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The Journal of Neuroscience, August 15, 2002, 22(16):6929-6938
VGF is Required for Obesity Induced by Diet, Gold
Thioglucose Treatment, and Agouti and is Differentially Regulated in
Pro-Opiomelanocortin- and Neuropeptide Y-Containing Arcuate
Neurons in Response to Fasting
Seung
Hahm1,
Csaba
Fekete3, 5,
Tooru M.
Mizuno1, 2,
Joan
Windsor1,
Hai
Yan7,
Carol N.
Boozer6,
Charlotte
Lee4,
Joel K.
Elmquist4,
Ronald M.
Lechan5,
Charles V.
Mobbs1, 2, and
Stephen R. J.
Salton1, 2
1 Fishberg Research Center for Neurobiology and
2 Department of Geriatrics, Mount Sinai School of Medicine,
New York, New York 10029, 3 Department of Neurobiology,
Institute of Experimental Medicine, Hungarian Academy of Sciences,
Budapest, 1083 Hungary, 4 Department of Neurology, Beth
Israel Deaconess Medical Center, and Program in Neuroscience, Harvard
Medical School, Boston, Massachusetts 02215, 5 Division of
Endocrinology, Diabetes, Metabolism, and Molecular Medicine, Tupper
Research Institute and Department of Medicine, New England Medical
Center, Boston, Massachusetts 02111, 6 Obesity Research
Center, St. Luke's-Roosevelt Hospital, Columbia University College of
Physicians and Surgeons, New York, New York 10025, and
7 Amgen Inc., Thousand Oaks, California 91320
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ABSTRACT |
Targeted deletion of the gene encoding the neuronal and
neuroendocrine secreted polypeptide VGF (nonacronymic) produces
a lean, hypermetabolic mouse. Consistent with this phenotype, VGF mRNA
levels are regulated in the hypothalamic arcuate nucleus in response to
fasting. To gain insight into the site(s) and mechanism(s) of action of
VGF, we further characterized VGF expression in the hypothalamus.
Double-label studies indicated that VGF and pro-opiomelanocortin were
coexpressed in lateral arcuate neurons in the fed state, and that VGF
expression was induced after fasting in medial arcuate neurons that
synthesize neuropeptide Y (NPY). Like NPY, VGF mRNA induction in
this region of the hypothalamus in fasted mice was inhibited by
exogenous leptin. In leptin-deficient ob/ob and
receptor-mutant db/db mice, VGF mRNA levels in the
medial arcuate were elevated. To identify neural pathways that are
functionally compromised by Vgf ablation, VGF mutant
mice were crossed with obese
Ay/a (agouti) and
ob/ob mice. VGF deficiency completely blocked the
development of obesity in
Ay/a mice, whereas
deletion of Vgf in ob/ob mice attenuated
weight gain but had no impact on adiposity. Hypothalamic levels of NPY and agouti-related polypeptide mRNAs in both double-mutant lines were
dramatically elevated 10- to 15-fold above those of wild-type mice.
VGF-deficient mice were also found to resist diet- and gold thioglucose-induced obesity. These data and the susceptibility of VGF
mutant mice to monosodium glutamate-induced obesity are consistent with
a role for VGF in outflow pathways, downstream of hypothalamic and/or
brainstem melanocortin 4 receptors, that project via the autonomic
nervous system to peripheral metabolic tissues and regulate energy homeostasis.
Key words:
VGF; neurotrophin; hypothalamus; obesity; agouti; POMC; NPY; leptin; melanocortin
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INTRODUCTION |
Hypothalamic neural pathways in the
CNS regulate feeding and energy expenditure. The state of peripheral
fat stores is relayed to the brain via circulating hormones such as
leptin, an adipocyte-synthesized protein that transduces its signal
through transport and binding to the leptin receptor system in the
hypothalamus (Ahima et al., 2000 ; Schwartz et al., 2000). Here
satiety-inducing melanocortin pathways decrease food intake through the
interplay of two peptides that compete for binding to the melanocortin
4 receptor (MC4-R),  -melanocyte stimulating hormone (-MSH), and
its antagonist agouti-related polypeptide (AGRP). Effects of these
leptin-responsive circuits on energy balance are mediated by
projections within the hypothalamus to the brainstem, spinal cord, and
cerebral cortex, and ultimately to autonomic pathways that
innervate peripheral metabolic tissues (Ahima et al., 2000).
Analyses of several obese mouse models indicate that leptin and the
melanocortin pathway play critical roles in the regulation of energy
balance. Mice with mutations in the leptin (ob/ob) or leptin
receptor (db/db) genes develop early onset obesity that is
associated with a reduced metabolic rate, increased food intake, diabetes, and decreased fertility (Friedman and Halaas, 1998). Mice
with defective signaling in the melanocortin pathway, caused by
targeted deletion of the hypothalamic MC4-R or its agonist -MSH
[pro-opiomelanocortin (POMC) knock-out] or by overexpression of the
melanocortin receptor antagonists agouti
(Ay/a) or the agouti-related
protein [AGRP or agouti-related transcript (ART)], develop a maturity
onset obesity syndrome that is associated with hyperphagia,
hyperinsulinemia, and hyperglycemia (Barsh, 1999; Salton et al.,
2000a). Inbreeding of these obese mice with other strains that harbor
additional genetic mutations, such as mahogany (Gunn et al., 1999;
Nagle et al., 1999) or the neuropeptide Y (NPY) knock-out (Erickson et
al., 1996), and analysis of the phenotypes of the resulting offspring
has been an extremely useful technique to better define the molecular
components of the leptin and melanocortin pathways.
Targeted deletion of the neurotrophin-regulated gene product called
VGF (nonacronymic) produces a lean, hypermetabolic, and hyperactive mouse (Hahm et al., 1999). This gene encodes a secreted polypeptide that is expressed throughout the CNS and PNS, selectively in neurons in which it can be rapidly induced by neurotrophins, as well
as in several endocrine and neuroendocrine tissues (Salton et al.,
2000b). VGF expression is regulated in the brain by activity and
injury, in the hypothalamic arcuate nucleus in response to fasting, and
in the dorsal motor nucleus of the vagus (DMV) and nucleus tractus
solitarius (NTS) in response to gastric manipulation (Salton et al.,
2000b). The phenotype of the VGF mutant mouse suggests that this
protein or one of its processed peptides regulates energy balance;
however, the precise bioactive fragment(s) and mechanism of action of
VGF have remained elusive. To identify potential site(s) of VGF action,
we examined VGF expression in the hypothalamus and determined which
central and peripheral neural pathways are impacted by targeted
deletion of the Vgf gene. We report that Vgf gene
ablation blocks the development of obesity from select environmental
and genetic causes, suggesting that VGF functions in outflow pathways,
downstream of hypothalamic or brainstem melanocortin receptors, to
regulate energy expenditure.
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MATERIALS AND METHODS |
Mouse strains used. Targeted deletion of the mouse
Vgf gene and initial characterization of VGF mutant mice has
been described previously (Hahm et al., 1999). Chimeric VGF knock-out
males were directly crossed to 129/SvJ or repetitively backcrossed to
C57BL/6 strains for 10 generations; homozygous VGF-deficient offspring of F10 and F1 heterozygotes
on either background were phenotypically indistinguishable. For the
experiments described here, mixed background VGF mutant mice from
F2 and F3 generations were
used. To generate double-mutant mice, fertile heterozygous
Vgf+/Vgf female mice or bilaterally
ovariectomized C57BL/6 × 129/SvJ females (The Jackson Laboratory,
Bar Harbor, ME) that had been grafted with
Vgf/Vgf ovaries were used. These were mated
with Ay/a (agouti) males or
with fertile ob/ob males, both on C57BL/6 backgrounds (obtained from The Jackson Laboratory), that had been rescued by a course of intraperitoneal leptin. Recombinant murine leptin (20 µg/gm body weight), generously provided by Amgen Inc. (Thousand Oaks, CA), was delivered to ob/ob
males twice daily for 7 d and then approximately every other day
for 30-60 d.
Fasting and leptin replacement study. Wild-type and
db/db mice were fasted for 48 hr and killed (fasted
group). For the leptin replacement study, ad libitum fed and
fasting mice were injected intraperitoneally every 12 hr with saline or
leptin (0.5 µg of recombinant murine leptin/gm body weight; R & D
Systems, Minneapolis, MN). The final, fifth injection was administered
30 min before the mice were killed, 2 hr after lights on.
Chemical lesioning. Mice, 3-4 months of age, were
administered a single intraperitoneal injection of gold thioglucose
(GTG) (0.8 mg/gm body weight) (Bergen et al., 1998), after which mice were weighed at regular intervals and food intake was measured. Two
weeks after GTG (or saline) injection, mice were killed and tissues
were removed for analysis. Separate groups of wild-type and VGF mutant
mice were killed 3 d after GTG injection, and brains were removed
and examined histologically by Nissl staining (Hahm et al., 1999) to
confirm that hypothalamic lesions developed in mice of each genotype.
Monosodium glutamate (MSG) injections were performed by minor
modification of previous methods (Pizzi and Barnhart, 1976). Mice
received the following daily subcutaneous injections starting at
postnatal day 2 (P2) until P12: 2.5, 2.8, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, and 4.8 mg MSG per gram of body weight (in PBS). Mice were
weighed weekly and killed at 9 months of age, hypothalamic RNAs were
isolated, and organ and tissue weights were determined.
Northern blot, blood, and serum analyses. Northern blot
analysis was performed as described previously (Mizuno et al., 1996b), using probes to NPY (Mizuno et al., 1996a), POMC (Mizuno et al., 1998),
and AGRP (Mizuno and Mobbs, 1999); relative mRNA levels were determined
by densitometric analysis of film autoradiograms.
Mice were anesthetized with avertin, and blood samples were collected
by cardiac puncture. Blood glucose levels were determined using a one
touch profile meter (Lifescan Inc., Milpitas, CA). Serum insulin and
leptin levels were determined by radioimmunoassay (ICN
Biomedicals, Inc., Costa Mesa, CA) or ELISA (Crystal Chem Inc.,
Chicago, IL).
Energy balance and body composition analyses. Food
consumption was measured daily using either a liquid diet or a
weighable solid pellet delivery system (Bio-Serv, Frenchtown, NJ) over
5 consecutive days and averaged. For the high-fat diet studies, mice
were fed for 5 weeks with standard chow or high-calorie chow (5.396 kcal/gm) with a high fat (35.5%) and high carbohydrate (35.4%)
content (#F2685; Bio-Serv). Body carcass analysis for total lipid,
protein, and water was performed as described previously (Chung et al.,
1998; Hahm et al., 1999).
Immunohistochemical characterization of VGF-containing neurons in
the arcuate nucleus. Adult male Sprague Dawley rats (280-300 gm;
Taconic Farms, Germantown, NY) were divided into three groups. To study
the colocalization of VGF and -MSH in the arcuate nucleus, animals
(n = 3) were anesthetized with sodium pentobarbital (50 mg/kg body weight, i.p.) and stereotaxically injected
intracerebroventricularly with 100 µg of colchicine in 6 µl of
0.9% saline 20 hr before perfusion fixation of the brain. To study the
colocalization of VGF and NPY in the arcuate nucleus of fed and fasted
rats, rats were implanted with a 22 gauge stainless steel guide cannula
(Plastics One Inc., Roanoke, VA) into the lateral cerebral ventricle
under stereotaxic control (coordinates from bregma:
anteroposterior, 0.8; lateral, 1.2; dorsoventral, 3.2) through
a burr hole in the skull. The cannula was secured to the skull
with three stainless steel screws and dental cement and temporarily
occluded with a dummy cannula. Bacitracin ointment was applied daily to
the interface of the cement and the skin. Animals were weighed daily,
and any animal showing signs of illness or weight loss after the third postoperative day was removed from the study and killed. One week after
intracerebroventricular cannulation, the animals were divided into two
groups. In the first group (n = 3), the animals were fed ad libitum. In the second group (n = 3),
animals were fasted for 64 hr. Both groups were treated with 100 µg
of colchicine in 6 µl of 0.9% saline through a 28 gauge needle that
extended 1 mm below the guide cannula, 20 hr before perfusion fixation of the brain.
All colchicine-treated animals were deeply anesthetized with sodium
pentobarbital and perfused transcardially with 20 ml of 0.01 M PBS, pH 7.4, containing 15,000 U/l heparin sulfate, 150 ml of 2% paraformaldehyde/4% acrolein in 0.01 M phosphate
buffer (PB), pH 7.4, followed by 30 ml of 2% paraformaldehyde in the same buffer. The brains were removed and cryoprotected in 30% sucrose
in PBS at 4°C overnight and then frozen on dry ice.
Immunofluorescence was performed essentially as described previously
(Fekete et al., 2000a). Coronal sections (25 µm thick) were incubated
in mixtures of either sheep anti--MSH (1:5000) (Elias et al., 1998)
or sheep anti-NPY antiserum (1:1000) (Peninsula Laboratories, Belmont,
CA) and rabbit anti-VGF antisera (1:400) (Salton et al., 1995), rinsed
in PBS, incubated in Texas Red-conjugated donkey anti-sheep IgG and
FITC-conjugated donkey anti-rabbit IgG (both 1:40; Jackson
ImmunoResearch, West Grove, PA), and analyzed by Zeiss (Thornwood, NY)
Axioskop 2 epifluorescent microscopy.
Double-label in situ hybridization for POMC and
VGF. Double-label in situ hybridization was performed
as described previously (Elias et al., 1999; Fekete et al., 2000b).
Sprague Dawley rats (280-300 gm) were divided into two groups.
One-half of the animals (n = 4) had ad
libitum access to food. The other group (n = 4) was fasted for 64 hr before the perfusion. Under sodium pentobarbital anesthesia (50 mg/kg body weight, i.p.), animals were perfused by
intracardiac perfusion with 20 ml of 0.01 M
PBS, pH 7.4, containing 15,000 U/l heparin sulfate followed by
150 ml of 4% paraformaldehyde in PBS. The brains were removed and
postfixed by immersion in the same fixative for 2 hr at room
temperature. Tissue blocks containing the hypothalamus were
cryoprotected in 20% sucrose in PBS at 4°C overnight and then frozen
on dry ice. Serial 20-µm-thick coronal sections through the
rostrocaudal extent of the arcuate nucleus were cut on a cryostat
(Reichert-Jung 2800 Frigocut-E; Leica Microsystems Inc.,
Bannockburn, IL), collected in freezing solution (30% ethylene glycol;
25% glycerol; 0.05 M PB) and stored at 20°C
until used. Serial sections were washed in 2× SSC, acetylated with
0.25% acetic anhydride in 0.9% triethanolamine for 20 min, and then
treated in graded solutions of acetone (50, 70, 90, and 100%),
chloroform, and a descending series of acetone (100, 90, 70, and 50%)
for 5 min each. After additional washes in 2×, 3×, and 4× SSC for 5 min each, the sections were hybridized with the mixture of
digoxigenin-labeled cRNA probe for POMC and
[35S]UTP-labeled cRNA probe for VGF.
The hybridization was performed for 16 hr at 56°C in 200 µl
polypropylene tubes in a hybridization buffer (50% formamide, 2× SSC,
10% dextran sulfate, 0.5% SDS, and 250 µg/ml denatured salmon sperm
DNA) that contained the digoxigenin-labeled POMC probe diluted 1:50 and
6× 105 cpm of
35S-labeled VGF cRNA probe. The slides
were washed in 1× SSC for 15 min and then treated with 25 µg/ml
RNase for 1 hr at 37°C. After additional washes in 0.1× SSC (four
times for 15 min) at 65°C, sections were washed in PBS, treated with
a mixture of 0.5% Triton X-100 and 0.5%
H2O2 for 15 min, and then
treated with 2% bovine serum albumin (BSA) in PBS for 20 min to reduce
the nonspecific antibody binding. The sections were incubated with
sheep anti-digoxigenin-peroxidase Fab fragments (1:100; Boehringer
Mannheim Corp., Indianapolis, IN) in 1% BSA in PBS for 2 d at
4°C. The sections were then rinsed in PBS and incubated in 0.1%
biotinylated tyramide and 0.01%
H2O2 in PBS for 10 min to
intensify the hybridization signal. After additional washes, the
sections were incubated in 7-amino-4-methylcoumarin-3-acetic acid
(AMCA) Avidin D (1:250; Vector Laboratories, Burlingame, CA) and
mounted on Superfrost/Plus glass slides (Fisher Scientific Co.,
Pittsburgh, PA). Slides were dipped into Kodak NTB2
autoradiography emulsion (Eastman Kodak, Rochester, NY), and the
autoradiograms were developed after 7 d of exposure at 4°C. The
specificity of hybridization was confirmed using sense probes, which
resulted in the absence of specific hybridization in the
paraventricular nucleus (PVN). To investigate regulation of VGF mRNA
levels by fasting, freshly dissected brains from 48 hr fasted and
ad libitum fed C57BL/6, ob/ob, and
db/db mice were rapidly frozen. Matching sections through
the hypothalami were processed for in situ hybridization, and 32P-labeled single-stranded mouse VGF
cDNA probes were used as described previously (Mizuno et al., 1998;
Hahm et al., 1999).
Real-time reverse transcriptase-PCR analysis. Total RNA was
isolated using TRIzol (Invitrogen, Gaithersburg, MD), and
first-strand cDNA synthesis was completed using the Superscript Choice
System (Invitrogen). RNA was hybridized for 10 min at 70°C with 100 pmol/µl oligo-dT24, and first-strand synthesis
was performed at 42°C for 60 min using Superscript II reverse
transcriptase (RT). Primers for real-time PCR were first examined by
standard PCR (94°C for 30 sec, 55°C for 1 min, and 72°C for 1 min × 30 cycles) and agarose gel electrophoresis for correct
product size and absence of primer/dimer formation. Real-time PCRs
(95°C for 15 sec, 60°C for 30 sec, and 72°C for 30 sec × 40 cycles) were performed in an ABI-prism 7700 sequence
detector (Applied Biosystems, Foster City, CA). Product accumulation
was measured in real-time, and mean cycle threshold (Ct), the cycle
when product is first detectable, was determined for replicate samples
(n = 4-8 independent reactions per primer pair and
cDNA sample) run on the same plate. Different cDNA samples were
normalized using primer sets to the housekeeping gene products glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. Primers were as follows: GAPDH, 5'-CTTGCTCAGTGTCCTTGCTG-3' and
5'-TGCGACTTCAACAGCAACTC-3'; actin, 5'-AGGTGACAGCATTGCTTCTG-3' and
5'-GCTGCCTCAACACCTCAAC-3'; NPY, 5'-AGCAGAGGACATGGCCAGAT-3' and
5'-AAATCAGTGTCTCAGGGCTGGA-3'; NPY Y5R, 5'-GAGAAGCACCTAACCGTTCCAG-3' and
5'-TGAGGGAACGCTTGACTCTCAT-3'; AGRP, 5'-TGACTGCAATGTTGCTGAGTTGTG-3'
and 5'-TAGGTGCGACTACAGAGGTTCGTG-3'; POMC,
5'-GCCCTCCTGCTTCAGACCTC-3' and 5'-CGTTGCCAGGAAACACGG-3'; cocaine
amphetamine-regulated transcript (CART), 5'-CTACTCTGCCGTGGATGATGC-3' and 5'-GGACTTCTTGCAACGCTTCG-3'; prohormone convertase 1 (PC1), 5'-AAGGGATGAGCAGGTACAAGGA-3' and 5'-GCTGAGCTTTGCACTTGGAGA-3'; MC4-R,
5'-GTGGGCGTTATGAATTGACATG-3' and 5'-TCTGATTTCGGCCACTACAGAA-3'; galanin,
5'-AAGAGAGGTTGGACCCTGAACAG-3' and 5'-TCAAAGCAGAGAACAGAGGATTGG-3'.
Statistical analyses. With the exception of the real-time
PCR results, which are presented as mean ± SD, data are presented as mean ± SEM. Data were analyzed by two-way ANOVA. When
indicated by the appropriate p value
(p < 0.05), groups were compared by Tukey-Kramer or Fisher's PLSD post hoc tests. A
p value of <0.05 was considered significant; p
and n values are noted in the figures and/or accompanying legends.
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RESULTS |
Immunohistochemical localization of VGF in the hypothalami of fed
and fasted rodents
Previous studies indicated that VGF mRNA was induced in the
hypothalamic arcuate nucleus by fasting (Hahm et al., 1999). To determine whether VGF was synthesized in POMC-expressing cells, we
mapped the distribution of neurons in the hypothalamus that make VGF
mRNA or polypeptide and either POMC mRNA or -MSH peptide using
double-label in situ hybridization or immunohistochemistry, respectively, and compared the patterns in the fed and fasted mouse and rat.
In fed rats, using double-label immunofluorescence, VGF was present in
94% of -MSH neurons in the retrochiasmatic area, 84% in the
anterior portion of the arcuate nucleus, 69% in the midarcuate nucleus, and 56% in the caudal arcuate nucleus (Fig.
1A-C). Using double-label in situ hybridization for VGF and POMC mRNAs,
fed animals were found to have an intense VGF signal in the
retrochiasmatic area and lateral arcuate nucleus that corresponded to
the location of the -MSH-containing cells (Fig.
1D). No VGF hybridization signal was detected in the
medial arcuate, where NPY-containing neurons are found. According to
double-label immunofluorescence, VGF was present in <10% of the
NPY-containing cells throughout the arcuate nucleus and retrochiasmatic
area (Fig. 1F). In summary, in the fed state, VGF and
POMC gene expression was found to significantly overlap in the lateral
arcuate nucleus.
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Figure 1.
Colocalization of VGF with
-MSH and NPY in the arcuate nucleus of fed and fasted rats.
Low-power micrographs of double-labeled immunofluorescence preparations
(A-C) demonstrate the colocalization
(yellow) of VGF (green) and
-MSH (red) in the retrochiasmatic region
(A) and in the rostral (B)
and caudal (C) part of the arcuate nucleus of fed
colchicine-treated rats (n = 3). Low-power
micrographs (D, E) show the colocalization
(yellow) of POMC mRNA (labeled with AMCA,
pseudocolored red) and VGF mRNA (dark-field
image of the silver grains, pseudocolored green) in the
arcuate nucleus of fed (D) (n = 4) and fasted (E) (n = 4)
rats. In colchicine-treated fed rats (F), VGF
immunoreactivity (green) and NPY immunoreactivity
(red) are localized in different populations of arcuate
neurons. In contrast, ~50% of NPY-immunoreactive neurons
(yellow), localized dorsally, cocontained VGF in
the arcuate nucleus of fasted colchicine-treated rats
(G) (n = 3).
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NPY is a potent stimulator of food intake, as is AGRP (or ART), an
-MSH antagonist that binds to the MC4-R, and these two orexigenic
peptides are coexpressed in hypothalamic medial arcuate neurons (Elias
et al., 1998, 1999). Expression of hypothalamic NPY and AGRP mRNAs
increase and POMC mRNA decreases with fasting (Hahn et al., 1998;
Mizuno et al., 1998, 1999). To investigate in which hypothalamic
neurons VGF expression is regulated during fasting, we colocalized POMC
and VGF mRNAs and NPY and VGF polypeptides after food deprivation for
64 hr (Fig. 1E,G). We noted a marked reduction in VGF
signal in the retrochiasmatic area and lateral arcuate nucleus in
POMC-containing neurons. Overall, the total density of silver grains in
the entire arcuate (medial and lateral groups) resulting from
hybridization to VGF was less in fasting animals than in fed animals by
~50%. However, we also observed increased VGF hybridization in the
medial part of the arcuate nucleus that is not seen in the fed animals.
Using double-label immunofluorescence, this increase was confined to
NPY-producing neurons and was dorsal to the NPY neurons seen in the fed
animals (Fig. 1G). In summary then, in the fasted state, VGF
and NPY expression was induced in a population of medial arcuate
neurons, but colocalization of VGF and POMC decreased.
Fasting-induced increases in VGF mRNA levels in the arcuate nucleus
are inhibited by leptin
VGF expression in the arcuate nucleus of the hypothalamus
increases with fasting, a state generally associated with decreased leptin levels. We therefore investigated whether exogenous leptin modulates hypothalamic VGF mRNA levels in wild-type mice and examined hypothalamic VGF mRNA expression in leptin-deficient (ob/ob)
and leptin-resistant (db/db) mice. Mice were ad
libitum fed or fasted as described in Materials and Methods, and a
subset of these mice was injected with leptin or with saline. VGF mRNA
was localized in the hypothalamus by in situ hybridization,
essentially as described previously (Hahm et al., 1999). VGF mRNA
levels were induced in fasted compared with fed wild-type mice in the
medial arcuate nucleus and somewhat decreased in regions lateral to
this, as described previously (Hahm et al., 1999) and shown above in
the rat, and the patterns of hybridization noted in the leptin-injected fasted mice were very similar to the fed controls (Fig.
2A-D). In
leptin-deficient ob/ob or leptin receptor-mutant
db/db mice, hypothalamic VGF mRNA levels in the fed state
resembled those of fasted wild-type mice, particularly in the arcuate
nucleus (Fig. 2E-H). Although VGF expression
is regulated by leptin in the hypothalamus of wild-type mice, VGF does
not appear to be an integral component of leptin signaling pathways,
because VGF mutant mice respond to exogenous leptin with weight loss,
decreased feeding, increased hypothalamic POMC expression, and
decreased adipose leptin expression (Fig. 2I).
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Figure 2.
Hypothalamic expression of VGF is regulated by
leptin. VGF expression was examined by in situ
hybridization in the hypothalami of ad libitum fed and
48 hr fasted wild-type, ob/ob, and db/db
mice (A-H). Wild-type mice were injected with
saline (sal) or leptin (lep) as
indicated (A-D). Photomicrographs of film
autoradiograms, representative of coronal sections examined from three
independent animals for each treatment group, are shown. Scale bar, 100 µm. VGF mutant mice respond to exogenous leptin
(I). VGF-deficient mice were treated with
leptin (20 µg/gm body weight, twice daily i.p. for 2 d), and
weight loss, feeding, hypothalamic POMC mRNA, and adipose leptin mRNA
were measured. Histograms identified by asterisks are
significantly different from one another (mean ± SE;
p < 0.05; ANOVA with Tukey-Kramer post
hoc comparisons; n = 4-6 mice per
treatment group).
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VGF is required for the development of diet-induced obesity
Wild-type mice fed high-calorie diets (35.5% fat, 35.4%
carbohydrate, 20% protein; 5.396 kcal/gm) for 5 weeks were all found to have substantially increased body weights, adiposity, and leptin levels in comparison with mice fed regular diets (Fig.
3). Ablation of the Vgf gene
blocked the metabolic effects of the high-fat diet (Fig.
3A), and although a small but significant increase in fat
pad weight was noted in knock-out mice fed the high-calorie diet (Fig.
3B), no significant changes in adipose leptin mRNA (Fig.
3C) or circulating leptin levels (data not shown) were
observed.
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Figure 3.
VGF mutant mice resist diet-induced obesity.
Wild-type (WT) (+/+) and VGF mutant (/) mice
were placed either on regular laboratory diets (Chow) or
high-fat diets (HF) for 5 weeks. Mice were then
weighed (A) and anesthetized, adipose tissues
were removed and weighed (B), and total RNA was
isolated and leptin mRNA levels were quantified by Northern blotting
(C). Histograms identified by different
letters are significantly different from one another
(mean ± SE; p < 0.05; ANOVA with
Tukey-Kramer post hoc comparisons;
n = 9-15 mice per treatment group).
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Ablation of the Vgf gene blocks GTG- but not
MSG-induced obesity
An alternative environmentally induced form of obesity is that
caused by lesions in the ventromedial hypothalamic area (Elmquist et
al., 1999). A particularly informative form of hypothalamic obesity is
that produced by GTG, which is taken up by and is toxic to
glucose-sensitive neurons primarily in the periarcuate area of the
ventromedial hypothalamus (Bergen et al., 1998). As with diet-induced
obesity, targeted deletion of the Vgf gene completely prevented the increase in body weight and hyperphagia produced by GTG
treatment (Fig. 4A,B).
This reduction in food intake in GTG-treated VGF mutant mice was also
quite remarkable, because ablation of the Vgf gene does not
influence food intake in wild-type mice. Thus, although GTG treatment
led to the formation of a characteristic hypothalamic lesion by Nissl
staining in all of the injected wild-type and VGF knock-out mice that
were examined (n = 3 for each group) (data not shown),
none of the GTG-injected VGF-deficient mice developed obesity.
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Figure 4.
VGF mutant mice resist obesity caused by GTG- but
not MSG-induced chemical lesions. Wild-type (+/+) and VGF-deficient
(/) mice were treated with GTG, and body weights
(A) and food consumption
(B) were determined (mean ± SE) at 16 weeks
of age. Wild-type and heterozygous and homozygous VGF-deficient mice
were treated with MSG, and body weights were measured (mean ± SE)
at 36 weeks of age (C). Histograms identified by
different letters are significantly different from one
another (p < 0.05; ANOVA with Tukey-Kramer
post hoc comparisons; n = 4-6 mice
per treatment group).
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Repetitive daily injections of MSG administered to neonatal mice from
P2-P12 result in obesity (Pizzi and Barnhart, 1976), most likely
through damage to the hypothalamus (Bergen et al., 1998; Morris et al.,
1998) and the sympathetic nervous system (Morris et al., 1998;
Tsukahara et al., 1998). NPY mRNA is virtually eliminated in the
arcuate nucleus (Bergen et al., 1998), NPY content is reduced in
several hypothalamic regions (Morris et al., 1998), and hypothalamic
POMC mRNA levels decrease (Bergen et al., 1998). In addition, decreased
glucose transporter protein-4 (GLUT4) transporter levels in the
brown adipose tissue (BAT) of MSG-lesioned mice (Morris et al., 1998)
and a decreased response of uncoupling protein (UCP) mRNA levels in BAT
to acute cold exposure (Tsukahara et al., 1998) suggest a possible
impairment of hypothalamic sympathetic input to brown fat that could
disrupt thermogenesis and lead to increased adiposity. We therefore
assessed whether Vgf gene ablation affected obesity caused
by MSG treatment. In contrast to all the other forms of obesity
examined, targeted deletion of the Vgf gene had little
influence on the ability of MSG treatment to increase body
weight (Fig. 4C). These results suggest that VGF functions in the hypothalamic/autonomic outflow pathways that innervate peripheral metabolic tissues such as BAT or white adipose tissue (WAT),
and that in the VGF mutant mice, toxic damage to these pathways blocks
the development of the hypermetabolic, lean phenotype.
VGF is required for the development of obesity in
Ay/a agouti but not
ob/ob mice
Agouti-mediated obesity results from ectopic overexpression of the
agouti polypeptide, a melanocortin receptor blocker that decreases
normal satiety signaling by -MSH, a peptide that is processed from
the POMC precursor (Fan et al., 1997; Barsh, 1999). To determine
whether VGF might play a functional role in the hypothalamic melanocortin system, we generated agouti mice, agouti mice that were
either heterozygous or homozygous for the targeted VGF mutation, and
VGF mutant mice. Ablation of the Vgf gene in
Ay/a mice completely suppressed
the obese agouti phenotype, including increased adiposity and body
weight (Fig. 5), and blocked increased linear growth [Ay/a, 9.86 ± 0.18 cm (n = 8);
Ay/a,Vgf/Vgf,
8.01 ± 0.18 (n = 3);
Vgf/Vgf, 7.88 ± 0.1 (n = 5)]. Targeted deletion of the Vgf gene, however, did not
block the effects of the Ay allele on coat
color, unlike mutations in the mahogany gene, which encodes a
transmembrane form of attractin that interacts with the agouti protein
and melanocortin receptor to modify coat color (Gunn et al., 1999;
Nagle et al., 1999). These rather dramatic differences in phenotype
between Ay/a and
Ay/a,Vgf/Vgf
mice suggested that VGF was required for the development of
agouti-induced maturity onset obesity, and that VGF might therefore function in pathways downstream to the MC4-R that project via the
autonomic nervous system to peripheral metabolic tissues.
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Figure 5.
VGF is required for the development of obesity in
agouti mice. Representative agouti
(Ay/a) and
double-mutant
(Vgf/Vgf,Ay/a)
mice are shown (A). Body weight
(B) and body composition
(C) measurements for the indicated genotypes are
shown (mean ± SE). Histograms identified by different
letters are significantly different from one another
(B) (p < 0.05; ANOVA
with Tukey-Kramer post hoc comparisons;
n = number of mice analyzed of the indicated
genotype).
|
|
Because VGF expression was upregulated in the hypothalami of
ob/ob and db/db mice (Fig. 2), we investigated
whether VGF was required for the development of obesity in
leptin-deficient ob/ob mice. Mice with mutations in either
the lepob or Vgf genes and
double mutants (ob/ob,Vgf/Vgf) were generated (Fig. 6A). Targeted
deletion of the Vgf gene completely blocked the effects of
leptin deficiency on hyperphagia and attenuated body weight gain in
double-mutant mice (Fig. 6). The effect that ablation of the
Vgf gene has on reduction of food intake in ob/ob mice is particularly striking, because VGF deficiency does not influence food intake in wild-type mice (Fig. 6D).
However, ablation of the Vgf gene did not prevent the
development of increased adiposity (Fig. 6C) or reduced body
temperature in ob/ob mice [ob/ob, 36.4 ± 0.6°C (n = 12);
ob/ob,Vgf/Vgf, 36.8 ± 0.6°C
(n = 6); wild type, 38.4 ± 0.5°C
(n = 9); Vgf/Vgf, 38.1 ± 0.5°C (n = 6)]. These results demonstrate that
products of the Vgf gene selectively influence specific
aspects of metabolic regulation.
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Figure 6.
VGF is required for the development of
hyperphagia but not increased adiposity in ob/ob mice.
Representative males of the following genotypes are shown: VGF mutant
(Vgf/Vgf), double-mutant
(Vgf/Vgf,ob/ob),
ob/ob, and wild type (A). Body
weight (B), body composition
(C), and daily food intake
(D) were measured for the indicated genotypes
(mean ± SE). Histograms identified by different
letters are significantly different from one another
(B, D) (p < 0.05; ANOVA with
Tukey-Kramer post hoc comparisons;
n = number of mice analyzed of the indicated
genotype).
|
|
Superinduction of hypothalamic NPY and AGRP expression in
double-mutant ob/ob,Vgf/Vgf, and
Ay/a,Vgf/Vgf
mice
Feeding and metabolic rate are controlled by a number of
hypothalamic neuropeptides, and their relative levels of expression are, in addition to weight and body fat stores, a sensitive indicator of the animal's state of energy balance. To determine whether the
observed failure of the obese phenotype to develop in
Ay/a,Vgf/Vgf
mice was associated with changes in hypothalamic gene expression,
real-time RT-PCR analysis was used to quantify mRNA levels for a number
of neuropeptides and receptors in VGF knock-out, agouti,
ob/ob, and double-mutant mice. Hypothalamic RNAs from each
genotype were pooled (n = 2-3), and cDNAs were synthesized as described in Materials and Methods. Each primer pair was
used to amplify hypothalamic cDNA by standard PCR methods, and agarose
gel electrophoresis was used to ensure that single DNA fragments of the
predicted size were obtained. Input amounts of cDNA used in each
reaction were normalized by quantifying the housekeeping gene products
actin and GAPDH, and the levels of the following mRNAs were quantified:
POMC, NPY, AGRP, galanin, CART, NPY Y5 receptor, MC4-R, and PC1.
In Table 1, the results of real-time PCR
analysis of cDNAs from wild-type,
Ay/a, VGF mutant, and
double-mutant
Ay/a,Vgf/Vgf
mice are shown. Similarity in the mRNA levels between VGF mutant and
double-mutant
Ay/a,Vgf/Vgf was
noted, in contrast to the values obtained for obese agouti mice. In
addition, NPY and AGRP mRNA levels were substantially higher in
Ay/a, Vgf/Vgf mice in comparison
with VGF mutant mice. Real-time PCR analysis of cDNAs from wild-type,
ob/ob, VGF-deficient, and double-mutant
ob/ob,Vgf/Vgf mice was also performed. VGF
mutant mice have low leptin levels, and ob/ob mice express
no leptin, so despite differences in weight and adiposity, similarities
in hypothalamic AGRP and NPY mRNA levels were noted (Table 1).
Double-mutant ob/ob,Vgf/Vgf mice were found,
like double-mutant
Ay/a,Vgf/Vgf
mice, to have even higher levels of AGRP and NPY mRNAs than either
ob/ob or VGF-deficient mice. In summary, we noted dramatic
increases in AGRP and NPY mRNA levels in both double-mutant strains,
which together with elevated levels in VGF mutant mice and the lack of
hyperphagia in each of these three strains point to functional
abnormalities in downstream neural pathways secondary to VGF
deficiency.
| |
DISCUSSION |
Suppression of the obese phenotype of ob/ob or agouti
mice through genetic inbreeding with other strains has been used to identify several molecular components of the leptin and melanocortin pathways. We found that inbreeding of
Ay/a (agouti) and VGF-deficient
mice suppressed maturity onset obesity. In contrast, VGF was not
required for the development of obesity in response to leptin
deficiency, but ablation of the Vgf gene did block the
development of hyperphagia in ob/ob mice. The inverse result, partial reversal of the ob/ob obesity syndrome but
no effect on the development of obesity in leptin-resistant
Ay/a mice, has been observed
previously by genetic removal of NPY (Erickson et al., 1996; Hollopeter
et al., 1998). VGF is therefore required for the development of
diet-induced obesity and obesity caused by decreased activity in the
melanocortin satiety pathway.
Where in the nervous system might VGF be functioning to regulate energy
expenditure? Previous studies have indicated that VGF is expressed in
peripheral sympathetic and parasympathetic neurons, widely expressed
throughout the brain, and regulated in the hypothalamus in response to
feeding (for review, see Salton et al., 2000b) (areas expressing VGF
are indicated by the speckled pattern in Fig.
7). Here we have shown that VGF is
primarily coexpressed with POMC in the hypothalamic arcuate nucleus in
the fed state, and that in the fasted state, VGF colocalization with
POMC decreases concomitant with VGF induction in populations of neurons
that express NPY and AGRP (Fig. 1). Recent studies using mice
transgenic for a cAMP response element (CRE)-lacZ construct
indicate that fasting activates reporter expression in the arcuate
nucleus in a subpopulation of neurons that express NPY but not POMC
(Shimizu-Albergine et al., 2001). Because several reports suggest that
CRE-binding protein (CREB) is likely to be a critical regulator of
Vgf gene expression in vitro and in
vivo (Hawley et al., 1992; Bonni et al., 1995; D'Arcangelo et
al., 1996; Di Rocco et al., 1997; Luc and Wagner, 1997), this
subpopulation of arcuate neurons that increase expression of NPY in
response to fasting are likely to be the same neurons that we have
shown upregulate VGF (Fig. 1). It is also of interest that leptin
administration to fasted CRE-lacZ mice significantly
attenuates both  -galactosidase and NPY expression in this part of
the hypothalamus. We also noted attenuation of VGF induction in the
medial arcuate nucleus in leptin-treated fasted mice and observed
elevated levels of VGF in the arcuate nuclei of ob/ob and
db/db mice compared with ad libitum fed wild-type mice (Fig. 2). Apparently then, the Vgf and Npy
genes are similarly regulated by CREB and by leptin in the medial
arcuate, and VGF, NPY, and AGRP are coexpressed in these neurons,
perhaps coreleased through a common secretory pathway, and may subserve
complementary or related downstream functions.
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Figure 7.
Schematic model indicating the sites where VGF may
regulate energy balance, based on genetic, histochemical, and lesion
data. Arcuate orexigenic and anorexigenic projections are shown as
gray and white, respectively. Regions of
the nervous system in which VGF mRNA or protein is abundantly expressed
and/or regulated by feeding or GI manipulation are indicated by a
speckled pattern. The dashed lines
identify areas susceptible to GTG- or MSG-induced injury.
Dark-gray projections outlined in
black, downstream of agouti effects on the MC4-R,
represent possible candidate circuits that might be regulated by
locally synthesized VGF (speckled areas).
ARC, Arcuate nucleus; DRG, dorsal root
ganglia; LH, lateral hypothalamus; VMN,
ventromedial nucleus.
|
|
Where do AGRP-, -MSH-, NPY-, and VGF-expressing arcuate neurons
project? Previous studies indicate that many NPY- and AGRP-containing arcuate neurons, a large number of which likely coexpress VGF (Fig. 1),
project to the PVN (Legradi and Lechan, 1999; Palkovits, 1999). In the
PVN, these processes terminate on thyrotropin-releasing hormone
neurons (Legradi and Lechan, 1999) and on neurons in the dorsal and
ventral parvocellular subdivisions, an area rich in MC4-R mRNA
(Mountjoy et al., 1994), that project to parasympathetic and
sympathetic centers in the brainstem and spinal cord (Sawchenko and
Swanson, 1982). Use of transneuronal pseudorabies virus tract tracers
and analysis of sympathetic nerve activity indicate that the PVN is the
major forebrain source and that the raphe pallidus is the major
brainstem source of innervation to preganglionic sympathetic neurons
that project to BAT (Bamshad et al., 1999; Morrison, 2001a,b). Thus VGF
release from PVN-projecting arcuate neurons may have direct effects in
the PVN, or the absence of VGF may indirectly alter release of NPY,
AGRP, and/or -MSH from these neurons.
The genetic data presented above argue that the predominant site(s) of
VGF action is likely to be downstream of hypothalamic, brainstem, or
spinal cord MC4-Rs (Mountjoy et al., 1994; van der Kraan et al., 1999),
and these data do not support a critical role for VGF in NPY and/or
AGRP secretion. Obesity in Ay/a
mice results from blockade of the MC4-R by the agouti protein, preventing -MSH binding and leading to inhibition of
satiety-inducing pathways (Barsh et al., 2000). Because the agouti
protein is ubiquitously expressed and secreted in
Ay/a mice (Miller et al.,
1993), it seems unlikely that VGF deficiency would inhibit agouti
release or binding to melanocortin receptors. The inbreeding of VGF and
MC4-R mutant mice that is underway should indirectly clarify this
issue. Regions of the brain in which melanocortin receptor blockade
could lead to alterations in energy balance include the hypothalamus
and brainstem (Fig. 7). In the brainstem, feeding and body weight are
controlled by the DMV (Grill et al., 1998), a region of high MC4-R
density that receives afferent projections from the gastrointestinal
(GI) tract and is located adjacent to the NTS, a site of POMC
synthesis. Consistent with a role for VGF in the brainstem, VGF mRNA
levels are regulated in the NTS and DMV in response to gastroduodenal
irritation (Kanemasa et al., 1995a,b).
Failure of Vgf/Vgf,ob/ob mice to
phenocopy Npy/Npy,ob/ob mice
(Erickson et al., 1996) is an indication that NPY synthesis or release
is unlikely to be dramatically decreased by targeted deletion of VGF.
That double-mutant ob/ob,Vgf/Vgf and
Ay/a,Vgf/Vgf
mice are not hyperphagic despite robust increases in hypothalamic NPY
and AGRP mRNA levels suggests that feeding pathways in VGF-deficient
mice are abnormal. Consistent with this possibility, VGF mutant mice
have increased hypothalamic NPY and AGRP mRNA levels; however, despite
decreased energy stores, these mice are hypermetabolic and consume
amounts of food similar to what is consumed by as control mice (Hahm et
al., 1999). However, comparable feeding responses of control and VGF
mutant mice after fasting (Hahm et al., 1999) and to
intracerebroventricular injection of NPY or AGRP (S. Hahm et al.,
unpublished data) do not indicate gross feeding deficiencies in the latter.
Both GTG and MSG treatments have been shown previously to lead to
obesity in NPY-deficient mice (Hollopeter et al., 1998) and to
decreased hypothalamic NPY levels in normal mice (Bergen et al., 1998),
indicating that obesity in these lesioned animals is not attributable
to NPY overexpression. Lower levels of hypothalamic POMC mRNA (Bergen
et al., 1998) in GTG-treated mice suggest rather that a decrease in
satiety signaling, caused by reduced activity and -MSH release from
injured glucose-responsive neurons in the lateral arcuate and
ventromedial hypothalamus leads to GTG-induced obesity. VGF mutant mice
fail to develop hyperphagia and obesity after GTG treatment, similar to
the phenotype of
Ay/a,Vgf/Vgf
double-mutant mice, indicating that VGF may be a required component in
either parallel or downstream pathways that are active when satiety
signaling is decreased. Neonatal MSG treatment leads to an extensive
degenerative lesion in the arcuate nucleus, reduced hypothalamic POMC
mRNA, and virtual elimination of hypothalamic NPY mRNA and AGRP- and
NPY-immunoreactive neurons and nerve fibers (Bergen et al., 1998;
Legradi and Lechan, 1998; Legradi et al., 1998). Unlike our results
with GTG, we noted that MSG treatment led to obesity in VGF mutant
mice. This could have resulted from the additional damage to
sympathetic postganglionic fibers that innervate BAT (Fig. 7), as
suggested by previous studies that have demonstrated impaired UCP
induction in response to cold exposure (Tsukahara et al., 1998) and
decreased GLUT4 (Morris et al., 1998) transporter levels in BAT from
MSG-treated obese mice.
VGF deficiency may therefore lead to increased sympathetic activity to
peripheral metabolic tissues including BAT, WAT, and skeletal muscle,
and damage to these pathways by MSG might be responsible for failure of
the hypermetabolic, lean phenotype to develop.
Vgf/Vgf mice are normothermic (Hahm et al.,
1999), double-mutant ob/ob,Vgf/Vgf mice are
equally as hypothermic as ob/ob mice, and preliminary
studies suggest that UCP1 mRNA levels are not elevated in
Vgf/Vgf BAT (E. Watson, T. Mizuno, Hahm, S. Salton, unpublished data). VGF might consequently function in
leptin-independent, melanocortin-regulated sympathetic projections to
WAT that control lipolysis. Consistent with this hypothesis, lipoprotein lipase and and -3 adrenergic receptor mRNA levels are significantly elevated in Vgf/Vgf WAT
(Watson, J. Windsor, Salton, unpublished data). Additional studies of
sympathetic nervous system activity, adrenergic receptor signaling, and
UCP expression in lean VGF mutant mice are warranted, and these should
result in a better understanding of the mechanisms responsible for the selective resistance of VGF mutant mice to lesion-induced,
diet-induced, and genetically induced obesity.
| |
FOOTNOTES |
Received Jan. 31, 2002; revised May 6, 2002; accepted May 15, 2002.
This work was supported by grants from the National Institutes of
Health, the Culpeper Foundation, and Amgen Inc. and by a Career
Scientist Award from the Irma T. Hirschl and Monique Weill-Caulier Trusts.
Correspondence should be addressed to Stephen R. J. Salton,
Fishberg Research Center for Neurobiology, Box 1065, Mount Sinai School
of Medicine, One Gustave L. Levy Place, New York, NY 10029. E-mail:
stephen.salton{at}mssm.edu.
S. Hahm's present address: Tularik Inc., Two Corporate Drive, South
San Francisco, CA 94080.
| |
REFERENCES |
-
Ahima RS,
Saper CB,
Flier JS,
Elmquist JK
(2000)
Leptin regulation of neuroendocrine systems.
Front Neuroendocrinol
21:263-307[ISI][Medline].
-
Bamshad M,
Song CK,
Bartness TJ
(1999)
CNS origins of the sympathetic nervous system outflow to brown adipose tissue.
Am J Physiol
276:R1569-R1578.
-
Barsh G
(1999)
From Agouti to Pomc: 100 years of fat blonde mice.
Nat Med
5:984-985[ISI][Medline].
-
Barsh GS,
Farooqi IS,
O'Rahilly S
(2000)
Genetics of body-weight regulation.
Nature
404:644-651[Medline].
-
Bergen HT,
Mizuno TM,
Taylor J,
Mobbs CV
(1998)
Hyperphagia and weight gain after gold-thioglucose: relation to hypothalamic neuropeptide Y and proopiomelanocortin.
Endocrinology
139:4483-4488[Abstract/Free Full Text].
-
Bonni A,
Ginty DD,
Dudek H,
Greenberg ME
(1995)
Serine 133-phosphorylated CREB induces transcription via a cooperative mechanism that may confer specificity to neurotrophin signals.
Mol Cell Neurosci
6:168-183[ISI][Medline].
-
Chung WK,
Belfi K,
Chua M,
Wiley J,
Mackintosh R,
Nicolson M,
Boozer CN,
Leibel RL
(1998)
Heterozygosity for Lep(ob) or Lep(rdb) affects body composition and leptin homeostasis in adult mice.
Am J Physiol
274:R985-R990.
-
D'Arcangelo G,
Habas R,
Wang S,
Halegoua S,
Salton SR
(1996)
Activation of codependent transcription factors is required for transcriptional induction of the vgf gene by nerve growth factor and Ras.
Mol Cell Biol
16:4621-4631[Abstract].
-
Di Rocco G,
Pennuto M,
Illi B,
Canu N,
Filocamo G,
Trani E,
Rinaldi AM,
Possenti R,
Mandolesi G,
Sirinian MI,
Jucker R,
Levi A,
Nasi S
(1997)
Interplay of the E box, the cyclic AMP response element, and HTF4/HEB in transcriptional regulation of the neurospecific, neurotrophin-inducible vgf gene.
Mol Cell Biol
17:1244-1253[Abstract].
-
Elias CF,
Saper CB,
Maratos-Flier E,
Tritos NA,
Lee C,
Kelly J,
Tatro JB,
Hoffman GE,
Ollmann MM,
Barsh GS,
Sakurai T,
Yanagisawa M,
Elmquist JK
(1998)
Chemically defined projections linking the mediobasal hypothalamus and the lateral hypothalamic area.
J Comp Neurol
402:442-459[ISI][Medline].
-
Elias CF,
Aschkenasi C,
Lee C,
Kelly J,
Ahima RS,
Bjorbaek C,
Flier JS,
Saper CB,
Elmquist JK
(1999)
Leptin differentially regulates NPY and POMC neurons projecting to the lateral hypothalamic area.
Neuron
23:775-786[ISI][Medline].
-
Elmquist JK,
Elias CF,
Saper CB
(1999)
From lesions to leptin: hypothalamic control of food intake and body weight.
Neuron
22:221-232[ISI][Medline].
-
Erickson JC,
Hollopeter G,
Palmiter RD
(1996)
Attenuation of the obesity syndrome of ob/ob mice by the loss of neuropeptide Y.
Science
274:1704-1707[Abstract/Free Full Text].
-
Fan W,
Boston BA,
Kesterson RA,
Hruby VJ,
Cone RD
(1997)
Role of melanocortinergic neurons in feeding and the agouti obesity syndrome.
Nature
385:165-168[Medline].
-
Fekete C,
Legradi G,
Mihaly E,
Huang QH,
Tatro JB,
Rand WM,
Emerson CH,
Lechan RM
(2000a)
-Melanocyte-stimulating hormone is contained in nerve terminals innervating thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and prevents fasting-induced suppression of prothyrotropin-releasing hormone gene expression.
J Neurosci
20:1550-1558[Abstract/Free Full Text].
-
Fekete C,
Mihaly E,
Luo LG,
Kelly J,
Clausen JT,
Mao Q,
Rand WM,
Moss LG,
Kuhar M,
Emerson CH,
Jackson IM,
Lechan RM
(2000b)
Association of cocaine- and amphetamine-regulated transcript-immunoreactive elements with thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus and its role in the regulation of the hypothalamic-pituitary-thyroid axis during fasting.
J Neurosci
20:9224-9234[Abstract/Free Full Text].
-
Friedman JM,
Halaas JL
(1998)
Leptin and the regulation of body weight in mammals.
Nature
395:763-770[Medline].
-
Grill HJ,
Ginsberg AB,
Seeley RJ,
Kaplan JM
(1998)
Brainstem application of melanocortin receptor ligands produces long-lasting effects on feeding and body weight.
J Neurosci
18:10128-10135[Abstract/Free Full Text].
-
Gunn TM,
Miller KA,
He L,
Hyman RW,
Davis RW,
Azarani A,
Schlossman SF,
Duke-Cohan JS,
Barsh GS
(1999)
The mouse mahogany locus encodes a transmembrane form of human attractin.
Nature
398:152-156[Medline].
-
Hahm S,
Mizuno TM,
Wu TJ,
Wisor JP,
Priest CA,
Kozak CA,
Boozer CN,
Peng B,
McEvoy RC,
Good P,
Kelley KA,
Takahashi JS,
Pintar JE,
Roberts JL,
Mobbs CV,
Salton SR
(1999)
Targeted deletion of the Vgf gene indicates that the encoded secretory peptide precursor plays a novel role in the regulation of energy balance.
Neuron
23:537-548[ISI][Medline].
-
Hahn TM,
Breininger JF,
Baskin DG,
Schwartz MW
(1998)
Coexpression of Agrp and NPY in fasting-activated hypothalamic neurons.
Nat Neurosci
1:271-272[ISI][Medline].
-
Hawley RJ,
Scheibe RJ,
Wagner JA
(1992)
NGF induces the expression of the VGF gene through a cAMP response element.
J Neurosci
12:2573-2581[Abstract].
-
Hollopeter G,
Erickson JC,
Palmiter RD
(1998)
Role of neuropeptide Y in diet-, chemical-, and genetic-induced obesity of mice.
Int J Obes Relat Metab Disord
22:506-512[ISI][Medline].
-
Kanemasa K,
Okamura H,
Kodama T,
Ibata Y
(1995a)
Induction of VGF mRNA in neurons of the rat nucleus tractus solitarius and the dorsal motor nucleus of vagus in duodenal ulceration by cysteamine.
Brain Res Mol Brain Res
32:55-62[Medline].
-
Kanemasa K,
Okamura H,
Kodama T,
Kashima K,
Ibata Y
(1995b)
Time course of the induction of VGF mRNA in the dorsal vagal complex in rats with cysteamine-induced peptic ulcers.
Brain Res Mol Brain Res
34:309-314[Medline].
-
Legradi G,
Lechan RM
(1998)
The arcuate nucleus is the major source for neuropeptide Y-innervation of thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.
Endocrinology
139:3262-3270[Abstract/Free Full Text].
-
Legradi G,
Lechan RM
(1999)
Agouti-related protein containing nerve terminals innervate thyrotropin-releasing hormone neurons in the hypothalamic paraventricular nucleus.
Endocrinology
140:3643-3652[Abstract/Free Full Text].
-
Legradi G,
Emerson CH,
Ahima RS,
Rand WM,
Flier JS,
Lechan RM
(1998)
Arcuate nucleus ablation prevents fasting-induced suppression of ProTRH mRNA in the hypothalamic paraventricular nucleus.
Neuroendocrinology
68:89-97[ISI][Medline].
-
Luc PV,
Wagner JA
(1997)
Regulation of the neural-specific gene VGF in PC12 cells. Identification of transcription factors interacting with NGF-responsive elements.
J Mol Neurosci
8:223-241[ISI][Medline].
-
Miller MW,
Duhl DM,
Vrieling H,
Cordes SP,
Ollmann MM,
Winkes BM,
Barsh GS
(1993)
Cloning of the mouse agouti gene predicts a secreted protein ubiquitously expressed in mice carrying the lethal yellow mutation.
Genes Dev
7:454-467[Abstract/Free Full Text].
-
Mizuno TM,
Mobbs CV
(1999)
Hypothalamic agouti-related protein messenger ribonucleic acid is inhibited by leptin and stimulated by fasting.
Endocrinology
140:814-817[Abstract/Free Full Text].
-
Mizuno TM,
Bergen H,
Kleopoulos S,
Bauman WA,
Mobbs CV
(1996a)
Effects of nutritional status and aging on leptin gene expression in mice: importance of glucose.
Horm Metab Res
28:679-684[ISI][Medline].
-
Mizuno TM,
Bergen H,
Funabashi T,
Kleopoulos SP,
Zhong YG,
Bauman WA,
Mobbs CV
(1996b)
Obese gene expression: reduction by fasting and stimulation by insulin and glucose in lean mice, and persistent elevation in acquired (diet-induced) and genetic (yellow agouti) obesity.
Proc Natl Acad Sci USA
93:3434-3438[Abstract/Free Full Text].
-
Mizuno TM,
Kleopoulos SP,
Bergen HT,
Roberts JL,
Priest CA,
Mobbs CV
(1998)
Hypothalamic pro-opiomelanocortin mRNA is reduced by fasting and [corrected] in ob/ob and db/db mice, but is stimulated by leptin.
Diabetes
47:294-297[Abstract].
-
Mizuno TM,
Makimura H,
Silverstein J,
Roberts JL,
Lopingco T,
Mobbs CV
(1999)
Fasting regulates hypothalamic neuropeptide Y, agou
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ABSTRACT
INTRODUCTION
