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NEUROENDOCRINOLOGY |
Departments of Physiology and Biophysics (A.J., S.M.K., R.A.S.), Obstetrics and Gynecology (D.L., L.L.J., D.N.T., D.K.C., R.A.S.), and Zoology (R.A.S.), and Graduate Program in Neurobiology and Behavior (M.J.C.), University of Washington, Seattle, Washington 98195-7290
Address all correspondence and requests for reprints to: Dr. Robert A. Steiner, Department of Physiology and Biophysics, University of Washington, Box 357290, Seattle, Washington 98195-7290. E-mail: steiner{at}u.washington.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Galanin-like peptide (GALP) was recently isolated from pig hypothalamus, and its cDNA was cloned from the brain of the pig, rat, and human (12). GALP is a 60 amino acid peptide that is structurally related to galanin. Thirteen of GALP’s amino acid residues (9–21) are identical to the N-terminal portion (1–13) of galanin, which is required to activate galanin receptors. GALP recognizes both the GalR1 (IC50 = 4.3 nM) and GalR2 (IC50 = 0.24 nM) receptors with high affinity, with a modest preference for the GalR2 receptor subtype (12). In contrast to galanin’s wide distribution in the brain, GALP mRNA-expressing cells in the rat are restricted to the arcuate nucleus (Arc) of the hypothalamus, most notably in its medial and caudal aspects, plus the median eminence and infundibular stalk (13, 14). In the rat, GALP mRNA is also found in the posterior pituitary (13, 15) but not in dorsal root ganglion (16). Recently, Takatsu et al. (17) used antibodies directed to the N-terminal (1–10) part of GALP to map the distribution of GALP-containing nerve fibers in the rat brain. GALP immunostaining was observed in the parvocellular division of the paraventricular hypothalamic nucleus, preoptic area, bed nucleus of stria terminalis, and lateral septum (17). In the posterior pituitary, GALP mRNA has been shown to be dramatically up-regulated by salt loading and dehydration in pituicytes as well as in nerve terminals, suggesting that GALP may be involved in the regulation of vasopressin (and perhaps oxytocin) release (15).
Leptin is a satiety hormone secreted from adipocytes, which acts on the brain to regulate feeding behavior, metabolism and pituitary function (18). The obese (ob/ob) mouse has a mutation in the leptin gene that prevents production of functional leptin protein (19). The phenotype of the ob/ob mouse includes severe obesity, infertility, and insulin resistance, and administration of recombinant leptin to these animals quickly ameliorates these disorders (20, 21, 22, 23, 24). In the rat, GALP gene expression in the Arc is reduced by fasting and reversed by systemic administration of leptin, suggesting that GALP may be involved in mediating leptin’s effects on the regulation of energy balance or in the regulation of pituitary function (13). The finding that the vast majority of the GALP cells in the Arc coexpress the leptin receptor, Ob-Rb (17), lends further credence to the idea that the GALP gene is involved in these processes. The purpose of the present investigation was first to clone and map the distribution of GALP mRNA in the brain and pituitary of the mouse; second, to compare the pattern and magnitude of its expression in the leptin-deficient ob/ob mouse with that of wild-type controls; and third, to examine the effects of leptin delivered centrally on the expression of GALP mRNA in the mouse brain. Here, we report the sequence of a mouse GALP cDNA and demonstrate that leptin acts centrally to induce the expression of GALP mRNA in the Arc of the ob/ob mouse.
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Materials and Methods |
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Intracerebroventricular (ICV) injections
Male age-matched ob/ob (n = 12) and WT (n =
6) mice were housed in individual cages. Body weight and food intake
were monitored for 1 wk before the start of the experiments. Animals
were anesthetized for injection with isoflurane delivered by a
vaporizer (Veterinary Anesthesia Systems, Bend, OR). For
freehand ICV injections, a small hole was bored in the cranium at 1 mm
posterior to bregma and 0.5 mm lateral to bregma. Afterward, the
animals were allowed to recover for 4 d during which time the
animals were handled daily. Three experimental groups of animals
(n = 6 each) were given daily freehand ICV injections into the
lateral ventricle at 1600 h, as previously described
(25). One group comprised vehicle-treated WT mice, and the
other two groups comprised ob/ob mice that received either
vehicle or human recombinant leptin (0.02 nmol/d). Body weight and food
intake were measured each day after the injection. The animals were
killed after 7 d, 1 h after the last injection. The mice were
rapidly decapitated, and the brains were quickly removed and frozen on
dry ice.
Cloning of a partial mouse GALP cDNA
Four adult C57BL/6 mice were injected for 2 d, twice daily
(0900 and 1600 h) with 6 nmol recombinant human leptin (sc), to
up-regulate GALP gene expression. The animals were killed 1 h
after the last injection, and the hypothalami were removed and
immediately frozen on dry ice. Total RNA was extracted with a Totally
RNA kit (Ambion, Inc., Austin, TX). Reverse transcription
of mRNA was performed with M-MLV reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD), and oligo-dT primers at
42 C for 1 h. The enzyme was inactivated by heating at 92 C for 10
min.
PCR cloning was performed with the CloneAMP pAMP system (Life Technologies, Inc.) and deoxy UMP-containing primers designed from the rat GALP sequence, (forward primer: 5'-CAUCAUCAUCAUCCAAGCATCTGGTCCTCTTC-3'; reverse primer: 5'-CUACUACUACUATCTATGGCCTTCCACAGGTC-3'). A low-stringency PCR program with an initial melting temperature set at 95 C followed by 35 cycles at 94 C for 30 sec, 45 C for 30 sec, 72 C for 60 sec, and a final extension at 72 C for 10 min was used to obtain PCR product. PCR was performed with Taq DNA polymerase (Life Technologies, Inc.) in a total reaction volume of 50 µl. A single band was detected at the expected size of 205 bp on a 1% agarose gel. The PCR product was purified with a QIAquick gel extraction kit (QIAGEN, Valencia, CA). The purified cDNA fragment was then used as template in a second round of PCR under the same conditions. After gel purification, the amplified PCR product was cloned into the pAMP1 plasmid (Life Technologies, Inc.), which was transformed into JM-109 cells (Promega Corp., Madison, WI) and cultured overnight. Plasmids were isolated with a Plasmid Maxiprep kit (QIAGEN). The insert was sequenced and shown to be a 205-bp cDNA fragment sharing 95% sequence identity to rat GALP cDNA.1
In situ hybridization
Antisense and sense mouse GALP 33P-labeled
cRNA probes were transcribed from linearized pAMP plasmid containing
the mouse GALP cDNA insert with SP6 and T7 RNA polymerases
(Roche, Indianapolis, IN), respectively. Two separate
in situ hybridization assays for GALP mRNA were performed,
one for the analysis of GALP mRNA distribution in the brain of WT
animals and another for assessing the regulation of GALP mRNA by leptin
in the ob/ob mouse. The assays were performed on separate
sets of coronal sections (20 µm) of mouse brain, cut through the
hypothalamus on a cryostat and thaw mounted onto Superfrost Plus slides
(VWR Scientific, West Chester, PA). Sections were
collected in a 1:4 series from the diagonal band of Broca, caudally
through the mammillary bodies as described previously
(26). In brief, tissue sections were fixed, acetylated,
and delipidated. A hybridization solution containing denatured,
radiolabeled GALP cRNA (0.3 pmol/ml) and yeast tRNA (2 mg/ml) in
hybridization buffer was applied to the tissue (100 µl/slide). The
slides were coverslipped, placed in horizontal slide racks, and
incubated overnight in humid chambers at 55 C. The next day slides were
treated with RNase A (Sigma, St. Louis, MO) and washed
under conditions of increasing stringency with two final hot washes at
60 C. The tissue was dehydrated in ethanol and finally dipped in NTB-3
emulsion (Kodak, Rochester, NY). The slides were exposed
for 6 d, developed, and counterstained with cresyl violet
(Sigma).
Quantitative analysis
Slides were assigned a random three-letter code and read in
alphabetical order, with an automated image analysis system by an
operator unaware of the experimental group to which the animal
belonged. The total number of cells and silver grains/cell were
determined on nine anatomically matched sections taken through the
rostrocaudal extent of the Arc with a grain-counting program, as
previously described (27). Differences among groups were
assessed by ANOVA. When the ANOVA indicated significant differences,
Fisher’s post hoc test was used to determine whether individual groups
were significantly different from one another. Differences were
considered significant when P < 0.05.
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Results |
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Distribution of GALP mRNA in the brain of the mouse
The 205-bp GALP cDNA was used to transcribe sense and antisense
riboprobes for in situ hybridization. In the brain of the
mouse, as in the rat, GALP mRNA-expressing cells were restricted to the
Arc and median eminence (Fig. 1
);
however, the anatomical distribution of GALP mRNA-containing cells
within the Arc of the mouse was different from that of the rat. In the
rat, GALP cells are located medially and close to the third ventricle,
whereas in the mouse, GALP cells are found more laterally and ventrally
within this nucleus. Moreover, the rostral-caudal distribution of GALP
mRNA-containing cells is different between the two rodent species. In
the rat, the majority of the GALP mRNA-containing cells are found in
the caudal part of the Arc (13), and this is also true in
the mouse. However, a larger fraction of the total number of GALP
mRNA-expressing cells are found in the rostral portion of the Arc in
the mouse, compared with the rat. No significant signal was detected
with the sense control probe.
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). Food intake in WT animals was not
significantly different from the ob/ob + leptin group;
however, the WT animals ate significantly less than the
ob/ob + vehicle group (data not shown). Compared with WT
mice, leptin-deficient ob/ob mice expressed very low levels
of GALP mRNA in the hypothalamus, with the number of identifiable cells
expressing GALP mRNA being reduced by 70% in ob/ob mice,
compared with WT mice (P < 0.01) (Fig. 3). In the few identifiable GALP
mRNA-containing cells present in the ob/ob animals, the
number of grains/cell (reflecting the amount of mRNA expressed by each
individual cell) was 20% lower in the ob/ob mice, compared
with WT controls, although this difference was not statistically
significant (Fig. 3). The group of ob/ob mice injected with
leptin showed a nearly 5-fold induction in the number of identifiable
GALP mRNA-containing neurons in the Arc (P < 0.0003),
with 40% higher levels of GALP mRNA signal (grains/cell), compared
with vehicle-treated ob/ob mice (Figs. 3 and 4).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The cloned mouse GALP cDNA was used for generation of radiolabeled cRNA probes for in situ hybridization to map mRNA-expressing cells in the mouse hypothalamus. GALP mRNA-containing cells were found throughout the rostrocaudal extent of the Arc of the hypothalamus similar to the distribution observed in the rat (13, 14, 17). GALP mRNA-expressing cells were also found in the median eminence of the mouse; however, in this region there were fewer cells observed in the mouse, compared with the rat. Cells expressing GALP mRNA within the Arc of the mouse appeared to have a more lateral distribution than the rat. In this regard, the pattern of GALP mRNA distribution in the mouse Arc resembled that of galanin (30). In the rat, the location of GALP-expressing cells in the Arc is highly shifted toward the caudal part of the nucleus. GALP cells in the Arc of the mouse are spread more uniformly throughout the full extent of the nucleus, with the exception of the most rostral part, where very few GALP cells are found. These discrepancies could be attributed to differential organization of the arcuate nucleus between the rat and mouse or to GALP serving different physiological functions in the two species.
Our second objective was to examine whether GALP mRNA was regulated by leptin in the mouse and, if so, to determine whether the effect of leptin was attributable to its central (vs. peripheral) action. We have previously shown in the rat that GALP gene expression in the Arc is reduced by fasting and up-regulated in fasted animals given peripheral (sc) administration of leptin (13). This experiment left unanswered the question of whether the induction of GALP mRNA was owing to a direct effect of leptin in the brain or to secondary mechanisms triggered by leptin acting on peripheral targets. We hypothesized that, compared with WT mice, leptin-deficient ob/ob mice would have relatively reduced levels of GALP mRNA in the hypothalamus, and this indeed proved to be the case. We also postulated that if GALP cells in the Arc were direct targets for leptin in the brain, infusion of leptin into the lateral ventricles should induce GALP gene expression in ob/ob mice, which again proved to be the case, both in terms of cell number and message content per cell.
Although our results demonstrate that leptin acts on the mouse brain to induce GALP gene expression, we cannot be absolutely certain that leptin directly targets GALP neurons to mediate this effect. In the rat, it is clear that most GALP neurons (> 85%) in the Arc express the leptin receptor, lending some credence to the inference that in the mouse, leptin acts directly on GALP neurons to induce GALP gene expression (17); however, this remains to be proven. In this regard, it would also be of interest to determine whether leptin activates signaling mechanisms downstream of the leptin receptor in GALP neurons, such as Fos and SOCS-3, as has been demonstrated in POMC neurons (31, 32).
The inductive effect of leptin on GALP mRNA in the Arc of the
ob/ob mouse is similar to leptin’s effect on the mRNAs
coding for POMC and cocaine- and amphetamine-regulated transcript
(CART) (33, 34) and opposite to its effect on NPY and
agouti-related protein (35, 36). On the basis of these
observations, we might infer that the induction of GALP may play a
parallel role with POMC or CART in the mediation of leptin’s effect on
satiety and metabolism or in the regulation of pituitary function. In
the rat, GALP does not appear to be colocalized with 
-MSH, NPY,
agouti-related protein, somatostatin, or galanin, indicating that GALP
neurons in the Arc might represent a novel population of
leptin-responsive neurons, distinct from those previously characterized
(17). However, because GALP neurons in the mouse appear
not to be restricted to the periventricular region of the Arc (unlike
the rat), the possibility of GALP being coexpressed with one or more of
these peptides in the mouse cannot be excluded. Galanin neurons located
in the lateral part of the Arc, in contrast to GALP neurons, are
neither regulated by leptin in the ob/ob mouse nor do they
express the signaling form of the leptin receptor Ob-Rb, which would
argue that galanin neurons in the Arc do not directly participate in
leptin-mediated events in the brain (30).
In summary, we report the cloning of a partial cDNA for mouse GALP and the anatomical distribution of GALP mRNA-containing cells in the brain of the mouse. We have also demonstrated that GALP mRNA in the Arc is a target for central regulation by leptin in the ob/ob mouse. These findings suggest that GALP neurons in the Arc of the mouse represent a novel target for leptin signaling and mark this newly discovered peptide as a possible molecular link coupling leptin and its effects on satiety, metabolism, and pituitary function.
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Acknowledgments |
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Footnotes |
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Abbreviations: Arc, Arcuate nucleus; CART, cocaine- and amphetamine-regulated transcript; GALP, galanin-like peptide; ICV, intracerebroventricular; ob/ob, obese; WT, wild-type.
Received June 7, 2001.
Accepted for publication August 13, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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