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(Received for publication, April 3, 1997)
From the ABSTRACT OB (leptin) has been identified as a factor that
suppresses appetite and stimulates metabolism. Attention has focused on
the hypothalamus as its potential site of action, but OB could also act
on other brain regions. In addition, the paradox of high OB levels in
obese humans remains unresolved. Here we show in mice that both the
long and short form of the OB receptor are expressed not only in the
hypothalamus but also in the amygdala and pituitary. Recombinant murine
OB elicited the release of corticotropin-releasing factor from
superfused brain slice preparations containing hypothalamus or
amygdala. Because corticotropin-releasing factor inhibits appetite and
stimulates metabolism, it may be a key mediator of central OB effects.
Recombinant OB also induced pituitary release of adrenocorticotrophic hormone. Because adrenocorticotrophic hormone-induced elevation of
plasma glucocorticoid levels can inhibit corticotropin-releasing factor
release via negative feedback, the OB effects on pituitary adrenocorticotrophic hormone release may be pertinent to human obesity,
which combines increased plasma glucocorticoid levels with elevated
levels of OB. An imbalance between the effects of OB on
corticotropin-releasing factor release from the hypothalamus and on
adrenocorticotrophic hormone release from the pituitary could
contribute to obesity.
INTRODUCTION Obesity is a cause of serious health problems, and new research on
the ob gene, which encodes an adipose tissue-derived hormone (OB or leptin) that suppresses appetite and stimulates metabolism (1,
2), raises hopes for therapeutical intervention. Little is known about
the molecular factors mediating OB effects. Increasing evidence
supports a central site of action for OB. Infusion of OB into the
lateral ventricles of normal or OB-deficient
ob/ob mice decreases feeding (3). Although the
hypothalamus has been identified as a potential site of action of OB
(4), OB may also act on other regions. Short and long forms of the OB
receptor (OB-R)1 differing in the length of
their intracellular domain are formed by differential splicing, but
only the long OB-R form can activate janus kinase (JAK) and
lead to phosphorylation of signal transducers and activators of
transcription (STAT) proteins. The JAK/STAT pathway has been proposed
to mediate the effects of OB on body weight (5). The function of the
short OB-R form has not been identified yet, but this form could also
have an important role in mediating central effects of OB.
Although neuropeptide Y has been implicated as a potential central
mediator of OB effects (4), recent evidence indicates that additional
factors may be involved (6). OB-deficient ob/ob mice treated with OB show strong Fos protein immunoreactivity in the
paraventricular nucleus (PVN) (7). It is interesting in this context
that corticotropin-releasing factor (CRF) acting at the PVN inhibits
food intake and stimulates metabolic rate in genetically obese animals
and lean controls (8-10). Furthermore, conditions that increase
hypothalamic CRF production also diminish food intake (11-16), and
reduction in central CRF activity has been suggested to contribute to
the development of obesity (17, 18). This suggests a possible OB-CRF
interaction.
The development and maintenance of obesity is associated with profound
endocrine disturbances, including increased activity of the
hypothalamic-pituitary-adrenal (HPA) axis (19-22). In genetically obese animals, hyperphagia and excessive weight gain are eliminated by
adrenalectomy and are restored by treatment with glucocorticoids (23-25). Glucocorticoids inhibit afferent input to the PVN (26), and
the inhibitory actions of corticosteroids on hypothalamic CRF synthesis
and/or release may contribute to obesity.
The amygdala is involved in stress-related reactions (27) and in the
regulation of the HPA axis (28, 29). The amygdala contains high levels
of CRF (30), and CRF-containing fibers have been traced from the
amygdala to the lateral hypothalamus and may directly innervate
CRF-containing neurons within the PVN (31). As yet, no reports have
appeared on the expression of OB-R in the amygdala. If expressed, OB-R
might mediate CRF release by OB within the amygdala.
The OB receptor is a member of the extended cytokine receptor family
and resembles gp130, the common signal-transducing subunit of a group
of cytokine receptors that includes receptors for IL-6. Receptors for
IL-1, IL-2, and IL-6 have been demonstrated in the pituitary of several
species, and direct stimulation of pituitary ACTH release by these
cytokines has been reported (32). OB might also directly act at the
level of the pituitary to modulate ACTH release.
The aim of the present work was to determine whether OB-R mRNA is
expressed in the hypothalamus, amygdala, and pituitary and whether OB
can modulate the release of CRF from the hypothalamus or the amygdala
and of ACTH from the pituitary to identify potential central mediators
of OB effects.
MATERIALS AND METHODS C57BL/6J mice 8-12 weeks of age (Jackson
Laboratories, Bar Harbor, ME) were used in all experiments. All mice
were housed at no more than four per cage under conditions of constant
temperature (18 °C), light from 6.00 a.m. to 6.00 p.m.,
and access to food and water ad libitum. The mice were
killed by decapitation between 10:00 a.m. and 11:00 a.m. to avoid
circadian variation. The hypothalamus, amygdala, and pituitary were
dissected as described (33).
The tissues were snap-frozen in
liquid nitrogen and stored at To measure hypothalamic CRF and arginine
vasopressin (AVP) release and pituitary ACTH release, dissected tissues
were placed onto a Brinkmann tissue chopper, and 300-µm slices were
prepared. Slices representing one entire hypothalamus, amygdala, or
pituitary were placed into individual chambers and superfused using an
in vitro superfusion system (Brandel, Gaithersburg, MD)
(33). Basal release of CRF and AVP from hypothalamus stabilizes after
100 min (35), and release of ACTH from the pituitary stabilizes after
180 min (36). These times were chosen as the start of the first 15-min
basal period for all subsequent experiments to determine the effect of
OB on hypothalamic CRF and AVP and pituitary ACTH release. During the
sample collection period, fractions were collected at 15-min intervals,
placed on dry ice, and stored at All data are expressed as means ± S.E. For
comparisons of multiple means, analysis of variance was used, followed
by Tukey-Kramer posthoc test when appropriate. A probability value of
less than 0.05 was considered significant.
RESULTS AND DISCUSSION To determine if OB may act on other regions besides the
hypothalamus, we looked for expression of the short and long forms of
OB-R mRNA in the amygdala and pituitary, regions that are involved in the regulation of the hypothalamus (28, 39). Both forms of the OB-R
were constitutively expressed not only in the hypothalamus, but also in
the amygdala and pituitary of mice (Fig. 1).
To determine the role of CRF in the central actions of OB, the effect
of recombinant mouse OB on the release of CRF from the hypothalamus and
amygdala was investigated in superfused brain slice preparations.
Superfusion with purified recombinant OB significantly increased CRF
release from hypothalamic slices (Fig. 2A).
In contrast to CRF release, hypothalamic AVP release was not affected
by superfusion with OB at 100 pM, 1 nM, or 10 nM (data not shown). To ensure that the effect of OB on
hypothalamic CRF release was due specifically to OB and not to an
impurity in the recombinant OB preparation, tissues were superfused
with OB in the presence of neutralizing antibodies. Neutralizing OB
antibodies blocked the OB-induced hypothalamic CRF release (Fig.
2B), indicating that the effect on CRF release was indeed
caused by OB. These findings are consistent with the postulate that CRF
may function as a mediator of OB effects.
The above observations support a key role for CRF in OB biology and
confirm the hypothalamus as an important site of OB actions. In
addition, our study revealed that OB has specific effects on other
brain regions. OB stimulated CRF release also from the amygdala (Fig.
3). In combination with the expression of OB-R mRNA
in the amygdala (Fig. 1), this finding suggests that the amygdala may play a role in centrally mediated OB effects. The amygdala, which contains high levels of CRF, is involved in the expression of fear and
anxiety (40) and could mediate behavioral effects of OB.
The development and maintenance of obesity is associated with an
increased activity of the HPA axis (22). To determine if pituitary OB-R
might play a role in HPA axis activation by inducing ACTH release, the
effect of OB on ACTH release from the pituitary was assessed.
Superfusion with recombinant OB significantly increased ACTH release
from pituitary slices (Fig. 4A). This effect
of OB on ACTH release was specific, because it was blocked by anti-OB antibodies (Fig. 4B).
In humans, serum OB concentrations correlate positively with the
percentage of body fat (41, 42). The expression of the short and long
forms of the OB-R in the pituitary and the OB-induced ACTH release from
the pituitary identified here may play an important role in the
development and maintenance of obesity. OB-induced increases in ACTH
release could be directly responsible for the elevated glucocorticoid
levels often found in obesity (22). Glucocorticoid receptors are
colocalized in CRF cells in the PVN of the hypothalamus (26), and
glucocorticoids can inhibit hypothalamic CRF synthesis/release via
negative feedback (43). Thus, in the presence of severely elevated
plasma OB, as found in obese individuals, two major effects could
prevent OB from reducing body weight via enhanced CRF release (Fig.
5). First, because the pituitary is not shielded from
the systemic circulation by the blood-brain barrier, blood-derived OB
would enhance pituitary ACTH release, increase glucocorticoid levels
and, via negative feedback, decrease hypothalamic CRF release. Second,
direct stimulatory OB effects on hypothalamic CRF release depend on
efficient transport of OB across the blood-brain barrier, and there is
evidence that this process may become increasingly inefficient as
plasma OB levels rise above a critical level. A saturable transport
system for OB from blood to brain has been described (44), and the
ratio of cerebrospinal fluid to plasma OB levels shows also signs of saturability when plasma OB levels rise to levels seen in obesity (45,
46).
In addition to the data presented here, a number of other studies also
support the disturbed negative feedback circuitry proposed in Fig. 5.
In obese fa/fa rats with high corticosterone
levels, hypothalamic CRF content and portal secretion of CRF are
reduced, and adrenalectomy in these animals results in enhanced portal CRF secretion (17, 43). Adrenalectomy also eliminates hyperphagia and
excessive weight gain in obese fa/fa rats (23) as
well as in obese rats with ventromedial hypothalamic lesions (24) and ob/ob mice (25). Lastly, treatment of such
adrenalectomized models with glucocorticoids restores the obese
phenotype (23-25).
In conclusion, we have demonstrated expression of both the short and
long form of the OB-R in the hypothalamus, as well as in the amygdala
and pituitary. In addition, OB was shown to induce CRF release from the
hypothalamus and amygdala and ACTH release from the pituitary. These
data indicate that CRF and ACTH may function as key mediators of OB
effects and emphasize the need to consider the differential effects of
OB on multiple regions of the central nervous system in the design of
OB-targeted treatments for obesity.
FOOTNOTES ACKNOWLEDGEMENTS We thank Stephen Ordway and Gary Howard for
editorial assistance.
REFERENCES
Volume 272, Number 24,
Issue of June 13, 1997
pp. 15057-15060
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
COMMUNICATION:
§, and
Gladstone Molecular Neurobiology Program and
the Department of Neurology, University of California, San Francisco,
California 94141-9100 and the ¶ Department of Immunology, The
Scripps Research Institute, La Jolla, California 92037
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
70 °C until RNA was extracted, and
RNase protection assay was performed as described previously (34). OB-R
and L32 mRNA levels were determined by RNase protection assay using
antisense riboprobes for the short and long forms of the OB-R and
murine L32, respectively. cDNA templates for the short (1250-1474,
GenBankTM accession number U42467[GenBank]) and the long (3040-3396,
GenBankTM accession number U46135[GenBank]) OB-R probes were subcloned from a
mouse hypothalamus cDNA library.
70 °C until determination of the
concentration of CRF and ACTH in the media by radioimmunoassay (33,
35). OB recombinant protein was produced in and purified from an
Escherichia coli expression system. In brief, the coding
region (65-619, GenBankTM accession number U18812[GenBank]) was cloned by
reverse transcription polymerase chain reaction using total RNA from
C57BL/6J white adipose tissue as a template. The coding region was
subcloned in an expression vector (pETM1) (37) to express a His-tagged recombinant mouse OB protein. The recombinant OB was expressed, purified, and refolded following a previously described procedure (38).
Polyclonal antibodies were raised by immunizing a rabbit with the
recombinant mouse OB and tested for biological activity to reduce body
weight in ob/ob and control mice as described
elsewhere.2
Fig. 1.
Expression of the long
(OB-RL) (A) and short
(OB-RS) (B) form of OB-R mRNA in
mouse pituitary, hypothalamus, and amygdala. Because restraint
stress has been reported to suppress food intake (47), OB-R mRNA
expression before (C) and after (R) 1 h of
restraint stress was also examined. 1 h of restraint stress did
not alter OB-R mRNA levels in the pituitary, hypothalamus, or
amygdala. To determine if additional time was required to detect alterations in OB-R mRNA expression, mice were returned to their cages after restraint stress. After 2 h (2) (and up to 24 h, data not shown), OB-R mRNA expression was not
significantly affected either. The L32 signal was used as a control for
RNA content/loading. U, undigested radiolabeled probes (no
mRNA or RNase added). The other lanes contained the same riboprobes
plus brain RNA samples digested with RNase.
[View Larger Version of this Image (50K GIF file)]
Fig. 2.
A, effect of OB on CRF release from
mouse hypothalamus. After a 100-min preperfusion, a base-line sample
was collected for 15 min. Superfusion was then carried out with medium
containing recombinant mouse OB protein (concentrations as indicated)
for two 15-min periods (between 115 and 145 min). n = 3-7 mice/dose. B, effect of neutralizing anti-OB antibodies
on OB-induced CRF release from mouse hypothalamus. After a 100-min
preperfusion, samples were collected during six consecutive 15-min
superfusion periods. Between 100 and 115 min, a base-line sample was
collected. Between 115 and 220 min, superfusion was carried out with
medium containing either non-immune serum or anti-OB antibodies (1:167 dilution). Recombinant mouse OB was present during six consecutive 15-min superfusion periods (between 130 and 220 min). The neutralizing anti-OB antibodies significantly inhibited CRF release; they also blocked the effect of recombinant mouse OB on food intake and body
weight (data not shown). n = 3 mice (non-immune serum);
n = 9 mice (anti-OB antibodies). *, p < 0.05 versus basal; **, p < 0.01 versus basal.
[View Larger Version of this Image (26K GIF file)]
Fig. 3.
Effect of OB on CRF release from mouse
amygdala. After a 100-min preperfusion, a base-line sample was
collected for 15 min. Superfusion was then carried out with medium
containing recombinant mouse OB protein (concentrations as indicated)
for two 15-min periods (between 115 and 145 min). n = 3-7 mice/dose. *, p < 0.05 versus basal;
**, p < 0.01 versus basal.
[View Larger Version of this Image (27K GIF file)]
Fig. 4.
Effect of OB on ACTH release from mouse
pituitary (A) and inhibition of this effect by neutralizing
anti-OB antibodies (B). A, after a 180-min
preperfusion, a base-line sample was collected for 15 min. Superfusion
was then carried out with medium containing recombinant mouse OB
protein during four subsequent periods (between 195 and 255 min).
n = 6-12 mice/dose. B, after a 180-min
preperfusion, a base-line sample was collected for 15 min. Between 195 and 255 min, superfusion was carried out with medium containing anti-OB
antibodies (1:167 dilution). OB was present during three consecutive
15-min superfusion periods between 210 and 255 min. n = 4-12 mice/condition. *, p < 0.05 versus
basal; **, p < 0.01 versus basal.
[View Larger Version of this Image (21K GIF file)]
Fig. 5.
Potential neuroendocrine interactions
involved in OB effects and obesity (see text for further details).
The effect of CRF on adipose tissue might be mediated by the
sympathetic nervous system, whose activity is increased by central CRF
treatment (48). Note that this diagram focuses primarily on effects
immediately pertinent to the current study. Because obesity is likely
multifactorial in etiology, many additional factors and interactions
may be involved.
[View Larger Version of this Image (21K GIF file)]
§
To whom correspondence should be addressed: Gladstone Molecular
Neurobiology Program, P.O. Box 419100, San Francisco, CA 94141-9100. Tel.: 415-695-3835; Fax: 415-826-6541; E-mail:
1
The abbreviations used are: OB-R, OB receptor;
JAK, janus kinase; STAT, signal transducers and activators
of transcription; PVN, paraventricular nucleus; CRF,
corticotropin-releasing factor; AVP, arginine vasopressin; HPA,
hypothalamic-pituitary-adrenal; ACTH, adrenocorticotrophic hormone; IL,
interleukin.
2
S. Chen, Y. Xia, and L. Feng, manuscript in
preparation. This manuscript describes the bioactivity of recombinant
OB and neutralizing capacity of anti-OB antibodies.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.
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