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The Journal of Neuroscience, February 15, 2001, 21(4):1238-1246
Downregulation of Fasting-Induced cAMP Response
Element-Mediated Gene Induction by Leptin in Neuropeptide Y
Neurons of the Arcuate Nucleus
Masami
Shimizu-Albergine,
Danielle
L.
Ippolito, and
Joseph A.
Beavo
Department of Pharmacology, School of Medicine, University of
Washington, Seattle, Washington 98195
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ABSTRACT |
States of increased metabolic demand such as fasting modulate
hypothalamic neuropeptide gene expression and decrease circulating leptin levels. This study tested the hypotheses that fasting stimulates gene induction mediated by cAMP response element (CRE)-dependent increases in gene transcription and that fasting-induced decreases in
leptin can regulate this CRE-mediated gene induction. Using C57BL/6J
mice transgenic for a CRE-lacZ construct, an
immunocytochemical study showed that fasting activated reporter gene
expression in the hypothalamic arcuate nucleus (Arc) in a small subset
of neurons and increased phosphorylation of CRE binding protein. The
increase of  -galactosidase expression caused by fasting was
inhibited by a protein kinase A inhibitor, Rp-8-Br-cAMPS, when the
compound was microinjected into the medial basal hypothalamus, and
enhanced by intraperitoneal injection of selective phosphodiesterase
inhibitors. In situ hybridization studies showed that
neuropeptide Y (NPY) mRNA levels increased in the Arc during fasting,
whereas proopiomelanocortin (POMC) mRNA levels decreased. Double
labeling of mRNA and -galactosidase immunoreactivity in the fasted
brain indicated that the subpopulation of the neurons expressing
-galactosidase all produced NPY but not POMC. To study the possible
involvement of decreased circulating leptin during starvation on
CRE-mediated gene induction, leptin was administered intraperitoneally
to fasted mice. Leptin significantly attenuated both -galactosidase
expression and NPY gene expression stimulated by fasting, suggesting
that leptin inhibits fasting-stimulated NPY gene expression at least in
part through downregulation of CRE-mediated gene induction in the Arc.
Leptin-induced modification of CRE-mediated gene induction in the Arc
may play an essential role in the central regulation of feeding
behavior and energy expenditure.
Key words:
arcuate nucleus; cAMP; CRE; CREB; leptin; NPY; PKA; PDE
inhibitor; POMC
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INTRODUCTION |
During starvation, expression
of various hypothalamic neuronal genes is regulated to change
behavioral, hormonal, and metabolic responses leading to a state of
positive energy balance. Fasting has been shown to increase the
expression of the orexigenic peptide neuropeptide Y (NPY) and decrease
the expression of the anorexigenic peptide proopiomelanocortin (POMC)
in the arcuate nucleus (Arc) (Kalra et al., 1991 ; Bergendahl et al.,
1992; Kim et al., 1996). The molecular mechanisms of cellular
processing required to regulate gene expression during fasting,
however, are not understood. A role for cAMP response element
(CRE)-mediated gene induction in response to starvation was suggested
by a recent study showing that 48 hr of fasting in rats increased CRE
binding activity in hypothalamic nuclear extracts (Sheriff et al.,
1997). cAMP-dependent protein kinase A (PKA) plays a major role in
CRE-mediated gene induction (Habener et al., 1995). Interestingly,
central injection of a cAMP analog has been reported to increase NPY
protein levels in the Arc (Akabayashi et al., 1994). Several in
vitro studies have demonstrated that NPY gene expression is
activated by treatment with forskolin or cAMP analogs (Higuchi at al.,
1988; Magni and Barnea, 1992). Therefore, it is conceivable that the
CRE-mediated gene induction modulated by PKA may be involved in NPY
gene induction in the hypothalamus during starvation. To test this
hypothesis, we used mice transgenic for a CRE-reporter gene
(lacZ) construct developed by Impey et al. (1996). Using
these transgenic mice, we found that fasting stimulated CRE-mediated
gene induction in NPY-containing neurons and increased phosphorylation
of CRE binding protein (CREB) and NPY mRNA levels in the Arc.
It is well established that fasting decreases circulating insulin
and leptin, two well known anorexigenic signals (Ahima et al., 1996;
Ahren et al., 1997). These changes might be responsible for the
regulation of CRE-mediated gene expression because leptin receptors are
highly expressed in the Arc (Mercer et al., 1996; Elmquist et al.,
1998; Håkansson et al., 1998) and leptin downregulates NPY gene
expression and upregulates POMC gene expression (Schwartz et al.,
1996; Thornton et al., 1997; Wang et al., 1997; Mizuno et al.,
1998). The long leptin receptor isoform found specifically in the
hypothalamus bears homology to the signaling subunits of the cytokine
receptor family and can activate the Janus kinase (JAK) family, causing
rapid phosphorylation of signal transducers and activators of
transcription (STATs) (Baumann et al., 1996; Ghilardi et al., 1996).
However, the involvement of the JAK-STAT signaling pathway in
leptin-induced regulation of hypothalamic neuropeptide gene expression
has not been determined. This study was designed to investigate whether
leptin modulates CRE-mediated gene transcription induced during
fasting. We demonstrate here that leptin downregulates
fasting-stimulated CRE-mediated gene induction and NPY gene induction
in the Arc, providing evidence that leptin may regulate hypothalamic
gene expression by modifying CRE-mediated transactivation.
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Materials and Methods |
Animals. Mice containing an insertion of a
CRE-lacZ construct were obtained from Drs. S. Impey and D. Storm
(Impey et al., 1996). The construct contained six tandem CREs upstream
from a minimal Rous sarcoma virus promoter that drives the expression of -galactosidase. Physiological relevance of a reporter gene expression in CRE-lacZ mice has been demonstrated both in
vivo and in vitro (Impey et al., 1996; Obrietan et al.,
1999). Mice were bred by mating a male transgenic heterozygous animal
with a C57BL/6J wild-type female obtained from a commercial vendor and
were genotyped by PCR for lacZ. The mice were maintained on a 12 hr
light/dark schedule with lights on at 6:00 A.M. and unlimited access to
food and water. All animal use procedures were in strict accordance
with institutional guidelines for the Care and Use of Laboratory
Animals at the University of Washington (Seattle, WA). Male mice aged
10-14 weeks were assigned to control or fasting groups when their
weights reached 24.0-29.0 gm. At this time there were no significant
differences in body weights between the two groups. The mice were fed
ad libitum or fasted for 16-48 hr with free access to
water. To assess effects of phosphodiesterase (PDE) inhibitors, mice
were injected intraperitoneally with saline or either the PDE3-specific
inhibitor milrinone or the PDE4-specific inhibitor rolipram (1 mg/kg
body weight, three times during a 48 hr fast) (Calbiochem, La Jolla,
CA) during fasting. The dosages of the PDE inhibitors were determined
by previous animal studies (Wachtel, 1983; Sweet et al., 1988;
O'Donnell and Frith, 1999). For treatment with leptin, recombinant
murine leptin (PeproTech, Rocky Hill, NJ) was injected
intraperitoneally (2 mg/kg body weight, three times during a 48 hr
fast). The dosage of leptin used was based on previous studies in which
leptin (3-5
mg · kg 1 · d1)
was subchronically injected in mice (Ahima et al., 1996; Schwartz et
al., 1996; Thornton et al., 1997; Mizuno and Mobbs, 1999). For
experiments involving the injection into the medial basal hypothalamus
(the area above the Arc), a 24 gauge stainless steel guide cannula was
stereotaxically implanted unilaterally 0.3 mm above the dorsolateral
border of the Arc. The stereotaxic coordinates were 1.6 mm posterior to
the bregma, 0.4 mm lateral to the midsagittal sinus, and 5.3 mm ventral
to the surface of the skull. The cannulas were permanently anchored to
the skull with dental acrylic. To maintain patency, a 30 gauge
stainless steel obturator was inserted into the lumen of each cannula.
After animals were allowed at least 10 d of postoperative
recovery, they were injected with the Rp-isomer of 8-bromo-adenosine
3', 5'-cyclic monophosphorothioate (Rp-8-Br-cAMPS; 4 nmol dissolved in
0.3 µl of saline) (Calbiochem) or saline and fasted for 48 hr. For
intracerebroventricular injection of leptin, the stereotaxic
coordinates were 0.5 mm posterior to the bregma, 1.6 mm lateral to the
midsagittal sinus, and 2.4 mm ventral to the surface of the skull.
Murine leptin (2 µg) was injected three times during a 48 hr fast.
Tissue preparation. Animals destined for immunocytochemistry
were anesthetized with phenobarbital and perfused with heparin PBS and
4% paraformaldehyde in 0.1 M phosphate buffer,
pH 7.4. After perfusion, brains were removed from the skull, and the
posterior cerebrum was excised. The tissues were post-fixed overnight
in the same 4% paraformaldehyde fixative and then cryoprotected by incubating with consecutive concentrations of sucrose (10, 20, and
30%) in PBS buffer. All tissues were then frozen in OCT compound and
stored at  70°C until cryostat sectioning at 20°C. For in situ hybridization, animals were decapitated, and the posterior cerebrum was rapidly placed in 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and fixed for 24 hr
at 4°C. The tissues were then cryoprotected in sucrose buffer as
described above.
Immunocytochemistry. A mouse brain atlas (Franklin and
Paxinos, 1996) was used as a guide to match coronal brain sections across animals. The medial Arc area was anatomically distinguished by
the appearance of median eminence and the elongation of the third
ventricle (bregma 1.3 mm to 2.4 mm). Each brain region was
sectioned into a series of 7-10 coronal sections containing the Arc (a
30-µm-thick section from every 80-100 µm). Free-floating sections
were preincubated in PBS containing 5% goat serum, 1 mg/ml BSA, and
0.05% Triton X-100 for 1 hr and incubated with anti--galactosidase
antibody (1:500; 5'Prime 3'Prime, Boulder, CO) or anti-phosphorylated
CREB antibody (1:500; New England BioLabs, Beverly, MA) in PBS
containing 1% goat serum, 1 mg/ml BSA, and 0.05% Triton X-100
overnight at 4°C under continuous gentle agitation on a rotary
shaker. All PBS solutions for labeling of phosphorylated CREB contained
NaF (1 mM). For detection of -galactosidase
immunoreactivity (IR), the sections were washed in PBS containing
0.05% Tween (three times for 20 min each), then incubated in goat
anti-rabbit IgG conjugated to Alexa-Fluor 488 (1:500; Molecular
Probes, Eugene, OR) for 2 hr at room temperature. After additional
washes in PBS, the sections were counterstained with propidium iodide.
For amplification of phospho-CREB-IR, the sections were incubated with
a goat anti-rabbit IgG biotin-conjugated antibody (1:400; Vector
Laboratories, Burlingame, CA) for 2 hr at room temperature, and
antigen-antibody reactions were visualized using Vectastain ABC
(Vector). Control and treatment groups were analyzed together under the
same experimental conditions.
In situ hybridization.
35S-Labeled cRNA probes for NPY mRNA (a
527 bp fragment encoding entire mouse NPY subcloned from mouse EST
385331) and POMC mRNA (a 377 bp fragment encoding part of mouse POMC
exon 3 subcloned from mouse EST 388641) were synthesized by in
vitro transcription with T7 or SP6 polymerase (Stratagene, La
Jolla, CA). A series of 8-10 coronal sections (30 µm thick) containing the Arc (bregma 1.3 mm to 2.4 mm) were cut through each
brain as described above and saved in ice-cold PBS, pH 7.4. Free-floating sections were acetylated with tetraethylammonium and
acetic anhydrate, prehybridized for 3 hr at 60°C, and hybridized for
16 hr at 60°C using a probe concentration of 0.1 ng · µl1 · Kb1
in the hybridization buffer (consisting of 50% deionized formamide, 10% dextran sulfate, 0.3 M NaCl, 1× Denhardt's
solution, 100 mM DTT, 1 mM
EDTA, 1 mg/ml yeast tRNA, 100 µg/ml synthetic polyA RNA, and 10 mM Tris HCl, pH 8.0). The sections were treated
with RNaseA at 37°C for 30 min and washed with 50% formamide
containing 2× SSC at 60°C for 30 min and 0.1× SSC at 60°C for 1 hr. Then the sections were mounted on slides, dried, and exposed to
Kodak BioMax MR (Eastman Kodak, Rochester, NY) for 8-20 hr.
Hybridization with sense probes for both NPY and POMC was included to
confirm the specificity of the procedure and verify that background was at a negligible level.
Combination of immunocytochemistry with in situ
hybridization. Free-floating sections from the fasted mice were
first processed for immunocytochemical detection of -galactosidase
using the ABC method described above, except that all solutions were
made with diethylpyrocarbonate-treated water. For visualization, the chromagen 3,3'-diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide were used. Acetylation, prehybridization, and hybridization were performed as described above. The sections were mounted, dried,
and dipped in NTB2 nuclear emulsion (Kodak). After a 2 d exposure,
the slides were developed in D19 developer for 4 min at 15°C and
fixed in rapid fixer for 5 min.
Quantification of images. Analysis for -galactosidase-IR
was performed with MetaMorph software by measuring immunofluorescence signals from images taken on a Bio-Rad confocal microscope. Before a
measurement was made, the background fluorescence intensity of each
image was determined from the gray scale histogram ranging from 0 (black) to 255 (white). This value usually ranged from 63 to 73. In
most cases, the area of -galactosidase-IR within the Arc was
thresholded by the background value and marked with a red thresholding
overlay as a visual indicator of the thresholded areas. Each pixel
exceeding this threshold was counted, and the results were expressed as
a percentage of the total pixels in the defined Arc area. The total Arc
area was anatomically defined by propidium iodide counterstaining. In a
few cases, the gray scale value of each thresholded pixel was
determined and expressed as a percentage of the total gray scale pixel
value in the defined Arc area. Essentially the same results were
obtained. Phospho-CREB-IR images were taken by a positive 35 mm film
and converted to computerized images. Percentages of threshold area
were measured by MetaMorph software. Both -galactosidase-IR and
phospho-CREB-IR induced by fasting tended to accumulate more in the
medial-caudal region of the Arc (bregma 1.8 mm to 2.4 mm) than the
rostral-medial region (bregma 1.3 mm to 1.8 mm). The mean
percentage threshold area for each animal, therefore, was obtained by
computing the percentage threshold area of four to seven sections that
showed a small variation (<20%) within the Arc collected in the
experiment (bregma 1.3 mm to 2.4 mm). These means were used to
calculate the overall mean ± SEM for each group. Significant
differences were determined using a two-tailed unpaired Student's
t test or the Wilcoxon-Mann-Whitney test. For analysis of
NPY and POMC mRNA levels, x-ray film exposures were captured to a
computer using a CCD camera (Sierra Scientific, Sunnyvale, CA), and
relative levels of mRNA were measured by NIH image software. NPY and
POMC mRNA levels were assessed by determining the total pixel area occupied on the film image by the hybridization signal from labeled cells within the Arc area. Pixel number in each animal was averaged from 8-10 sections (bregma 1.3 mm to 2.4 mm), and these averaged numbers were used to calculate the overall mean ± SEM for each group. NPY mRNA signals tended to concentrate in the medial-caudal region of the Arc, whereas POMC mRNA signals were relatively highly distributed in the rostral-medial region of the Arc. The averaged number, therefore, provided the representative value of the mRNA level
of each animal in the same Arc area rather than its maximal number.
Significant differences between treatment groups were determined using
either a two-tailed unpaired Student's t test or one-way
ANOVA, followed by Dunnett's multiple comparison test. A p
value of <0.05 was considered significant. To analyze colocalization of -galactosidase-IR and NPY or POMC mRNA signals, NPY- or
POMC-containing neurons were identified as a grain cluster if they
exceeded background levels determined by scanning each section with
dark-field optics. Under high-power, bright-field optics, if the grain
cluster (>10 grains per cell) was verified as associating with DAB
staining (-galactosidase-IR), this cell was counted as a
-galactosidase-expressing and NPY- or POMC-containing neuron. The
mean number of both -galactosidase-IR and mRNA signal-expressing
cells was computed for each animal (four to five sections of the
intermediate aspect of the Arc of each animal), and these means were
used to calculate the overall mean ± SEM for each group.
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RESULTS |
Activation of CRE-mediated gene induction by fasting in the
mouse hypothalamus
In the fed transgenic mice, the expression of the CRE reporter
gene, -galactosidase, was negligible in the hypothalamic area (Fig.
1A). After a 48 hr
fast, there was a significant increase in the number of positively
labeled cells specifically in the Arc, and some labeled cells were
observed in the dorsomedial hypothalamic nucleus (DMH) (Fig.
1B,C). A similar -galactosidase
expression pattern in the hypothalamus under fasting condition was seen
in two independent breeder lines of the transgenic mice. Levels of CREB
phosphorylation, the putative event preceding CRE-mediated gene
expression, were also determined in the fasted animals by using a
specific antibody for phosphorylated CREB (Fig.
1D-G). Fasting increased the intensity of
phospho-CREB-IR in the area of the Arc (Fig.
1F,G). It is important to note that
there was no difference in phospho-CREB-IR between control and fasting
in the ventromedial hypothalamic nucleus (VMH), an adjacent area to the
Arc, suggesting that the increased phosphorylation of CREB observed in
the Arc is not artificial but a significant phenomenon. There was no
change in the levels of phospho-CREB-IR in the DMH despite an
increase in -galactosidase expression caused by fasting in some
cells. Quantification of both -galactosidase-IR and phospho-CREB-IR of the Arc area indicated that the enhancement of the signals was
dependent on the duration of fasting (16-48 hr). -Galactosidase-IR showed a maximal increase under 48 hr fasting, whereas the phospho-CREB level was higher after 24 hr of fasting compared with 48 hr (Fig. 1H). It is probable that fasting activated
CRE-mediated gene induction by increasing phosphorylation of CREB in
the Arc.
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Figure 1.
Immunocytochemical demonstration of
fasting-induced CRE-mediated gene induction (A-C) and
CREB phosphorylation (D-G) in the hypothalamus of CRE
transgenic mice. Representative coronal sections through the
hypothalamic region containing the arcuate nucleus
(Arc) are shown. -Galactosidase-IR in the Arc and the
DMH were induced by fasting for 48 hr (B,
C). Control animals fed ad libitum showed
little or no -galactosidase-IR (A). The
distance posterior to bregma, according to the mouse brain map
described by Franklin and Paxinos (1996), is indicated in each coronal
section. Fasting for 48 hr increased CREB phosphorylation in the
Arc (E, G). Control animals fed ad
libitum showed only a weak basal phospho-CREB-IR in the Arc
(D, F). F and
G represent a higher magnification of phospho-CREB
immunostaining in fed and fasted animals, respectively.
Fasting-induced changes in -galactosidase-IR and
phospho-CREB-IR were quantified in the sections of the hypothalamic
region containing the Arc of mice fasted for 16-48 hr as described in
Materials and Methods (H). The intensity
of -galactosidase-IR and phospho-CREB-IR for each fasting interval
is plotted as the mean (±SEM; n = 3-7 animals per
group) percentage of the maximal number observed for
-galactosidase-IR (48 hr) and phospho-CREB-IR (24 hr), respectively.
3V, Third ventricle; ME, median eminence;
DMH, dorsomedial hypothalamic nucleus;
VMH, ventromedial hypothalamic nucleus.
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To determine whether PKA plays a significant role in fasting-induced
CRE-mediated gene induction and CREB phosphorylation, the
membrane-permeable PKA inhibitor Rp-8-Br-cAMPS (4 nmol/0.3 µl) was
injected into one side of the medial basal hypothalamus with the
contralateral side serving as a control. Rp-8-Br-cAMPS significantly
inhibited fasting-induced -galactosidase expression by 74% on the
injected side of the Arc compared with the uninjected contralateral
side (Fig.
2A,C).
Saline had no effect on -galactosidase-IR in the injected side of
the Arc (Fig. 2B,C) (102% of the
uninjected side), suggesting that the inhibitory effect of
Rp-8-Br-cAMPS did not result from a damage of surrounding tissue by
cannulation. Rp-8 Br-cAMPS also reduced the phospho-CREB-IR increased
by fasting as compared with the uninjected side (Fig.
2D). Saline injection had no effect on the
phospho-CREB-IR (Fig. 2E). It remains a possibility, however, that PKA inhibition in other hypothalamic regions may affect
fasting-induced effects in the Arc because the drug is expected to
diffuse to an area surrounding the Arc.
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Figure 2.
Effect of PKA inhibitor on fasting-induced
CRE-mediated gene induction (A) and
phosphorylation of CREB (D). A stainless steel
guide cannula was stereotaxically implanted unilaterally in the medial
basal hypothalamus (0.3 mm above the dorsolateral border of the Arc; an
asterisk indicates the hemisphere that was injected).
Mice were injected with Rp-8-Br-cAMPS (4 nmol dissolved in 0.3 µl of
saline) (A, D) or saline
(B, E) and fasted for 48 hr. PKA
inhibition attenuated fasting-induced -galactosidase expression on
the injection side of the Arc relative to the uninjected side
(A). The intensity of -galactosidase-IR on the
microinjected side is represented as a percentage of the mean (±SEM;
n = 4 animals per group) -galactosidase-IR on
the uninjected side (C). Rp-8-Br-cAMPS also
attenuated the fasting-induced CREB phosphorylation on the injection
side (D). Saline injection had no effect on the
levels of CREB phosphorylation (E).
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Moreover, we observed that intraperitoneal injection of PDE inhibitors
milrinone (1 mg/kg, three times during a 48 hr fast) or rolipram (1 mg/kg, three times during a 48 hr fast) enhanced the fasting-induced
-galactosidase expression in the Arc by 53 ± 9% (mean ± SEM, n = 4 animals) and 65 ± 11%
(n = 4), respectively, relative to intraperitoneal
injection of saline (Fig. 3D).
Figure 3A-C shows typical images of
-galactosidase-IR in the Arc treated with saline, milrinone, or
rolipram. The treatment of fed mice with the PDE inhibitors did not
cause any significant changes in the -galactosidase-IR at the dosage
used.
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Figure 3.
PDE inhibitors increased -galactosidase and NPY
mRNA expression in the Arc of the fasted mice. Mice were injected
intraperitoneally with milrinone (MIL), rolipram
(RPM) (1 mg/kg body weight, three times during a
48 hr fast), or saline (sal). PDE inhibitors
enhanced fasting-induced CRE-mediated gene expression in the Arc
(A-C). Levels of -galactosidase-IR in milrinone- and
rolipram-treated groups were increased by 53 ± 9% (mean ± SEM; n = 4 animals) and 65 ± 11%
(n = 4), respectively, relative to saline treatment
(D). Neither milrinone nor rolipram had any
effects on -galactosidase-IR in mice fed ad libitum.
E represents the effects of PDE inhibitors on NPY and
POMC mRNA levels in fed and fasted animals. Animals were treated as
described above. In situ hybridization was performed
using 35S-labeled cRNA probes for NPY and POMC and analyzed
as described in Materials and Methods. Each value shows the mean
(±SEM; n = 3 animals per group) number of pixels
of 35S-labeled area. The data represent a typical result
among three repeated experiments. *p < 0.05, **p < 0.01.
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Changes in Arc NPY gene expression observed during fasting and
effects of PDE inhibition
Activation of Arc NPY neurons has been implicated as a strong
stimulator of feeding. It has been reported consistently that a state
of negative energy balance, such as fasting, upregulates NPY gene
expression in the Arc, whereas it downregulates POMC gene expression
(Bergendahl et al., 1992; Ahima et al., 1996; Mizuno et al., 1998).
Consistent with these findings, we observed an increase in mRNA levels
of NPY and a decrease in mRNA levels of POMC in the Arc of mice fasted
for 48 hr (Fig. 3E). If the stimulation of NPY gene
expression by fasting requires CRE-mediated gene transactivation
regulated by the cAMP-PKA signaling pathway, it was expected that PDE
inhibitors may modify the NPY mRNA levels during fasting. To test this
hypothesis, the mice were injected intraperitoneally with milronine (1 mg/kg, three times during a 48 hr fast) or rolipram (1 mg/kg, three
times during a 48 hr fast) during 48 hr fasting, and the NPY and POMC
mRNA levels were analyzed. Consistent with our hypothesis, both PDE
inhibitors significantly enhanced the fasting-induced increases in mRNA
for NPY but not for POMC (Fig. 3E). Treatment with the PDE
inhibitors had no effect on either NPY or POMC mRNA levels in fed animals.
CRE-mediated gene induction in NPY neurons by fasting
Fasting increased CRE-mediated gene induction and NPY mRNA levels
in the Arc. Both changes were amplified by treatment with PDE
inhibitors. A site resembling a CRE has been shown to exist on the
promoter region of the rat NPY gene (Larhammar et al., 1987; Higuchi et
al., 1988). Thus, fasting-induced CREB phosphorylation could
conceivably enhance NPY gene levels through CRE transactivation in the
Arc. To test this possibility, brain sections of fasted mice were
double-labeled for NPY mRNA and -galactosidase-IR (Fig. 4A-D).
Figure 4E depicts the number of cells expressing
-galactosidase in one side of the Arc that were colabeled with NPY
or POMC radiolabeled cRNA probes. Ninety-seven percent of
-galactosidase-expressing cells colocalized with NPY mRNA signals
(Fig. 4A,B), whereas
-galactosidase-expressing cells did not contain POMC mRNA signals
(Fig. 4C,D). The same analysis in
milrinone-treated animals under fasting showed that enhancement of
fasting-induced -galactosidase expression by the PDE inhibitor
occurred in NPY neurons but not POMC neurons (data not shown).
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Figure 4.
Fasting activates Arc NPY neurons.
Photomicrographs of coronal Arc sections of mice fasted for 48 hr were
treated to detect -galactosidase-IR (brown staining)
and either NPY mRNA (A, B) or POMC mRNA (C,
D) signals detected by autoradiographic grains. High-power
magnification photographs (B, D) show
colocalization of -galactosidase-IR and NPY mRNA (B,
arrows) but not POMC mRNA (D,
arrowhead). Graph illustrates the mean
(±SEM; n = 4 animals per group) numbers of cells
on one side of the Arc in which -galactosidase expression
colocalized with either NPY mRNA or POMC mRNA in mice fed ad
libitum or fasted for 48 hr (E). The
total number of -galactosidase-expressing cells on one side of the
Arc is 61 ± 6 (mean ± SEM; n = 4 animals) and 2 ± 1 (n = 4 animals) in fasted
and fed mice, respectively.
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Effects of leptin on fasting-induced increases of CRE-mediated gene
induction and NPY mRNA expression
Either central or peripheral administration of leptin has been
reported to downregulate the expression of NPY genes under starvation
(Wang et al., 1997; Mizuno and Mobbs, 1999). Immunocytochemical studies
have shown that leptin receptors were localized in NPY and POMC neurons
in the Arc (Mercer et al., 1996; Cheung et al., 1997; Baskin et al.,
1999; Horvath et al., 1999). Therefore, we hypothesized that leptin
might regulate NPY levels by modulating intracellular signaling to CRE.
To assess this possibility, mice were injected intraperitoneally with
leptin (2 mg/kg, three times during a 42 hr fast), and
-galactosidase expression (Fig.
5A-D), and NPY
mRNA levels (Fig. 5E-H) of the Arc were
qualitatively and quantitatively assessed. Peripheral administration of
leptin attenuated the fasting-induced increases of -galactosidase
expression (Fig. 5D,I) and
NPY mRNA levels (Fig. 5H,J).
In fed mice, leptin did not have significant effects on either
-galactosidase expression or NPY mRNA levels (Fig.
5B,F).
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Figure 5.
Leptin downregulates fasting-induced CRE-mediated
gene expression and NPY gene induction. Expression of -galactosidase
(A-D) and NPY mRNA (E-H) in the
Arc in fed or fasted mice treated with leptin (2 mg/kg body weight,
i.p., three times during a 42 hr fast) (B,
D, F, H) or saline
(A, C, E,
G). Mean intensity (±SEM; n = 8 animals for each treatment group) of -galactosidase-IR in the Arc in
fasted mice treated with either leptin or saline is plotted as a
percentage of saline-treated controls (I).
Intensity of autoradiography is plotted as the number of pixels
(mean ± SEM; n = 3 animals for each treatment
group) labeled for NPY mRNA in the Arc of mice fed or fasted (42 hr)
and treated with either saline or leptin
(J). Data represent a typical result among
four repeated experiments. **p < 0.01 relative to
saline-treated controls.
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To ascertain whether the effect of leptin on fasting-induced
CRE-mediated gene induction was mediated by hypothalamic leptin receptors and not peripheral effects mediated by leptin, (e.g., effects
on insulin secretion and fatty acid metabolism), leptin (2 µg,
i.c.v., three times during a 48 hr fast) was centrally administered
during fasting. Central administration of leptin also inhibited
fasting-induced CRE-mediated gene induction in the Arc [78 ± 6%
mean inhibition of saline-injected control, (±SEM), four animals per
treatment group], suggesting that leptin-induced attenuation of
fasting-induced CRE-mediated gene induction resulted from stimulation
of hypothalamic leptin receptors. These data support a role for leptin
in negative regulation of NPY gene expression as shown in previous
reports and suggest that at least part of the mechanism of this effect
is by downregulation of CRE-mediated gene induction stimulated by
fasting in the Arc.
| |
DISCUSSION |
Our results show that fasting increases phosphorylation of CREB
and stimulates CRE-mediated gene induction in the Arc. CRE-mediated gene expression induced by fasting was decreased by inhibition of PKA
and enhanced by PDE inhibitors, suggesting that the cAMP-PKA signaling
pathway is required in CRE-mediated gene expression during fasting. Our
observation that fasting increased NPY mRNA expression in the Arc is
consistent with previous reports showing that fasting augmented NPY
gene expression in the Arc and increased NPY release to the
paraventricular nucleus (PVN) (Kalra et al., 1991; Marks et al.,
1992; Mizuno et al., 1998). The double-labeling experiments (Fig. 4)
provided evidence that NPY neurons, but not POMC neurons, were
activated during fasting and supported a possible role for CRE in
stimulation of Arc NPY gene expression. Indeed, a site resembling a CRE
has been shown to exist on the 5'-flanking region of the rat NPY gene
(Larhammar et al., 1987; Higuchi et al., 1988). Several in
vitro studies have also shown that NPY gene expression was
activated by treatment with forskolin or cAMP analogs in PC12 cells,
N18TG-2 neuroblastoma cells, and rat brain dissociated cells (Higuchi
at al., 1988; Magni and Barnea, 1992). Moreover, an in vivo
study recently reported that NPY protein levels were significantly
increased in the Arc and the PVN when rats were
intracerebroventricularly injected with dibutyryl-cAMP (Akabayashi et
al., 1994). Coupling these reports with the results of the present
study, we conclude that CRE-mediated gene induction regulated by PKA is
likely to be involved in increasing NPY gene expression in the Arc
during starvation.
Leptin has been implicated in regulating feeding and energy expenditure
(Campfield et al., 1995; Halaas et al., 1995, 1997; Ahima et al.,
1996). During fasting, circulating leptin levels are markedly
decreased, suggesting that decreased leptin may be interpreted at the
level of the Arc as a cue to enhance CRE-mediated gene induction. The
idea that NPY neurons may be one of the targets of leptin action has
been supported by studies in which genetically obese ob/ob
and db/db mice exhibited increased NPY mRNA in the Arc and
NPY levels in various hypothalamic sites (Stephens et al., 1995;
Schwartz et al., 1996; Woods et al., 1998). Furthermore, leptin
receptors are expressed in NPY neurons in the Arc (Mercer et al., 1996;
Baskin et al., 1999; Horvath et al., 1999). To examine whether
CRE-mediated gene induction and NPY mRNA gene expression stimulated by
fasting are a result of decreased circulating leptin levels, we
administered leptin to fasted animals. The data showed that exogenously
administered leptin attenuated both fasting-induced increases of
-galactosidase expression and NPY mRNA expression, suggesting that
leptin modulates NPY gene levels by downregulation of CRE-mediated
transactivation. However, because leptin did not completely counteract
the fasting-induced effects, other physiological changes such as
decreased insulin and/or glucose levels also might be involved in the
regulation of gene induction during starvation.
Current evidence has strongly implicated increased NPY activity in the
Arc as a key central mediator of hunger. Central administration of NPY
stimulated feeding in both fasted and satiated animals (Clark et al.,
1985), whereas immunoneutralization of NPY decreased food intake in
rats (Dube et al., 1994) and selective antagonists of Y1 and Y5
receptors, which are highly expressed in the brain attenuated feeding
(Kanatani et al., 1996; Schaffhauser et al., 1997). These support the
idea that leptin plays a key physiological role in controlling food
intake by regulating the NPY gene. However, NPY neurons contain
agouti-related transcripts that also produce orexigenic effects by
inhibiting melanocortin receptors (Ollmann et al., 1997; Shutter et
al., 1997; Broberger et al., 1998: Hahn et al., 1998), and the mRNA
levels of this transcript are increased under starvation and decreased
by leptin (Mizuno and Mobbs, 1999). Therefore, it remains a possibility
that agouti-related transcripts or another unknown molecule expressed
in NPY neurons, or both, also might be regulated by CRE under
starvation and may be targets of leptin.
What is the basis of leptin-induced downregulation of CRE-mediated gene
induction? Sheriff et al. (1997) reported that fasting for 48 hr
increased phospho-CREB levels in nuclear extracts from the rat
hypothalamus, an effect reversed by refeeding. These results suggest
that decreased CREB phosphorylation and increased CREB dephosphorylation, or both, may negatively modulate gene
expression responsible for the initiation of feeding. One of the
possible cellular mechanisms of the negative regulation of leptin on
CRE-mediated gene induction might be an attenuation of CREB
phosphorylation. We have reported previously that leptin can decrease
cAMP levels by the activation of a specific PDE isoform, PDE3B, thereby
inhibiting insulin secretion stimulated by glucagon-like peptide-1 in
pancreatic cells (Zhao et al., 1998) and inducing insulin-like
signaling in primary hepatocytes through activation of
phosphatidylinositol 3-kinase and protein kinase B (Zhao et al., 2000).
Our immunocytochemical study also showed that PDE3B is highly expressed
in the mouse hypothalamus (data not shown). Therefore, leptin may
negatively regulate cAMP levels through activation of PDE in the brain
as well as cells, thereby attenuating phosphorylation of CREB. The
precise molecular point of cross-talk between leptin and cAMP signal
transduction in the brain, however, remains to be elucidated. Attenuation of CREB phosphorylation by leptin may depend on the modulating activity of a protein phosphatase. Morishita and colleagues (1998) showed that central injection of leptin in rats increased hypothalamic gene expression of protein phosphatase type 2B, [an enzyme implicated in the dephosphorylation of CREB (Enslen et al.,
1994)].
Studies in cells transfected with leptin receptors showed that leptin
stimulated JAK2 activity and phosphorylation of STATs, suggesting a
role for the JAK-STAT signaling pathway downstream of the leptin
receptor (Baumann et al., 1996; Ghilardi et al., 1996; Bjørbæk et
al., 1997). Recent in vivo studies demonstrated that
peripheral administration of leptin specifically increased STAT3
tyrosine phosphorylation in the hypothalamus (Vaisse et al., 1996;
McCowen et al., 1998) and increased mRNA expression of suppressors of
cytokine signaling-3 (SOCS-3) induced by the activation of the JAK-STAT
signaling pathway (Bjørbæk et al., 1998). The increase of CRE binding
activity as well as the increased CREB phosphorylation in hypothalamic
nuclear extracts of fasted mice has also been reported by Sheriff et
al. (1997), suggesting that CREB-CRE binding may be regulated by other
gene transcription factors such as STATs under conditions requiring
different energy demands. The decreased circulating leptin levels
observed during fasting may enhance CRE binding activity caused by
released suppression of the STATs, resulting in an increase in
CRE-mediated transcription. This idea may be supported by recent
evidence showing that the STAT motif plays a role in cell type-specific
transcription of the vasoactive intestinal peptide gene regulated by
CRE (Hahm and Eiden, 1998) and that transcriptional regulation in the
junB and interferon regulatory factor-1 genes is uniquely
accomplished by both a CRE-like site and a STAT-binding site in the
interferon-6 response element (Kojima et al., 1996; Ichiba et al.,
1998).
Another possible mechanism of leptin-induced downregulation on
CRE-mediated gene induction may be attributable to an indirect regulation of NPY neurons by leptin through other neuronal groups. Arc
POMC neurons express leptin receptors (Cheung et al., 1997; Håkansson
et al., 1998), and POMC and NPY neurons are synaptically linked in the
Arc (Horvath et al., 1992; Broberger et al., 1997). Thus, gene
expression in NPY neurons may be antagonistically modulated by POMC
neurons during fasting and leptin treatment. Our results suggest that
fasting does not elicit CRE-mediated gene induction in POMC neurons,
although the POMC gene contains a motif resembling a CRE on its
promoter (Kraus and Höllt, 1995), suggesting the existence of
separate transcriptional pathways that regulate CRE-mediated gene
induction in discrete neuronal populations. Indeed, Elias et al. (1999)
has recently demonstrated that leptin induced SOCS-3 mRNA expression in
both NPY and POMC neurons but Fos expression in only POMC neurons. It
is also conceivable that leptin may act on other hypothalamic nuclear
groups, such as the DMH and the VMH that were found to be directly
activated by leptin using the c-fos method (Elmquist et al., 1997).
Leptin has been reported to inhibit the depolarization-induced release
of noradrenaline and dopamine from isolated hypothalamic neuronal
endings (Brunetti et al., 1999), suggesting a possibility that leptin
may modulate the activity of presynaptic neurons that innervate Arc NPY neurons.
The study presented here provides the first evidence that activation of
Arc NPY neurons but not POMC neurons results in stimulation of
CRE-mediated gene induction during fasting. Fasting-stimulated CRE-mediated gene induction in NPY neurons requires CREB
phosphorylation and activation, at least partly, by PKA. More
importantly, administration of leptin downregulated fasting-induced
CRE-mediated gene induction and NPY mRNA levels in the Arc. These
results suggest that CREB-CRE is one of the important molecular loci
associated with the regulatory effects of leptin on hypothalamic gene
expression in NPY neurons. Downregulation of CRE-mediated gene
induction may be an important component in the molecular mechanism
underlying the central effect of leptin on regulating feeding behavior.
| |
FOOTNOTES |
Received July 17, 2000; revised Oct. 6, 2000; accepted Nov. 30, 2000.
This study was supported by National Institutes of Health Grant DK
21723. We thank Drs. S. Impey and D. R. Storm, Department of
Pharmacology, University of Washington, for providing the CRE-lacZ transgenic mice, and Dr. R. A. Steiner, Departments of Obstetrics and Gynecology, University of Washington, for reviewing this
manuscript. We also thank Cari Ostenson for technical assistance.
Correspondence should be addressed to Joseph A. Beavo, Department of
Pharmacology, Box 357280, University of Washington, School of Medicine,
Seattle, WA 98195. E-mail:
beavo{at}u.washington.edu.
| |
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B. Holst, M. Lang, E. Brandt, A. Bach, A. Howard, T. M. Frimurer, A. Beck-Sickinger, and T. W. Schwartz
Ghrelin Receptor Inverse Agonists: Identification of an Active Peptide Core and Its Interaction Epitopes on the Receptor
Mol. Pharmacol.,
September 1, 2006;
70(3):
936 - 946.
[Abstract]
[Full Text]
[PDF]
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ABSTRACT
INTRODUCTION
