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Endocrinology Vol. 144, No. 6 2634-2643
Copyright © 2003 by The Endocrine Society

Changes in Hypothalamic Expression Levels of Galanin-Like Peptide in Rat and Mouse Models Support That It Is a Leptin-Target Peptide

Satoshi Kumano, Hirokazu Matsumoto, Yoshihiro Takatsu, Jiro Noguchi, Chieko Kitada and Tetsuya Ohtaki

Discovery Research Laboratories I, Pharmaceutical Research Division, Takeda Chemical Industries, Ltd., Tsukuba, Ibaraki 300-4293, Japan

Address all correspondence and requests for reprints to: Tetsuya Ohtaki, Ph.D., Wadai 10, Tsukuba, Ibaraki 300-4293, Japan. E-mail: ohtaki_tetsuya{at}takeda.co.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Galanin-like peptide (GALP) is a novel peptide that has been isolated from the porcine hypothalamus. The expression of GALP mRNA is localized to the hypothalamic arcuate nucleus and is thought to be under the regulation of leptin. First, we confirmed by real-time PCR analysis that sc administration of leptin to Wistar rats under food-deprived conditions resulted in a 1.5-fold increase in hypothalamic GALP mRNA levels. Next, GALP mRNA levels were found to be reduced by 50% in 11-wk-old male Zucker obese rats compared with age-matched Zucker lean rats, whereas neuropeptide Y mRNA levels were increased by 55% and proopiomelanocortin mRNA levels were reduced by 53% in Zucker obese rats. Analysis using a two-site enzyme immunoassay revealed a lower level of hypothalamic GALP immunoreactivity in 11-wk-old Zucker obese rats (5.9 fmol/mg protein) than in age-matched Zucker lean rats (19.6 fmol/mg protein). Immunohistochemical studies demonstrated that Zucker obese rats (11 wk old) had a reduced number of GALP immunoreactivity-positive cells (29.4 cells/3 slices) in the arcuate nucleus compared with age-matched Zucker lean rats (115 cells/3 slices). Furthermore, Zucker obese rats showed increased sensitivity to intracerebroventricularly administered GALP compared with Zucker lean rats, in that a lower dose of GALP increased plasma LH levels in male Zucker obese rats, but not in male Zucker lean rats. In addition, a reduction in the level of hypothalamic GALP mRNA was found in db/db and ob/ob mice. The result supports the hypothesis that the hypothalamic GALP gene expression is controlled by leptin signals and suggests possible involvement of GALP in the reproductive abnormalities of the Zucker obese rat.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GALANIN-LIKE peptide (GALP) was originally isolated from the porcine hypothalamus after the discovery of its agonistic activity for galanin receptor subtype GALR2 (1). To date, the amino acid sequence of GALP has been determined in pigs, humans, rats (1), and macaque monkeys (2) by molecular cloning studies. It is of particular interest that the sequence of GALP-(9–21), GWTLNSAGYLLGP, is conserved in all of these species and is identical to that of galanin-(1–13). GALP has a high affinity for GALR2, but a lower affinity for GALR1, whereas galanin has a high affinity for both receptor subtypes (1). However, it remains to be elucidated whether GALP serves as a physiological ligand of GALR2 and/or a ligand of a GALP-selective receptor that is yet to be identified.

Recent in situ hybridization and immunohistochemical studies have localized GALP-expressing neurons to the hypothalamic arcuate nucleus (ARC) in the rat (3, 4, 5, 6), mouse (7), and macaque monkey (2). More than 85% of GALP-positive neurons in the rat ARC were double-labeled with a leptin receptor antibody, indicating coexpression of GALP and leptin receptors (6). Moreover, Jureus et al. (3) reported that the number of GALP-expressing neurons in the ARC was decreased under fasting conditions and restored to beyond the fed level by the administration of leptin. Their subsequent studies demonstrated that genetically leptin-deficient ob/ob c57BL/6 mice have a decreased number of hypothalamic GALP-expressing cells compared with wild-type c57BL/6 mice (7). Also, leptin administration to ob/ob mice was shown to lead to a compensative increase in the number of GALP-expressing cells (7). Taken together with these results, it has been suggested that GALP is located downstream of the hypothalamic leptin-signaling pathway.

In addition to the ARC, brain GALP gene expression has been found in the external zone of the median eminence (3, 5, 6) and infundibular stalk (3, 6). In the pituitary gland, GALP gene expression was found in the neural lobes but not in the anterior pituitary (5, 8). It has been shown that the expression of pituitary GALP mRNA is localized to the pituicytes, glia-like cells, and is increased under dehydrated conditions or salt-loading conditions (8, 9). Kastin et al. (10) found a higher concentration (>400 pg/ml) of GALP-like immunoreactivity in mouse plasma, which was reduced after 48 h of food deprivation.

The intracerebroventricular (icv) administration of GALP increased plasma LH levels in male rats via a mechanism that was suggested to involve the activation of GnRH neurons in the medial preoptic area (MPA) (11). The MPA together with the anterior part of the paraventricular nucleus (PVN), the bed nucleus of the stria terminalis, and the lateral septal nucleus are enriched with GALP-positive nerve fibers (6). In the MPA, GALP-positive nerve fibers have been found in close apposition with the cell bodies and dendrites of GnRH neurons (6). On the other hand, it has been shown that GALP induces food intake more potently than does galanin (12). Lawrence and colleagues (13) recently reported that icv injection of GALP significantly stimulated acute food intake at 1 h after injection, whereas a higher injected dose decreased food intake for 24 h.

GALP is now being established as a new member of the leptin-regulated peptides. Although the level of GALP gene expression has been shown to change in leptin-administered rats (3) and in ob/ob mice (7), the possible changes in other obese animal models with an impaired leptin-signaling pathway have not been reported. In the present study we first established a real-time RT-PCR method for easier determination of the GALP mRNA level and confirmed the reported changes in hypothalamic GALP mRNA levels (3, 7). We then applied this method to other animal models, the Zucker obese rat and db/db mouse, and found decreased hypothalamic GALP mRNA levels in these obese animals compared with their lean littermates. Furthermore, we demonstrated that the chronic decrease in GALP mRNA in Zucker obese rats is actually accompanied by decreases in hypothalamic GALP immunoreactivity (ir-GALP) and the number of GALP-immunoreactive cells in the ARC. We further found that male Zucker obese rats show increased sensitivity of the plasma LH response to icv-administered GALP. These findings suggest that the Zucker obese rat is a good model for studying the physiological function of GALP. Here we describe our findings and discuss the physiological significance of GALP with reference to a possible leptin downstream signaling molecule.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Wistar rats (Charles River Japan, Kanagawa, Japan; or Japan SLC, Shizuoka, Japan), Zucker obese (fa/fa) and lean (Fa/fa or Fa/Fa) rats (Japan SLC), C57BL/6J-ob/ob mice and their lean littermates C57BL/6J-ob/m+ (The Jackson Laboratory, Bar Harbor, ME), and C57BL/KsJ-db/db mice and their lean littermates C57BL/KsJ-db/m+ (CLEA Japan, Inc., Tokyo, Japan) were housed in a light (12-h light, 12-h dark cycle; lights on at 0800 h)- and temperature-controlled (23 C) environment. All experimental procedures were performed in accordance with institutional guidelines for animal care at Takeda Chemical Industries Ltd. (Osaka, Japan).

Preparation of hypothalamic cDNAs from Wistar rats
Male Wistar rats (8 wk old; Charles River Japan) were deprived of food for 72 h (from 1600 h on d 0 to 1600 h on d 3). During this period, sc administration of recombinant rat leptin (3 mg/kg body weight; R\|[amp ]\|D Systems, Inc., Minneapolis, MN) or vehicle was performed six times (at 2000 and 0800 h on each day) (n = 5/group). At 1600 h on d 3, the rats were killed by decapitation, and the brain was quickly removed. Trunk blood was collected for measurement of the plasma leptin level. The hypothalamus was dissected out following a previous method (14) with slight modification. Briefly, the brain was placed ventral side up, and a 4-mm-thick coronal slice was cut caudal to the optic chiasm. The slice was then dissected laterally up to the hypothalamic sulci and dorsally up to the mammilothalamic tract. Total RNA was extracted from individual hypothalamic dissections using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD). After treatment of total RNA with deoxyribonuclease I (Life Technologies, Inc.), cDNA samples were synthesized from 500 ng total RNA with oligo(deoxythymidine) primer at 42 C using the SuperScript first strand cDNA synthesis system (Life Technologies, Inc.). Hypothalamic cDNA was also prepared from ad libitum-fed or 72-h food-deprived male Wistar rats (8 wk old, n = 5/group; Charles River Japan) or ad libitum-fed male Wistar rats (11 wk old, n = 6; Charles River Japan) as described above.

Preparation of hypothalamic cDNAs from Zucker obese and lean rats
Male Zucker obese and lean rats (11 wk old, n = 6 for obese and n = 5 for lean) were killed by decapitation. Trunk blood was collected for measurement of the plasma insulin level. The brain was quickly removed, and hypothalamic dissection was carried out as described above. Total RNA extraction and cDNA synthesis were performed as described above.

Preparation of hypothalamic cDNAs from ob/ob and db/db mice
The mouse hypothalamus was dissected as described for the rat brain, except that the slice thickness was 2 mm. Male C57BL/6J-ob/ob mice and their lean littermates C57BL/6J-ob/m+ (8 wk old, n = 5 for ob/ob and n = 4 for ob/m+), and C57BL/KsJ-db/db mice and their lean littermates C57BL/KsJ-db/m+ (8 wk old, n = 6/group) were used. Total RNA extraction and cDNA synthesis were performed as described above.

Male C57BL/6J-ob/ob mice received a total of six sc administrations of recombinant mouse leptin (3 mg/kg body weight; Genzyme Techne, Cambridge, MA) or vehicle (8 wk old, n = 4/group) at 2000 and 0800 h on each of 3 consecutive days under ad libitum-fed conditions. At 1600 h on d 3, mice were killed by decapitation and prepared for dissection of the hypothalamus. Total RNA extraction and cDNA synthesis were performed from individual dissections as described above.

Quantification of the gene transcripts in rat cDNAs by real-time RT-PCR
Quantification of GALP, neuropeptide Y (NPY), proopiomelanocortin (POMC), and galanin mRNA was carried out by real-time PCR (TaqMan) using the ABI PRISM 7700 (PE Applied Biosystems, Foster, CA). TaqMan PCR was performed in a 96-well optical tray according to the manufacturer’s instructions with minor alterations. The TaqMan primers (JBioS, Saitama, Japan) and probes (PE Applied Biosystems) were designed with the assistance of PrimerExpress software as follows: rGALTMF (5'-ACCTGTGGAAGGCCATAGATG-3'), rGALTMR (5'-GTTTCTCCCATTGACCTTTTGG-3'), and rGALTM1 [5'-6-carboxy- fluorescein (Fam)-TCCCTTATTCCCGCTCTCCAAGGATG-6-carboxy-tetramethyl-rhodamine (Tamra)-3'] for rat GALP; rNPYTMF (5'-ACATGGCCAGATACTAC-3'), rNPYTMR (5'-GCTGGATCTCTTGCCATATCTC-3'), and rNPYTM (5'-Fam-TGCGACACTACATCAATCTCATCACCAGAC-Tamra-3') for rat NPY; rPOMCTMF (5'-TTCAGACCTCCATAGACGTGTGG-3'), rPOMCTMR (5'-ATCTCCGTTGCCTGGAAACA-3'), and rPOMCTM (5'-Fam-AAAGCAACCTGCTGGCTTGCATCC-Tamra-3') for rat POMC; rGANTMF (5'-GCCCACATGCCATTGACAA-3'), rGANTMR (5'-GTGGTAACTCCCTCTTGCCTGT-3'), and rGANTM1 (5'-Fam-CACAGATCATTTAGCGACAAGCATGGCC-Tamra-3') for rat galanin; and mGALPTMF (5'-GGTCCTCTTCCTCACCATCTTG-3'), mGALPTMR (5'-CCAGGAGGTAACCAGCACTATTG-3'), and mGALTM1 (5'-Fam-TGAGCCTGGCAGAAACACCGGAAT-Tamra-3') for mouse GALP. ß-Actin mRNA and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA were also quantified to normalize RNA concentration. Primers and probes used were rßACTMF (5'-ATGAGCTGCCTGACGGTCAG-3'), rßACTMR (5'-GGAAGGCTGGAAGAGAGCCT-3'), and rßACTM (5'-Fam-CATCACTATCGGCAATGAGCGGTTCC-Tamra-3') for rat ß-actin; mßACTMF (5'-CATCACTATTGGCAACGAGCG-3'), mßACTMR (5'-GATTCCATACCCAAGAAGGAAGG-3'), and mßACTM (5'-Fam-TTCCGATGCCCTGAGGCTCTTTTCCA-Tamra-3') for mouse ß-actin. TaqMan rodent GAPDH control reagents for both rats and mice were purchased from PE Applied Biosystems. Standard curves were obtained with each standard DNA solution of known copy number (10–106 copies). The number of copies in each sample was determined from a standard curve and is shown in copies per nanograms of total RNA. The amount of total RNA was determined by OD at 260 nm and was normalized by multiplying the average of two coefficients, k1 and k2. The k1 of each sample was calculated by dividing the content of ß-actin mRNA (copies per nanograms of total RNA) of each sample by that of a standard total RNA sample. The k2 was obtained in the same manner for GAPDH mRNA.

Preparation of individual hypothalamic extract samples
Brains were obtained from male Zucker obese rats, Zucker lean rats (11 wk old, n = 5 in each), and Wistar rats (11 wk old, n = 5; Japan SLC). The hypothalamic dissections were carried out as described above, and the specimens were boiled in 0.5 ml distilled water for 5 min, cooled on ice, and then homogenized. The protein concentration of the homogenate was determined using the Coomassie Protein Assay Reagent (Pierce Chemical Co., Rockford, IL). The homogenate was mixed with a 1:17 volume of acetic acid, stirred, and centrifuged for 15 min at 15,000 rpm. The supernatant was concentrated using a Sep-Pak column (C18-ODS, 6 cc/500 mg, Waters Corp., Milford, MA) and then lyophilized with 200 mg BSA. The lyophilized powder was dissolved in 0.2 ml 0.05% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)-containing buffer C (15), and each 0.01-ml (for Wistar and Zucker lean rats) or each 0.04-ml (for Zucker obese rats) aliquot was subjected to two-site enzyme immunoassay (EIA; see below).

Preparation of HPLC-fractionated samples
Dissected hypothalamic tissue (total, 0.58 g) from five Wistar rats (Japan SLC) was boiled in 5 ml distilled water for 10 min, cooled on ice, and then homogenized. The tissue extract was prepared as described above and was concentrated using a Sep-Pak Plus C18 cartridge (Waters Corp.). The eluate from the Sep-Pak was lyophilized, dissolved, and then injected into a TSK gel Super ODS column (Tosoh, Tokyo, Japan). Elution was performed by a linear gradient increase in the acetonitrile concentration from 0–60% in 0.1% trifluoroacetic acid for 60 min at a flow rate of 1 ml/min at 40 C. The eluate was collected in 1-ml fractions and lyophilized. Each lyophilized fraction was dissolved in 0.25 ml CHAPS-containing buffer C (15), and a 0.1-ml aliquot was subjected to two-site EIA (see below). After completion of the hypothalamic extract analysis, recombinant rat GALP was analyzed by HPLC under the same conditions, and the eluate was subjected to two-site EIA.

Immunoassays
Plasma insulin and leptin levels were determined using a Shionoria insulin RIA kit (Shionogi, Osaka, Japan) and a rat leptin RIA kit (Linco Research, Inc., St. Charles, MO), respectively. The concentration of galanin in hypothalamic tissue extract samples was determined using a rat galanin RIA kit (Peninsula Laboratories, Inc., Belmont, CA).

Two-site EIA for immunoreactive GALP
A GR-1Na antibody (IgG fraction) (6) was labeled with horseradish peroxidase (HRP), as described previously (16). Standard rat GALP peptide ranging between 1 x 10-6 and 1 x 10-12 M, or tissue extract dissolved in 100 µl 0.1% CHAPS-containing buffer EC (15) was added to a GR-1Ca [obtained as described previously (9)] antibody-coated microtest plate (96 wells, Nunc, Naperville, IL), and incubated at 4 C for 24 h. Subsequently, after five washes with PBS, 100 µl HRP-labeled GR-1Na antibody diluted to 2000-fold with buffer C (15) were added to the plate and incubated at 4 C for 22 h. The plate was washed with PBS five times, and the bound enzyme activity was measured using a tetramethylbenzidine microwell peroxidase system (Kirkegaard \|[amp ]\| Perry Laboratories, Inc., Gaithersburg, MD). A 100-µl aliquot of HRP substrate was added to the plate, and after the reaction was complete, it was stopped by adding 100 µl 1 M phosphate buffer. The immunoreaction was detected at 450 nm by a plate reader, and ir-GALP was quantified from a standard curve.

Immunohistochemical staining of GALP-positive cells in rat hypothalamus
Zucker obese and lean rats (11 wk old, n = 5/group) were anesthetized with an ip injection of sodium pentobarbital (75 mg/kg; Dainippon Pharmaceutical, Osaka, Japan) and perfused transcardially with 100 ml 2% sodium nitrite in saline, followed by 400 ml Mildform 10 N (Wako Pure Chemical Industries, Ltd., Osaka, Japan). The brain was removed, blocked, and immersed in the same fixative for 2 h, then immersed in 18% sucrose for 24 h at 4 C, and rapidly frozen on dry ice. Coronal sections (16 µm) were cut on a cryostat at -17 C and mounted on SuperFrost glass slides (Matsunami Glass Industries Ltd., Osaka, Japan). The sections were treated with a 1% H2O2-methanol solution for 15 min, then washed in PBS containing 0.3% Triton X-100. After preincubation in 10% normal horse serum for 30 min, the sections were incubated with GR-2Ca mouse monoclonal antibody (9) at 5 µg/ml overnight at 4 C. The sections were washed in PBS three times and incubated with biotinylated antimouse immunoglobulin G (1:200; Vector Laboratories, Inc., Burlingame, CA) for 30 min at room temperature. After three washes in PBS, the sections were incubated in avidin-biotin-peroxidase complex (ABC Elite, Vector Laboratories, Inc.) for 30 min at room temperature. The immunolabeling was visualized with a mixture of diaminobenzidine and H2O2 in 0.05 M Tris-buffered saline solution. The sections were dehydrated and mounted with Canada Balsam (Sigma-Aldrich Corp., St. Louis, MO). Tyramide signal amplification (6) was unnecessary for the present immunochemistry using GR-2Ca (9). Three slices were prepared from the anterior (-3.6 to -3.8 mm from the bregma according to the stereotaxic coordinates of Paxinos and Watson) or posterior (-4.0 to -4.5 mm) parts of individual rat brains. The number of ir-GALP-positive cells was determined, and the mean count of GALP-positive cells per three slices was obtained.

The icv administration of GALP
The icv administration of GALP was performed as described previously (11). Briefly, recombinant rat GALP (17) was dissolved in distilled water at 1 mM, diluted twice with 2-fold concentrated PBS, and further diluted 5-fold with PBS. Ten microliters of GALP solution (0.5 or 0.1 mM) or vehicle (PBS) were administered into the third ventricle of male Zucker obese and lean rats (11 wk old, n = 5/group) through steel cannulas during the early light phase (0900–1100 h). At 0, 10, 20, 30, and 60 min after the administration, blood samples were taken from the venous catheter and then mixed with aprotinin and EDTA. The plasma samples were prepared and then subjected to RIA (Amersham Pharmacia Biotech, Arlington Heights, IL) to determine the LH concentration. The increase in the plasma LH level ({Delta} plasma LH) was calculated by subtracting the LH level at 0 min in each animal.

Statistical analysis
All results are given as the mean ± SEM. Probabilities of chance differences between two groups were calculated using a two-tailed t test. Statistical analysis in the LH release experiment was performed using one-way ANOVA for repeated measurements, followed by Dunnett’s multiple test. The level of statistical significance was set at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Up-regulation of GALP mRNA expression in leptin-administered rats
We tested the effect of 72 h of food deprivation on hypothalamic GALP, NPY, and POMC mRNA levels using real-time RT-PCR analysis. Although food-deprived rats showed a 33% increase in the NPY mRNA level (P < 0.05) and a 27% decrease in the POMC mRNA level (P < 0.05) compared with ad libitum-fed rats (8 wk old, n = 5/group), there was no significant difference (P = 0.50) in GALP mRNA levels between the food-deprived and ad libitum-fed rats (33.2 ± 2.7 and 36.9 ± 4.6 copies, respectively; Fig. 1AGo). In another set of experiments we determined GALP, NPY, and POMC mRNA levels in rats that had received an sc injection of leptin or vehicle during 72 h of fasting (8 wk old, n = 5/group). During this treatment, body weight dropped from 319.5 ± 6.6 to 267.3 ± 6.1 g in leptin-treated rats or from 318.8 ± 7.2 to 266.0 ± 7.4 g in vehicle-treated rats. At the end of the experimental period, the leptin level was 6.7 ± 1.2 ng/ml in leptin-treated rats and 0.36 ± 0.065 ng/ml in vehicle-treated rats. As summarized in Fig. 1BGo, leptin-treated rats showed a 49% increase in the GALP mRNA level (P < 0.05) compared with vehicle-treated rats (49.7 ± 4.0 and 33.3 ± 3.3 copies, respectively). The leptin-treated rats also showed (statistically insignificant) tendencies for a decrease in the NPY mRNA level (P = 0.07) and an increase in the POMC mRNA level (P = 0.33) compared with vehicle-treated rats.



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  		Figure 1. GALP, NPY, and POMC mRNA levels in the hypothalamus of food-deprived Wistar rats. A, GALP, NPY, and POMC mRNA levels in the hypothalamus of ad libitum-fed or 72-h fasted Wistar rats (8 wk old, males, n = 5/group). B, GALP, NPY, and POMC mRNA levels in the hypothalamus of Wistar rats that had received vehicle or leptin under 72-h food-deprived conditions (8 wk old, males, n = 5/group). *, P < 0.05; **, P < 0.01 (by t test). Bars represent the mean ± SEM.

 
Decreased expression of GALP mRNA in the hypothalamus of Zucker obese rats
We next determined GALP mRNA levels in the Zucker obese rat, a genetically obese animal model that has a Gln269Pro mutation in the leptin receptor gene (18) and exhibits leptin resistance. Male Zucker obese (fa/fa) rats (11 wk old, n = 6) showed a severe obese phenotype and hyperinsulinemia, whereas age-matched Zucker lean (Fa/fa or Fa/Fa,) rats (n = 5) had an almost normal phenotype (Table 1Go). In Zucker obese rats, hypothalamic GALP mRNA levels were decreased by 50% (P < 0.01), NPY mRNA levels were increased by 55% (P < 0.05), and POMC mRNA levels were decreased by 53% (P < 0.01) compared with Zucker lean rats. No significant difference was found in galanin mRNA levels between the two groups of rats. GALP, NPY, and POMC mRNA levels in Zucker lean rats (Table 1Go) were comparable to those (63.8 ± 13.1, 319 ± 29, and 2330 ± 180 copies/ng total RNA, respectively) in 11-wk-old Wistar rats (377 ± 25 g body weight, n = 6).


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  		Table 1. GALP, NPY, POMC, and galanin mRNA levels in the hypothalamus of 11-wk-old male Zucker rats

 
Decreased ir-GALP in the hypothalamus of Zucker obese rats
For the specific and sensitive detection of endogenous GALP in the hypothalamus, we established a two-site EIA with monoclonal antibodies GR2–1Na (6) and GR-1Ca. This EIA detected authentic rat GALP-(1–60) at the concentration range of 0.3–100 fmol/well and did not cross-react with galanin even at 3000 fmol/well (Fig. 2EGo). The average content of ir-GALP in the hypothalamus of male Wistar rats (11 wk old, n = 5) was 97.7 fmol/mg protein (Table 2Go). This value was about one eighth that of galanin (818 pmol/mg protein) in the same preparations (Table 2Go). The hypothalamic extract was further analyzed by HPLC and subjected to EIA. The analysis provided a single peak of GALP immunoreactivity in fraction 52 (Fig. 2CGo), where the authentic rat GALP-(1–60) was eluted under the same conditions (Fig. 2DGo).



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  		Figure 2. HPLC analysis of ir-GALP in the rat hypothalamus. Extracts of rat hypothalamus (A) and synthetic rat GALP-(1–60) (B) were analyzed by reverse phase HPLC under the same conditions. Every 1-ml fraction of the eluate was collected and analyzed by two-site EIA. The ir-GALP in the extract of rat hypothalamus (C) and in authentic rat GALP-(1–60) (D) is shown. Standard curves for rat ({circ}), pig ({triangleup}), and human GALP ({square}) in two-site EIA are shown (E).

 

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  		Table 2. Determination of ir-GALP and galanin in the rat hypothalamus

 
Next, we determined the ir-GALP concentration in the hypothalamus of Zucker rats. As shown in Table 2Go, the average hypothalamic ir-GALP concentration in Zucker obese rats (11 wk old, n = 5) was decreased to 30% (P < 0.01) of that in Zucker lean rats (n = 5). The ir-GALP levels in Zucker lean rats were lower than those in Wistar rats (Table 2Go). In contrast, there was no significant difference in galanin concentration among these three groups of rats (Table 2Go).

Decreased number of ir-GALP-positive cells in the ARC of Zucker obese rats
Immunohistochemical analysis was carried out to quantify the decrease in the number of ir-GALP-positive cells in the ARC of Zucker obese rats. The ir-GALP-positive cells were counted in both the anterior (bregma level, -3.6 to -3.8 mm) and posterior (-4.0 to -4.5 mm) part of the ARC. More GALP-immunoreactive cells were found in the caudal direction (Fig. 3Go), as was the case in Wistar rats (6). In both the anterior and posterior ARC, Zucker obese rats at 11 wk of age had 73% and 74% fewer ir-GALP-positive cells, respectively, than age-matched Zucker lean rats (Fig. 3Go).



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  		Figure 3. ir-GALP-positive cells in the ARC of Zucker obese and lean rats. The ir-GALP-positive cells were visualized in the ARC (caudal part) of Zucker obese (A and B) and lean (C and D) rats. Boxes in A and C were enlarged in B and D, respectively. The number of cells per three slices from the anterior (bregma level, -3.6 to -3.8 mm) and posterior (-4.0 to -4.5 mm) part of individual brains (n = 5/group) is shown (E). *, P < 0.05; **, P < 0.01 (by t test). Bars represent the mean ± SEM. 3V, Third ventricle.

 
Increased sensitivity to icv administered GALP in Zucker obese rats
The effect of icv administration of GALP on the plasma LH level was tested in male Zucker obese and lean rats (11 wk old, n = 5/group). As shown in Fig. 4Go, 1- and 5-nmol doses of GALP clearly provoked an increase in plasma LH levels to a similar extent in Zucker obese rats (P < 0.05 vs. vehicle-treated obese rats), whereas only the 5-nmol dose had a significant effect on the plasma LH level in Zucker lean rats (P < 0.05 vs. vehicle-treated lean rats). There was no significant difference in the responses to 5 nmol GALP in Zucker obese and lean rats.



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  		Figure 4. Increase in plasma LH level induced by icv administration of GALP. Rat GALP ({circ}, 5 nmol; {triangleup}, 1 nmol) or vehicle ({square}) was administered icv to male Zucker obese (A) or lean (B) rats (n = 5/group). The increase in the plasma LH level ({Delta} plasma LH) is shown. Data are the mean ± SEM. *, P < 0.05; **, P < 0.01 (by Dunnett’s multiple test).

 
Hypothalamic GALP mRNA in db/db and ob/ob mice
Mouse GALP cDNA was cloned using a method that has been described previously (1) and is available in GenBank (accession no. AY163546). Real-time PCR analysis was performed with hypothalamic total RNA prepared from male db/db mice and lean littermates db/m+ (8 wk old, n = 6/group). The db/db mice exhibited an obese phenotype (38.8 ± 0.2 g body weight) compared with db/m+ mice (25.4 ± 0.2 g body weight). As shown in Fig. 5Go, hypothalamic GALP mRNA levels in db/db mice were reduced by 37% (P < 0.05) compared with those in their lean littermates.



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  		Figure 5. Expression level of hypothalamic GALP mRNA in db/db and ob/ob mice. GALP mRNA levels in db/db and db/m+ lean littermates (A; 8 wk old, males, n = 6), ob/ob and ob/m+ lean littermate (B; 8 wk old, males, n = 5 and 4, respectively), and leptin- or vehicle-administered ob/ob mice (C; 8 wk old, males, n = 4) were determined by real-time RT-PCR. *, P < 0.05; **, P < 0.01 (by t test). Bars represent the mean ± SEM.

 
Similar analysis was carried out with male ob/ob mice (8 wk old, n = 5 for ob/ob and n = 4 for ob/m+) that exhibited obesity due to leptin deficiency. The ob/ob mice showed a 41% decrease in hypothalamic GALP mRNA levels (P < 0.01) compared with their lean littermates (Fig. 5Go). In another experiment we injected leptin (3 mg/kg body weight) or vehicle sc into male ob/ob mice (8 wk old, n = 4/group). This injection was repeated six times, once every 12 h for 3 d under ad libitum-fed conditions. At the commencement of leptin administration, the average body weights were 54.4 ± 1.4 g (leptin-administered group) and 54.9 ± 1.1 g (vehicle-administered group). At the end of the experimental period (68 h), body weights were 49.8 ± 1.7 g (leptin-administered group) and 55.5 ± 0.7 g (vehicle-administered group). Hypothalamic GALP mRNA levels in leptin-treated mice were increased 1.6-fold (P < 0.05) compared with those in vehicle-treated mice (Fig. 5Go).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The ARC is one of the hypothalamic nuclei that express the highest levels of the leptin receptor isoform, Ob-Rb (19). NPY/agouti-related peptide (AGRP) and POMC/cocaine- and amphetamine-regulated transcript (CART) neurons are well characterized ARC neuronal populations that are involved in energy homeostasis under the regulation of leptin. Leptin suppresses the neural tone of NPY/AGRP neurons and oppositely enforces that of POMC/CART neurons by regulating the transcription levels of these neural peptides (20, 21, 22, 23). A recent report has provided direct evidence that leptin acutely depolarizes POMC neurons in the ARC (24). These findings provide a molecular basis for the anorectic activity of leptin. In addition to these two neuronal populations, GALP-expressing neurons were recently localized to the ARC (2, 3, 4, 5, 6, 7). A double-labeling immunohistochemical study has demonstrated that GALP neurons coexpress the leptin receptor and constitute a novel neuronal population(s) that is distinct from NPY/AGRP and POMC/CART neurons (6). More recently, however, detailed double-labeling studies have indicated that 3–12% of GALP neurons are double-labeled with an {alpha}MSH antibody (25). Leptin downstream regulation of GALP neurons in rats has been implicated by evidence from in situ hybridization studies in which it was shown that the number of GALP cRNA-labeled cells was increased by leptin administration (3).

In the present study, real-time PCR analysis confirmed quantitatively that hypothalamic GALP mRNA levels were increased by sc leptin treatment under fasting conditions. The changes in GALP mRNA levels were more remarkable than the changes in NPY mRNA levels. This is partly due to the fact that hypothalamic GALP-expressing neurons in rats constitute an almost unique neuronal population that is localized to the ARC (3, 4, 5, 6), whereas hypothalamic NPY-expressing neurons comprise several neuronal populations with different localizations and leptin responses. Jureus et al. (3) reported that Sprague Dawley rats that had been fasted for 48 h showed a (statistically insignificant) tendency to have a decreased number of GALP-cRNA-labeled cells in the ARC. However in this study it was difficult to detect a significant difference between the hypothalamic GALP expression levels of Wistar rats fed ad libitum and those that had been fasted for 48 h (data not shown) or 72 h. In contrast, the hypothalamic POMC mRNA levels decreased significantly in the same food-deprived rats. We speculate that GALP-expressing neurons tend to be more sensitive to increases in leptin concentration above baseline levels, but less sensitive to its decrease. Ahima and colleagues (26) postulated that POMC and CART mRNA levels in the ARC changes only unidirectionally under physiological conditions. They reported that these mRNA levels decreased under fasting conditions, but did not respond to increases in plasma leptin levels within the physiological range (1.5- to 2-fold increase) (26). On the other hand, NPY mRNA in the ARC changed bidirectionally, responding to both increases and decreases in leptin levels (26). Detailed studies are required to reveal whether GALP mRNA levels change in response to increases in leptin levels within the physiological range.

The homozygous Zucker (fa/fa) obese rat has an impaired leptin-signaling pathway because of the dysfunctional leptin receptor (27) and exhibits an obese phenotype. It has been shown that Zucker obese rats have higher NPY mRNA levels in the hypothalamus (28) and ARC (29) than Zucker lean rats. NPY peptide levels are also shown to be increased in the ARC, PVN, and dorsomedial nucleus of Zucker obese rats (30, 31). The food intake and body weight of Zucker obese rats were reduced by icv injection of NPY antisera (32). Thus, it is believed that up-regulation of NPY gene expression largely contributes to hyperphagia and obesity in the Zucker obese rat. On the other hand, POMC mRNA levels in the ARC and {alpha}MSH peptide levels in the PVN were shown to be almost half those in lean rats (29, 33). We subjected hypothalamic RNA preparations from Zucker obese rats (11 wk old) to real-time RT-PCR and reproduced the reported changes in NPY and POMC mRNA levels. Using the same RNA preparations, we then determined the hypothalamic GALP mRNA levels. Also, we determined hypothalamic ir-GALP levels using a newly established EIA method that specifically and sensitively detected endogenous ir-GALP. On the basis of HPLC analysis, this was suggested to be GALP-(1–60) itself or peptides very similar to it. The level of hypothalamic GALP mRNA in Zucker obese rats was decreased to half that in Zucker lean rats, on the average. Furthermore, the hypothalamic ir-GALP level in Zucker obese rats was decreased to 30% that in Zucker lean rats. Immunohistochemical analysis demonstrated a similar decrease in the number of GALP-expressing cells in Zucker obese rats, providing histological evidence for decreased production of GALP in the ARC. The level of ir-GALP in Zucker lean rats was lower than that in Wistar rats, although the level of GALP mRNA in Zucker lean rats was almost comparable to that in age-matched Wistar rats (11 wk old) or slightly higher than that in 8-wk-old Wistar rats (Fig. 1Go). This could be due to metabolic differences between different rat strains, although the underlying mechanism remains unclear.

This result obtained from the Zucker obese rat supports the hypothesis that GALP expression is positively regulated by leptin. However, we cannot exclude the possibility that the hyperinsulinemia of Zucker obese rats may also contribute to their decreased GALP expression. The expression level of NPY is known to increase in streptozotocin-treated rats and is reduced to normal levels by insulin treatment in the same animals (34). Therefore, the increased insulin level may decrease the hypothalamic GALP mRNA level in Zucker obese rats. The effect of insulin on GALP expression has yet to be determined.

Furthermore, we have shown that db/db mice, which inherit the leptin receptor mutation, have a lower level of hypothalamic GALP mRNA level than heterozygous db/m+ mice. Recently, Jureus et al. (7) reported a decreased number of GALP-expressing cells in genetically leptin-deficient ob/ob mice compared with control mice. They further indicated a compensative increase in hypothalamic GALP-expressing cells in ob/ob mice that had been rescued by icv administered leptin (7). We confirmed their result quantitatively using real-time RT-PCR method. These results in mouse models also support the present hypothesis of GALP as a leptin target. It should also be noted that the level of hypothalamic GALP mRNA in mice lay within a much lower range than that in rats.

Central administration of galanin is well known to stimulate feeding behavior (35, 36, 37, 38). It has recently been reported that icv administration of GALP also induces acute food intake in rats (12, 13). By analogy with NPY and {alpha}MSH, however, leptin-dependent up-regulation of GALP gene expression suggests that GALP is an anorexigenic peptide. Because of this discrepancy, the physiological relevance of its orexigenic activity remains controversial. On the other hand, it is reported that icv administration of leptin decreases galanin mRNA levels in the hypothalamus (39). Although galanin gene expression is reportedly unchanged under food-deprived conditions (40), it is known that central administration of insulin decreases galanin expression in the PVN (41). In comparison with Zucker lean rats, Zucker obese rats fed ad libitum had higher galanin concentrations in the PVN, where the galanin cell bodies are localized, and decreased galanin concentrations in the median eminence, where galanin-immunoreactive fibers are concentrated (42). As GALP is not established as a physiological ligand for galanin receptor GALR2, the possibility that the orexigenic activity is a pharmacological effect caused by cross-reaction with galanin receptors cannot be excluded. However, Wang et al. (43), using partially subtype-selective galanin fragment peptides, have suggested that the orexigenic activity of galanin is mediated by GALR1 or an unidentified galanin receptor and not by GALR2 or GALR3. Alternatively, if GALP is considered as a feeding stimulatory peptide, GALP is at least less involved in vigorous feeding behavior under energy-deficient conditions, because food-deprived rats showed no remarkable changes, other than a tendency for reduced GALP mRNA levels. Furthermore, GALP is not responsive to hyperphagia and obese phenotypes in genetically obese animals. Therefore, the question arises as to the physiological significance of feeding stimulatory activity under high leptin levels. As increasing leptin levels are linked closely to reproductive functions (discussed below), the feeding stimulatory roles of GALP, if any, may also act to maintain reproductive functions. Establishment of GALP-Tg and knockout mice will be crucial for elucidating the physiological significance of GALP neurons in the regulation of feeding behavior.

Leptin was initially thought to be an anorexigenic hormone conveying information about energy storage from adipocytes to the brain. However, several lines of evidence have demonstrated that one of the crucial roles of leptin is the regulation of reproductive functions via the hypothalamus-pituitary-gonadal axis. It has been reported that leptin treatment reverses the delayed onset of puberty in food-restricted mice (44, 45) and the reproductive abnormalities in ob/ob mice (46) and accelerates sexual maturation and puberty in normal mice (47, 48). Moreover, the leptin transgenic skinny mice were recently shown to have accelerated onset of puberty (49). Although the precise mechanisms by which leptin regulates reproductive functions are not fully elucidated, one important mechanism is the control of pulsatile GnRH secretion in the hypothalamus. The icv administration of leptin antibody caused a marked decrease in LH pulsatility (50), and leptin administration to male monkeys or ovariectomized female rats reversed the suppression of LH pulsatility during fasting (51, 52). Because of the relative lack of coexpression of GnRH and Ob-R in the hypothalamus (53), it is believed that the leptin signal is mediated indirectly. The NPY/ARGP and POMC/CART neurons are candidates mediating reproductive information about leptin. The icv injection of NPY is known to suppress the pulsatile release of LH in the rhesus monkey (54); however, administration of {alpha}MSH does not significantly change the level of LH (55, 56). It is argued that POMC neurons do not mediate reproductive information of leptin, as a melanocortin receptor antagonist, SHU9119, attenuated the effect of leptin on food intake, but did not alter the stimulatory effect of leptin on the reproductive axis (57). Recently, we postulated that GALP neurons are another candidate based on evidence of close apposition of GALP terminals with GnRH neurons in the MPA (6).

Furthermore, we previously reported that icv administration of GALP raised the plasma LH level in male Wistar rats (11). As the GnRH receptor antagonist Cetrorelix blocked this biological event, it is suggested that GALP activates GnRH neurons to induce the liberation of LH from the pituitary gland (11). In the present study, we found that male Zucker obese rats showed more sensitive responses to a low dose (1 nmol) of GALP, which did not elicit a significant response in Zucker lean rats. A high dose (5 nmol) of GALP raised LH levels in Zucker obese and lean rats to a similar extent, but this was smaller than the response reported previously in Wistar rats (11). The increased sensitivity to exogenous GALP may be the result of decreased production of hypothalamic GALP. If GALP is physiologically involved in the regulation of LH levels, and a possible redundant pathway does not compensate for the decreased production, the sensitivity to GALP should be increased. Also, it is suggested that the abundant and totally unchanged galanin in the hypothalamus of Zucker obese rats does not compensate for the decreased production of GALP. This is compatible with the previous finding that the LH-increasing activity was specific to GALP and was not found with galanin (11). The molecular basis of this increased sensitivity, however, remains unknown. Cettour-Rose and Rohner-Jeanrenaud (58) recently reported that the leptin-like effect of the {alpha}MSH agonist MTII was more marked in Zucker obese rats than in Zucker lean rats. They suggested that decreased production of {alpha}MSH in Zucker obese rats increases melanocortin receptor density and thus causes the greater responsiveness to MTII.

It is well known that female Zucker obese rats are infertile (59), displaying several reproductive abnormalities, such as delayed puberty, aberrant estrous cyclicity, and inadequate reproductive behavior (60, 61). The mechanisms by which these abnormalities are induced have not yet been elucidated, but they are believed to be linked to a dysfunction of the leptin-signaling pathway. One possible explanation has been ascribed to NPY (32). As NPY is known to suppress the pulsatile release of LH (54), overexpression of NPY in the hypothalamus of Zucker obese rats may cause infertility. Therefore, we hypothesize that the decreased expression level of GALP also contributes to infertility in Zucker obese rats. To confirm this, the effect of GALP on pulsatile LH release in female rats and the effect of chronic icv infusion of GALP in Zucker obese rats should be elucidated in future studies.

In conclusion, all the genetically obese animal models tested in the present study, the Zucker obese rat, the db/db mouse, and the ob/ob mouse, have lower levels of hypothalamic GALP mRNA than their lean littermates. These findings support the hypothesis that hypothalamic expression of GALP gene is up-regulated by leptin signals.


   Acknowledgments
 
We are grateful to Drs. Y. Fujisawa, Y. Sumino, and M. Fujino for their helpful discussions.


   Footnotes
 
Abbreviations: AGRP, Agouti-related peptide; ARC, arcuate nucleus; CART, cocaine- and amphetamine-regulated transcript; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; EIA, enzyme immunoassay; Fam, 6-carboxy-fluorescein; GALP, galanin-like peptide; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HRP, horseradish peroxidase; icv, intracerebroventricular; ir-, immunoreactivity; MPA, medial preoptic area; NPY, neuropeptide Y; POMC, proopiomelanocortin; PVN, paraventricular nucleus; Tamra, 6-carboxy- tetramethyl-rhodamine.

Received October 25, 2002.

Accepted for publication February 11, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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