragment of MCH cDNA was performed at an annealing temperature of 55°C using 5⬘-GGAATTCGTCGAC AGGATGGGCGAAGATGAGCCTC-3⬘ (SalI tail) as sense and 5⬘-GAATTCAA GCTTCAGACTTGCCAACAGGGTCGGT-3⬘ (HindIII tail) as antisense primers. Both fragments were subcloned in pBluescript II KS vector (Stratagene) and verified by sequencing. Perifusion of hypothalamic neurons and experimental design. Two coverslips were placed, one face up and one face down, in a chamber maintained at 37°C and perifused with Krebs-Ringer buffer (15 mmol/l HEPES, 111 mmol/l NaCl, 4.7 mmol/l KCl, 2.5 mmol/l CaCl2, 1.2 MgSO4, 24.8 mmol/l NaHCO3, 1.2 mmol/l KH2PO4, and 11.1 mmol/l D-Glucose [Merck] containing 0.5% human serum albumin [ZLB Zentrallaboratorium]), which was kept at pH 7.4 by constant gassing with 95% O2 and 5% CO2. The flow rate was set at 100 ␮l/min, and fractions were collected every 5 min and assayed for CRH and NPY. CRH was quantified by immunoradiometric assay (IRMA) (37) and NPY was quantified by enzyme-linked immunosorbent assay (32). Effluent was collected in Nunc-Gibco tubes containing 1 ␮l of 10% Tween-20 (Pierce) and was stored at ⫺20°C until peptide measurements. Effects of galanin on CRH and NPY secretion. To evaluate the effects of galanin on neuropeptide secretion, neurons were stimulated for 30 min with graded concentrations of galanin (2 ⫻, 4 ⫻, and 8 ⫻ 10⫺5 mol/l) applied in random order. Galanin stimulations were followed by 30 min of recovery. In further experiments aimed at studying the potential for leptin to modulate the responsiveness of CRH and NPY neurons to galanin, cells were stimulated with 4 ⫻ 10⫺5 mol/l galanin. Effects of leptin on CRH and NPY secretion. Two different experimental paradigms were applied to test both the acute and chronic effects of leptin on basal as well as galanin-stimulated neuropeptide secretion. Acute effects of leptin were investigated using the stimulation of perifused neurons with galanin alone, followed by a similar galanin stimulation performed 30 min after the start of a 10⫺7 mol/l leptin perifusion. The choice of 10⫺7 mol/l leptin was based on our model of adrenal cell cultures (36,38). In the second paradigm, neurons were preincubated for 24 h in leptin-containing medium and then stimulated with galanin in the presence of leptin. The two paradigms were always applied in parallel columns. This allowed us to compare the 2667

GALANIN MODULATES CRH AND NPY SECRETION

FIG. 1. Photomicrography of an immunocytochemical analysis performed on 3-week-old neuronal cultures, using hematoxylin counterstaining. Shown are: CRH-positive neurons (A) with negative controls (B) and NPY-positive neurons (C) with negative controls (D). effects of galanin alone with those of galanin applied after leptin preincubations (between-column comparisons). NPY immunoneutralization. Because of the stimulation of NPY secretion exerted by galanin, the potential role of endogenously produced NPY in the galanin-stimulated secretion of CRH was also evaluated. In another series of experiments, neurons were pretreated for 1 h with a neutralizing anti-NPY antibody (39) used at a blocking concentration of 10⫺8 mol/l. This was followed by galanin stimulation (4 ⫻ 10⫺5 mol/l) for an additional hour in the presence of the same concentration of neutralizing antibody, and CRH was measured in the effluent. Data analysis. Cells positive for MAP2, GFAP, CRH, and NPY were directly counted under a light microscope. The length of neurites was also directly estimated under the light microscope. For dynamic experiments, a minimum of three independent perifusions were run on different days (n ⫽ 4 for all leptin experiments). Integrated secretion was estimated by calculating the areas under the secretory curve (AUCs) after galanin stimulation, using the trapezoidal method. All results were expressed as the means ⫾ SE, and comparisons were performed using Student’s t test or nonparametric tests (Van der Waerden test), when appropriate.

RESULTS

Characterization of primary hypothalamic neurons in culture. Within 48 h of plating, neuronal cells were recognizable by their growing neurites, often subdividing and reaching lengths of 5- to 10-fold the diameter of the 2668

cell body. After 1 week, cells appeared clearly differentiated: axonal prolongations were intertwining in a complex net, attaining a length of 20- to 40-fold the diameter of the cell body. Mechanical dispersion of tissue was limited to insure acceptable viability of cells. Therefore, there were remaining cellular clumps in the culture plates. Remarkably, some of the clump-forming cells remained alive in culture, because numerous axonal connections were observed between neurons in the clumps and neurons in the monolayer. After 3 weeks, immunocytochemical analysis showed 80 –90% MAP2-positive neurons and a remaining 10 –20% GFAP-positive glial cells. Strong, positive MAP2 staining was retained for up to 6 weeks, but axonal degradation became evident by that time. Expression of CRH, NPY, POMC, MCH, orexins, and the long isoform of the leptin receptor was demonstrated by Northern blot or RT-PCR analysis. In addition, the expression of CRH, NPY, gonadrophin-releasing hormone, and arginine vasopressin at the peptide level was confirmed by immunocytochemistry. The relative expression of CRH and NPY by these DIABETES, VOL. 50, DECEMBER 2001


FIG. 2. Dose-dependent effects of galanin to stimulate CRH (A) and NPY (B) secretion by hypothalamic neurons placed in a dynamic perifusion apparatus. Each graph displays a representative experiment, demonstrating the significant stimulation induced by various concentrations of galanin, followed by a return to baseline at the end of the stimulation. AUCs (insert panels) are calculated from three independent experiments and expressed as the mean ⴞ SE. *P < 0.05; **P < 0.01. Shaded areas represent the duration of galanin stimulation.

neurons is illustrated in Fig. 1. CRH immunoreactivity was present in relatively scattered neurons, principally observed in cell bodies, with a slight staining of the proximal portion of the dendrites that faded distally (Fig. 1A). In contrast, NPY immunoreactivity was found in a larger number of cells and also evidenced primarily in neuronal bodies (Fig. 1C). In addition, a fine network of axons and axon terminals was strongly stained with NPY. CRH and NPY mRNA expression was quantified weekly between weeks 2 and 5. Very strong NPY expression was present at all time points, but a trend toward a decrease was observed with time. In contrast, CRH mRNA levels remained stable throughout the study period. After these experiments, perifusions were always performed between weeks 3 and 4. Effects of galanin. CRH secretion by unstimulated neurons was around the detection limit of our IRMA method (0.48 fmol/ml). Galanin was found to stimulate CRH secretion in a dose-dependent manner, the minimal effective concentration being 2 ⫻ 10⫺5 mol/l. This concentration induced an approximately eightfold increase in mean CRH secretion (0.6 ⫾ 0.4 vs. 4.6 ⫾ 0.6 fmol/ml; P ⬍ 0.001), followed by a return to baseline levels at the end of the stimulation (Fig. 2A). Galanin also produced a dosedependent stimulation of NPY secretion (Fig. 2B), with mean hormone levels increasing from 1.1 ⫾ 0.2 fmol/ml at DIABETES, VOL. 50, DECEMBER 2001

baseline to 4.2 ⫾ 0.6 fmol/ml (P ⬍ 0.001) at the lowest galanin dose. The dose-dependency of the effect of galanin on the secretion of both neurotransmitters was demonstrated as well by the comparison of the AUCs for CRH and NPY (Fig. 2, inserts). When the galanin concentration was increased from 2 ⫻ 10⫺5 to 4 ⫻ 10⫺5 mol/l, the AUC for CRH increased from 28.8 ⫾ 1.8 to 46.4 ⫾ 4.2 fmol/pulse (P ⬍ 0.05). A further doubling of the galanin concentration, from 4 ⫻ 10⫺5 to 8 ⫻ 10⫺5 mol/l, resulted in a corresponding increase in the AUC for CRH, from 46.4 ⫾ 4.2 to 111.1 ⫾ 15.4 fmol/pulse (P ⬍ 0.01) (Fig. 2A, insert). The increases in the AUCs of NPY following the same increments in galanin concentration were slightly less pronounced (Fig. 2B, insert), the difference being statistically significant only between the 4 ⫻ 10⫺5 and 8 ⫻ 10⫺5 mol/l concentrations (28 ⫾ 8.6 vs. 69.2 ⫾ 18.9 fmol/pulse; P ⬍ 0.05). Further perifusion experiments were always performed at a galanin concentration of 4 ⫻ 10⫺5 mol/l. Of note, NPY immunoneutralization before galanin stimulation did not alter the CRH secretory response to galanin (data not shown), strongly suggestive of a direct galanin effect on CRH neurons present in the cultures. Effects of leptin. The acute and chronic effects of leptin on CRH and NPY secretion were then analyzed. Leptin administered 30 min before galanin had no effect on CRH or NPY secretion (data not shown). When administered over longer time periods (24-h preincubations), 10⫺7 mol/l leptin did not modify either basal or galanin-stimulated CRH secretion (Fig. 3A and B). In contrast, although basal NPY secretion was not affected by the 24-h leptin prein-

FIG. 3. Effect of 24 h preincubation with leptin (10ⴚ7 mol/l) on galanin-stimulated CRH (A and C) and NPY (B and D) secretion. A and B: Each graph displays a representative experiment, with shaded areas indicating the duration of galanin stimulation. Gray areas in the background represent the mean ⴞ SE of CRH (fmol/ml) or NPY (fmol/ml) secretion stimulated by galanin in the absence of leptin. C and D: Bar graphs represent the areas under the secretory curve for CHR (C) and NPY (D) (Mean ⴞ SE, n ⴝ 4 experiments). *P < 0.05. 2669


cubation, the responsiveness of NPY neurons to galanin was significantly altered by this treatment (Fig. 3C). The peak galanin-stimulated levels of NPY were decreased from 2.7 ⫾ 0.6 fmol/ml in controls to 1.7 ⫾ 0.6 fmol/l in the presence of leptin (P ⬍ 0.05). The interference of longterm leptin pretreatment with the stimulatory effect of galanin on NPY secretion was also evident when comparing the AUCs for NPY after galanin stimulation in the absence or presence of leptin (Fig. 3D), which showed a decrease from 6.1 ⫾ 1.5 fmol/pulse to 4.1 ⫾ 1.2 fmol/pulse (P ⬍ 0.05). DISCUSSION

We report here that galanin can directly stimulate NPY and CRH secretion from hypothalamic neurons. Galanin is known to increase feeding when injected centrally (40). This role in feeding regulation has been ascribed to the hypothalamic paraventricular nucleus (PVN) (9,41), but little is known about the mechanism(s) involved in this effect. The PVN being an important site of NPY and CRH expression, we hypothesized that the effects of galanin to stimulate food intake in vivo could be mediated via a modulation of the activity of CRH or NPY neurons. We have used hypothalamic neurons to explore in vitro the interplay between galanin, NPY, and CRH. These cells express many hypothalamic neurotransmitters in a differential way, suggesting that they reflect the physiological relations existing among different neuropeptides in vivo. Grown on coverslips, they develop a dense neuronal network and can be successfully used in dynamic perifusion experiments. Compared with static cultures (42), this perifusion model presents the advantage of minimizing potential autocrine or paracrine effects occurring in vitro, because the effluent is continuously washed out of the chamber. We were also able to measure neurotransmitter secretion from a much lower number of neurons than previously reported using hypothalamic neurons adsorbed onto Biogel beads (43). This difference suggests that preservation of the neuronal network upon transfer into the perifusion chambers may result in increased cellular viability and possibly better preservation of normal cellular functions. Given the strong inhibition on feeding exerted by CRH (21), our observation of a stimulation of CRH secretion by galanin could be somewhat surprising. However, it is fully consistent with the relative potencies of galanin and NPY to stimulate feeding in vivo: galanin was recently reported to be significantly less effective than NPY on a molar basis (13). Therefore, the present results on CRH and NPY secretion suggest that galanin expressed in the PVN may act as an integrator of feeding behavior, at least partially by balancing the respective endogenous tones of two opposite systems. However, the possibility that galanin may also modulate feeding independent of NPY or CRH neurons in vivo remains open. Finally, the requirement for relatively high doses of galanin could be inherent to this novel perifusion system, but it should be stressed that very little is known about the actual concentrations of neuropeptides present within synaptic connections in vivo. The influence of short- and long-term treatment with leptin (3) was also analyzed. In our system, long-term leptin treatment interfered downstream of the galanin 2670

signaling pathway, at the level of NPY secretion. In contrast, leptin affected neither the basal nor the galaninmediated induction of CRH secretion. The main physiological role of leptin is probably to function as a peripheral satiety signal to the central nervous system. As such, a considerable amount of data obtained in vivo concurs to demonstrate that hypothalamic NPY neurons represent an important, although not exclusive, target of leptin’s effects (rev. in 1). According to the prevalent hypothesis, increasing levels of circulating leptin induce a progressive decline in hypothalamic NPY expression associated with a resulting decrease in food intake. However, there is only one report demonstrating that leptin can affect hypothalamic NPY at the level of peptide secretion. In experiments using perifused hypothalamic fragments, Stephens et al. (44) showed that leptin decreases the secretion of NPY stimulated by glucocorticoids. Therefore, by providing evidence that leptin can also decrease the secretion of NPY stimulated by galanin, our results represent the second demonstration that leptin can directly modulate hypothalamic NPY secretion. Furthermore, the lack of effect of leptin on stimulated CRH secretion suggests that the anorexigenic effects of leptin observed in vivo are not mediated by a direct action on CRH neurons. Our observation that 24 h of exposure to leptin are required to modulate NPY secretion was somewhat puzzling. The possibility that stimulation with high concentrations of galanin may obliterate the acute effects of leptin could not be excluded, even though the dose of galanin used was of intermediate potency in our system. However, this time course is consistent with electrophysiological data obtained in rat hypothalamic slices (45). In this model, leptin induced a progressive hyperpolarization of hypothalamic neurons that was not reversible after leptin removal over the duration of the experiment (60 min). It could therefore indicate that the leptin-induced hyperpolarized state represents the trigger of a cascade of intracellular events, with a resulting modulation of neurotransmitter secretion occurring at later time points, as reported here. It is noteworthy that such a long time course has also been reported for the leptin modulation of adrenocortical cell function (36,38,46,47). Another similarity between the two models (adrenocortical cells and hypothalamic neurons) lies in the fact that leptin does not exert any measurable effect alone but modulates stimulated secretion. It is therefore tempting to speculate that leptin acts to alter the neuronal responsiveness to physiological stimuli, at least partly by activating ATP-sensitive K⫹ channels and thus depolarizing its target neurons (45). A better understanding of the leptin-induced modulation of neuronal function will probably involve the careful description of the cellular and molecular mechanisms participating in the transduction of the leptin signal (45,48,49). Our observation of a differential modulation of NPY and CRH secretion by leptin sheds a novel light on earlier data in the literature. It has been shown that leptin can prevent food intake stimulated by galanin in vivo (50), whereas it does not seem to interfere with CRH expression at high doses (27). Our results suggest that leptin may act downstream of galanin by blocking transmission of the portion of its feeding effects that is mediated via NPY, leaving CRH DIABETES, VOL. 50, DECEMBER 2001


secretion unaffected. Such a mechanism would result in a decrease of galanin-induced feeding, as has been observed in vivo (50). In addition, the present observations are also in accordance with data showing that NPY neurons respond across a broad range of variations in leptin levels (27). In conclusion, using our model of primary neurons in culture, we could generate data suggesting that hypothalamic CRH and NPY neurons can interact in a very subtle way to mediate the stimulation of food intake induced by galanin. In addition, we could demonstrate that leptin differentially affects the galanin-stimulated secretion of NPY and CRH, decreasing NPY while leaving CRH untouched. These results on neurotransmitter secretion can then be correlated to data obtained in vivo and dealing exclusively with measures of expression, thus providing a more comprehensive knowledge of integrated hypothalamic function. Such detailed knowledge probably represents a prerequisite for the design of novel therapeutic agents aimed at treating obesity and related disorders. ACKNOWLEDGMENTS

This work was supported by grants from the Swiss national science foundation (no. 31-50748.97 to R.C.G. and F.P.P. and no. 31.053256.98 to E.G.). F.P.P. is the recipient of a Research and Career Development Award from the Prof. Dr. Max Cloe¨ tta Foundation. We are grateful to Dr. Hughes Cadas for his help in establishing the neuronal cell culture system. REFERENCES 1. Schwartz MW, Woods SC, Porte D, Seeley RJ, Baskin DG: Central nervous system control of food intake. Nature 6778:661– 671, 2000 2. Edholm OG: Energy balance in man studies. J Hum Nutr 6:413– 431, 1977 3. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM: Positional cloning of the mouse obese gene and its human homologue. Nature 425– 432, 1994 4. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, Collins F: Effects of the obese gene product on body weight regulation in ob/ob mice. Science 540 –543, 1995 5. Considine RV, Sinha MK, Heiman ML, Kriauciunas A, Stephens TW, Nyce MR, Ohannesian JP, Marco CC, McKee L J, Bauer TL, Caro JF: Serum immunoreactive-leptin concentrations in normal-weight and obese humans. N Engl J Med 5:292–295, 1996 6. Boden G, Chen X, Mozzoli M, Ryan I: Effect of fasting on serum leptin in normal human subjects. J Clin Endocrinol Metab 9:3419 –3423, 1996 7. Clark JT, Kalra PS, Crowley WR, Kalra SP: Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 1:427– 429, 1984 8. Levine AS, Morley JE: Neuropeptide Y: a potent inducer of consummatory behavior in rats. Peptides 6:1025–1029, 1984 9. Kyrkouli SE, Stanley BG, Seirafi RD, Leibowitz SF: Stimulation of feeding by galanin: anatomical localization and behavioral specificity of this peptide’s effects in the brain. Peptides 5:995–1001, 1990 10. Leibowitz SF: Differential functions of hypothalamic galanin cell grows in the regulation of eating and body weight. Ann N Y Acad Sci 206 –220, 1998 11. Allen YS, Adrian TE, Allen JM, Tatemoto K, Crow TJ, Bloom SR, Polak JM: Neuropeptide Y distribution in the rat brain. Science 4613:877– 879, 1983 12. Gehlert DR: Role of hypothalamic neuropeptide Y in feeding and obesity. Neuropeptides 5:329 –338, 1999 13. Edwards CM, Abusnana S, Sunter D, Murphy KG, Ghatei MA, Bloom SR: The effect of the orexins on food intake: comparison with neuropeptide Y, melanin-concentrating hormone and galanin. J Endocrinol 3:R7–R12, 1999 14. Gruaz NM, Pierroz DD, Rohner-Jeanrenaud F, Sizonenko PC, Aubert ML: Evidence that neuropeptide Y could represent a neuroendocrine inhibitor of sexual maturation in unfavorable metabolic conditions in the rat. Endocrinology 4:1891–1894, 1993 15. Ahima RS, Prabakaran D, Mantzoros C, Qu D, Lowell B, Maratos-Flier E, DIABETES, VOL. 50, DECEMBER 2001

Flier JS: Role of leptin in the neuroendocrine response to fasting. Nature 250 –252, 1996 16. Hohmann JG, Clifton DK, Steiner RA: Galanin: analysis of its coexpression in gonadotropin-releasing hormone and growth hormone-releasing hormone neurons. Ann N Y Acad Sci 221–235, 1998 17. Sawchenko PE, Imaki T, Potter E, Kovacs K, Imaki J, Vale W: The functional neuroanatomy of corticotropin-releasing factor (Review). Ciba Found Symp 172:5–21 [discussion 21–29], 1993 18. Cone RD: The corticotropin-releasing hormone system and feeding behavior–a complex web begins to unravel. Endocrinology 8:2713–2714, 2000 19. Vaughan J, Donaldson C, Bittencourt J, Perrin MH, Lewis K, Sutton S, Chan R, Turnbull AV, Lovejoy D, Rivier C, Rivier J, Sawchenko PE, Vale W: Urocortin, a mammalian neuropeptide related to fish urotensin I and to corticotropin-releasing factor. Nature 378:287–292, 1995 20. Britton DR, Koob GF, Rivier J, Vale W: Intraventricular corticotropinreleasing factor enhances behavioral effects of novelty. Life Sci 4:363–367, 1982 21. Richard D, Huang Q, Timofeeva E: The corticotropin-releasing hormone system in the regulation of energy balance in obesity. Int J Obes Relat Metab Disord S36 –S39, 2000 22. Ceccatelli S, Eriksson M, Hokfelt T: Distribution and coexistence of corticotropin-releasing factor-, neurotensin-, enkephalin-, cholecystokinin-, galanin- and vasoactive intestinal polypeptide/peptide histidine isoleucine-like peptides in the parvocellular part of the paraventricular nucleus. Neuroendocrinology 3:309 –323, 1989 23. Wahlestedt C, Skagerberg G, Ekman R, Heilig M, Sundler F, Hakanson R: Neuropeptide Y (NPY) in the area of the hypothalamic paraventricular nucleus activates the pituitary-adrenocortical axis in the rat. Brain Res 1:33–38, 1987 24. Wang Q, Bing C, Al-Barazanji K, Mossakowaska DE, Wang XM, McBay DL, Neville WA, Taddayon M, Pickavance L, Dryden S, Thomas ME, McHale MT, Gloyer IS, Wilson S, Buckingham R, Arch JR, Trayhurn P, Williams G: Interactions between leptin and hypothalamic neuropeptide Y neurons in the control of food intake and energy homeostasis in the rat. Diabetes 3:335–341, 1997 25. Schwartz MW, Seeley RJ, Campfield LA, Burn P, Baskin DG: Identification of targets of leptin action in rat hypothalamus. J Clin Invest 5:1101–1106, 1996 26. Schwartz MW, Baskin DG, Bukowski TR, Kuijper JL, Foster D, Lasser G, Prunkard DE, Porte D Jr, Woods SC, Seeley RJ, Weigle DS: Specificity of leptin action on elevated blood glucose levels and hypothalamic neuropeptide Y gene expression in ob/ob mice. Diabetes 4:531–535, 1996 27. Ahima RS, Kelly J, Elmquist JK, Flier JS: Distinct physiologic and neuronal responses to decreased leptin and mild hyperleptinemia. Endocrinology 11:4923– 4931, 1999 28. Corder R, Pralong F, Turnill D, Saudan P, Muller AF, Gaillard RC: Dexamethasone treatment increases neuropeptide Y levels in rat hypothalamic neurons. Life Sci 23:1879 –1886, 1988 29. Brewer GJ, Torricelli JR, Evege EK, Price PJ: Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J Neurosci Res 5:567–576, 1993 30. Brewer GJ: Serum-free B27/neurobasal medium supports differentiated growth of neurons from the striatum, substantia nigra, septum, cerebral cortex, cerebellum, and dentate gyrus. J Neurosci Res 5:674 – 683, 1995 31. Riederer BM, Draberova E, Viklicky V, Draber P: Changes of MAP2 phosphorylation during brain development. J Histochem Cytochem 12: 1269 –1284, 1995 32. Grouzmann E, Aubert JF, Waeber B, Brunner HR: A sensitive and specific two-site, sandwich-amplified enzyme immunoassay for neuropeptide Y. Peptides 6:1049 –1054, 1992 33. Haefliger JA, Bergonzelli G, Waeber G, Aubert JF, Nussberger J, Gavras H, Nicod P, Waeber B: Renin and angiotensin II receptor gene expression in kidneys of renal hypertensive rats. Hypertension 5:733–737, 1995 34. Thompson RC, Seasholtz AF, Herbert E: Rat corticotropin-releasing hormone gene: sequence and tissue-specific expression. Mol Endocrinol 5:363–370, 1987 35. Fort P, Marty YL, Piechczyck M, el Sabrouty S, Dany C, Jeanteur P, Blanchard JM: Various rat adult tissues express only one major mRNA species from glyceraldehyde-3-phosphate-dehydrogenase multigenic family. Nucelic Acids Research 1431–1442, 1985 36. Pralong FP, Roduit R, Waeber G, Castillo E, Mosimann F, Thorens B, Gaillard RC: Leptin inhibits directly glucocorticoid secretion by normal human and rat adrenal gland. Endocrinology 139:4264 – 4268, 1998 37. Pralong FP, Corder R, Gaillard RC: The effects of chronic glucocorticoid excess, adrenalectomy and stress on neuropeptide Y in individual rat hypothalamic nuclei. Neuropeptides 4:223–231, 1993 2671


38. Cherradi N, Capponi AF, Gaillard RC, Pralong FP: Decreased expression of steroidogenic acute regulatory protein (StAR): a novel mechanism participating to the leptin-induced inhibition of glucocorticoid biosynthesis. Endocrinology 142:3302–3308, 2001 39. Grouzmann E, Cressier F, Walker P, Hofbauer K, Waeber B, Brunner HR: Interactions between NPY and its receptor: assessment using ant-NPY antibodies. Regul Pept 54:439 – 444, 1994 40. Kyrkouli SE, Stanley BG, Leibowitz SF: Galanin: stimulation of feeding induced by medial hypothalamic injection of this novel peptide. Eur J Pharmacol 1:159 –160, 1986 41. Akabayashi A, Koenig JI, Watanabe Y, Alexander JT, Leibowitz SF: Galanin-containing neurons in the paraventricular nucleus: a neurochemical marker for fat ingestion and body weight gain. Proc Natl Acad Sci U S A 22:10375–10379, 1994 42. Hellbach S, Gartner P, Deicke J, Fischer D, Hassan AH, Almeida OF: Inherent glucocorticoid response potential of isolated hypothalamic neuroendocrine neurons. FASEB J 2:199 –207, 1998 43. Krsmanovic LZ, Mores N, Navarro CE, Saeed SA, Arora KK, Catt KJ: Muscarinic regulation of intracellular signaling and neurosecretion in gonadotropin-releasing hormone neurons [see comments]. Endocrinology 10:4037– 4043, 1998 44. Stephens TW, Basinski M, Bristow PK, Bue-Valleskey JM, Burgett SG, Craft

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L, Hale J, Hoffmann J, Hsiung HM, Kriauciunas A, MacKellar W, Rosteck PR, Jr, Schoner B, Smith D, Tinsley FC, Zhang X-Y, Heiman M: The role of neuropeptide Y in the antiobesity action of the obese gene product. Nature 530 –532, 1995 45. Spanswick D, Smith MA, Groppi VE, Logan SD, Ashford ML: Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels. Nature 6659:521–525, 1997 46. Glasow A, Haidan A, Hilbers U, Breidert M, Gillespie J, Scherbaum WA, Chrousos GP, Bornstein SR: Expression of Ob receptor in normal human adrenals: differential regulation of adrenocortical and adrenomedullary function by leptin. J Clin Endocrinol Metab 12:4459 – 4466, 1998 47. Pralong FP, Gomez F, Guillou L, Mosimann F, Franscella S, Gaillard RC: Food-dependent Cushing’s syndrome: possible involvement of leptin in cortisol hypersecretion. J Clin Endocrinol Metab 10:3817–3822, 1999 48. El-Haschimi K, Pierroz DD, Hileman SM, Bjorbaek C, Flier JS: Two defects contribute to hypothalamic leptin resistance in mice with diet-induced obesity. J Clin Invest 12:1827–1832, 2000 49. Bjorbaek C, El-Haschimi K, Frantz JD, Flier JS: The role of SOCS-3 in leptin signaling and leptin resistance. J Biol Chem 42:30059 –30065, 1999 50. Sahu A: Leptin decreases food intake induced by melanin-concentrating hormone (MCH), galanin (GAL) and neuropeptide Y (NPY) in the rat. Endocrinology 11:4739 – 4742, 1998

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