ENERGY BALANCE-OBESITY

Central Nesfatin-1 Reduces Dark-Phase Food Intake

and Gastric Emptying in Rats: Differential Role of
Corticotropin-Releasing Factor2 Receptor

Andreas Stengel,* Miriam Goebel,* Lixin Wang, Jean Rivier, Peter Kobelt,
Hubert Mönnikes, Nils W. G. Lambrecht, and Yvette Taché
Department of Medicine (A.S., M.G., L.W., N.W.G.L., Y.T.), CURE, Center for Neurobiology of Stress,
Digestive Diseases Division, University of California, Los Angeles, Veterans Administration Greater Los
Angeles Healthcare System, Los Angeles, California 90073; Peptide Biology Laboratories (J.R.), Salk
Institute, La Jolla, California 92037; Clinic for Psychosomatic Medicine (P.K.), Charité-Universitätsmedizin
Berlin, D-10117 Berlin, Germany; and Department of Medicine and Institute of Neurogastroenterology
(H.M.), Martin-Luther-Hospital, D-14193 Berlin, Germany

Nesfatin-1, derived from nucleobindin2, is expressed in the hypothalamus and reported in one
study to reduce food intake (FI) in rats. To characterize the central anorexigenic action of nesfatin-1
and whether gastric emptying (GE) is altered, we injected nesfatin-1 into the lateral brain ventricle
(intracerebroventricular, icv) or fourth ventricle (4v) in chronically cannulated rats or into the
cisterna magna (intracisternal, ic) under short anesthesia and compared with ip injection. Nesfa-
tin-1 (0.05 ␮g/rat, icv) decreased 2–3 h and 3– 6 h dark-phase FI by 87 and 45%, respectively, whereas
ip administration (2 ␮g/rat) had no effect. The corticotropin-releasing factor (CRF)1/CRF2 antago-
nist astressin-B or the CRF2 antagonist astressin2-B abolished icv nesfatin-1’s anorexigenic action,
whereas an astressin2-B analog, devoid of CRF-receptor binding affinity, did not. Nesfatin-1 icv
induced a dose-dependent reduction of GE by 26 and 43% that was not modified by icv astressin2-B.
Nesfatin-1 into the 4v (0.05 ␮g/rat) or ic (0.5 ␮g/rat) decreased cumulative dark-phase FI by 29 and 60%
at 1 h and by 41 and 37% between 3 and 5 h, respectively. This effect was neither altered by ic
astressin2-B nor associated with changes in GE. Cholecystokinin (ip) induced Fos expression in 43% of
nesfatin-1 neurons in the paraventricular hypothalamic nucleus and 24% of those in the nucleus tractus
solitarius. These data indicate that nesfatin-1 acts centrally to reduce dark phase FI through CRF2-
receptor-dependent pathways after forebrain injection and CRF2-receptor-independent pathways af-
ter hindbrain injection. Activation of nesfatin-1 neurons by cholecystokinin at sites regulating food
intake may suggest a role in gut peptide satiation effect. (Endocrinology 150: 4911– 4919, 2009)

esfatin-1 is an 82-amino-acid peptide derived from
N posttranslational processing of the N-terminal frag-
ment of nucleobindin2 (NUCB2), a 396-amino-acid pro-
tein highly conserved across mammalian species (1). Post-
translational cleavage of NUCB2 results in the expression
of nesfatin-2 (85–163) and nesfatin-3 (166 –396) in addi-
tion to nesfatin-1 (1). Oh-I et al.’s (1) initial report indi-
cates that nesfatin-1 (named as acronym for NEFA/nucleo-
bindin2-encoded satiety- and fat-influencing protein) may
have physiological relevance in regulating food intake.
This was based on convergent observations that nesfatin-1
injected acutely into the third brain ventricle reduced food
consumption occurring during the dark phase, whereas
nesfatin-2 or nesfatin-3 had no effect (1). Likewise, con-
tinuous infusion of nesfatin-1 (5 pmol/d for 10 d into the
third brain ventricle) significantly decreased food intake
and body weight gain in rats (1). Conversely, third ven-
tricle infusion of a NUCB2 antisense oligonucleotide in-

ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: apPVN, Anterior parvocellular part of the PVN; CCK-8S, sulfated cholecys-
Printed in U.S.A. tokinin-8; CRF, corticotropin-releasing factor; GE, gastric emptying; ic, intracisternal; icv,
Copyright © 2009 by The Endocrine Society intracerebroventricular; ir, immunoreactive; NTS, nucleus of the solitary tract; NUCB2,
doi: 10.1210/en.2009-0578 Received May 19, 2009. Accepted July 29, 2009. nucleobindin2; PVN, paraventricular nucleus; SON, supraoptic nucleus; 4v, fourth ventricle.
First Published Online October 1, 2009
* A.S. and M.G. contributed equally.

Endocrinology, November 2009, 150(11):4911– 4919 endo.endojournals.org 4911

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 4912 Stengel et al. Nesfatin-1: Central Actions to Reduce Food Intake
creased food intake and body weight gain compared with
a missense NUCB2 oligonucleotide (1). In addition, a 24-h
fast decreased the expression of NUCB2 in the paraven-
tricular nucleus (PVN) (1). Lastly, HPLC of rat cerebro-
spinal fluid yields a peak corresponding to nesfatin-1, sug-
gestive of occurrence as secreted fragment (1), although
the nesfatin-1 receptor remains to be identified. Several
laboratories reproducibly showed abundant NUCB2/nes-
fatin-1 expression in relevant hypothalamic and medul-
lary sites involved in feeding regulation in rats, including
the arcuate nucleus, PVN, and the nucleus of the solitary
tract (NTS) (1–5), further supporting the assumption that
nesfatin-1 is involved in food intake regulation.
However, there is still a paucity of information on the
central action of nesfatin-1 to influence food intake out-
side the initial report (1). In the present study, we evalu-
ated nesfatin-1 action to modulate food intake response
upon injection into the forebrain at the level of the lateral
brain ventricle (intracerebroventricularly, icv) or the hind-
brain into the fourth ventricle (4v) or cisterna magna (in-
tracisternally, ic) in freely fed rats during the dark phase or
in response to fasting-refeeding during the light phase. A
recent in vitro study indicates that nesfatin-1 influences
the activity of a large proportion of PVN neurons includ-
ing those containing corticotropin-releasing factor (CRF)
(6). Activation of brain CRF signaling pathways by CRF
acting on CRF1 and CRF2 receptors and by selective en-
dogenous CRF2 agonists urocortin 2 or 3 (7) inhibits food
intake (8). Therefore, we investigated whether the central
action of nesfatin-1 on food intake may be mediated by
the activation of brain CRF receptors using astressin-B,
a CRF1 and CRF2 receptor antagonist, and astressin2-B, a
selective CRF2 receptor antagonist, compared with a
structurally related astressin2-B analog, devoid of CRF
antagonist activity (9). Because alterations of gastric tran-
sit influence the degree of fullness and can convey satiety
signals to reduce food intake (10, 11), we also examined
whether nesfatin-1 injected icv, into the 4v, or ic influences
gastric emptying (GE). Changes in circulating levels of
glucose are reflected in brain extracellular glucose levels
(12), which influence food intake and gastric motility (13,
14). Therefore, next we assessed whether the nesfatin-1
action is associated with variations in blood glucose levels.
To gain insight into the responsiveness of brain nesfatin-1
neurons to a stimulus known to inhibit food intake, we
explored whether an ip injection of sulfated cholecysto-
kinin-8 (CCK-8S) (15) activates neurons in the PVN and
NTS known to express nesfatin-1 immunoreactivity (1–3,
16) and to be involved in CCK satiety actions (17, 18).
Because a recent report showed a reduction of dark-phase
food intake after ip injection of nesfatin-1 in mice (19) and
we recently reported the expression of NUCB2/nesfatin-1
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Endocrinology, November 2009, 150(11):4911– 4919
in rat gastric endocrine cells (20), we also assessed whether
the orexigenic drive induced by fasting (21) influences
both the activity of nesfatin-1-containing neurons in the
brain and nesfatin-1 plasma levels.
Materials and Methods
Animals
Adult male Sprague Dawley rats (Harlan, San Diego, CA)
weighing 280 –350 g were housed under controlled illumination
(0600 –1800 h) and temperature (21–23 C). Rats had free access
to standard rodent chow (Prolab RMH 2500 LabDiet; PMI Nu-
trition, Brentwood, MO) and tap water. Protocols were ap-
proved by the Veterans Administration Institutional Animal
Care and Use Committee (99-127-07).
Peptides
Rat nesfatin-1 (1-82; Phoenix Pharmaceuticals, Burlingame,
CA) and CCK-8S (Bachem, Torrance, CA) were aliquoted in
distilled water and stored at ⫺80 C. Astressin-B, astressin2-B,
the astressin2-B analog cyclo(33–36) 关DPhe11, His12, Nle17,
C␣MeLeu13, 39, Glu33, Lys36兴 Ac-Sau (8 – 40) devoid of CRF re-
ceptor binding affinity (9), and amidated rat Ac-nesfatin-11– 44-
NH2, (Salk Institute, La Jolla, CA) were synthesized as previ-
ously described (9, 22) and kept in powder form at ⫺80 C.
Peptides were dissolved in distilled water immediately before the
experiments.
Brain injections
Implanting cannulas into the right lateral (icv) or 4v and ic
injections in 5 ␮l were performed as previously described (23,
24). Stereotaxic coordinates were obtained from Paxinos and
Watson’s brain atlas (25). After surgery, animals were housed
one per cage, had a 7-d recovery period, and were handled for
another 7 d to become accustomed to the injection procedure. At
the end of the experiments, the correct placement of the cannula
was verified by injecting dye (0.1% toluidine blue). Rats with
misplaced cannulas (five of 122 after icv and three of 20 after 4v
injection) were excluded retrospectively. The ic injection in 5 ␮l
was performed as previously reported (26) in rats under short
isoflurane anesthesia (2–3 min, 4.5% vapor concentration in
oxygen; VSS, Rockmart, GA). The accuracy of the ic injection
was ascertained before the injection by withdrawal of cere-
brospinal fluid into the Hamilton syringe. On average, rats
completely recovered from anesthesia within 7 min.
Food intake
Dark-phase food intake in conscious rats with chronic
cannula into the lateral and fourth brain ventricle
Rats maintained one per cage were injected with vehicle (py-
rogen-free distilled water) or nesfatin-1 either icv or into the 4v
(0.05 ␮g/rat), and preweighed rat chow was made available.
Food intake was monitored up to 24 h and calculated as g/300
g body weight. The 24-h body weight change was also deter-
mined. The dose of nesfatin-1 was based on previous dose-re-
sponse studies performed after third brain ventricle injection in
rats (1). In other studies, rats were injected icv with vehicle,
astressin-B (30 ␮g/rat), astressin2-B (30 ␮g/rat), or astressin2-B
 Endocrinology, November 2009, 150(11):4911– 4919
analog (30 ␮g/rat) immediately before the icv injection of vehicle
or nesfatin-1 (0.05 ␮g/rat), and cumulative food intake was mea-
sured for 6 h. The dose of CRF antagonists was selected based on
our previous dose-response studies showing blockade of icv CRF
or acute stress on gut function (23, 27). All injections were per-
formed at the onset of the dark phase in freely fed rats maintained
in their familiar housing cages, except a reduced amount of
bedding.
Dark-phase food intake in response to nesfatin-1
injected ic or ip in noncannulated rats
In freely fed rats housed two per cage until the start of the
experiment, nesfatin-1 was injected at the onset of the dark phase
either ic (0.5 ␮g/rat) under short isoflurane anesthesia or ip (2
␮g/rat) in conscious rats, or vehicle (pyrogen-free distilled water)
was injected under similar conditions. Afterward, rat chow was
made available and food intake monitored up to 24 h. In another
study, freely fed rats were injected ic at the onset of the dark phase
with vehicle, astressin-B (30 ␮g/rat), or astressin2-B (30 ␮g/rat)
before the ic injection of vehicle or nesfatin-1 (0.5 ␮g/rat), and
cumulative food intake was monitored for 6 h. Feeding experi-
ments were repeated in a crossover design, and rats were habit-
uated to the experimental conditions by training them for single
housing for 8 h/d three times before the experiments.
Blood glucose
Freely fed singly housed rats were injected icv with vehicle or
nesfatin-1 (0.05 ␮g/rat) at the onset of the dark phase. Blood was
collected by tail prick in conscious rats at 20 min, 1 h, and 2 h
after injection, and blood glucose levels were measured (One-
Touch Ultra; LifeScan, Milpitas, CA).
Behavior
Freely fed singly housed rats were injected icv with nesfatin-1
(0.05 ␮g/rat) or vehicle (n ⫽ 8 per group) at the onset of the dark
phase and placed back in their home cage with paper under the
cage divided into six equal squares. Locomotor activity (total
number of squares crossed) and grooming (washing, licking,
and/or scratching) were monitored throughout the third hour
after injection. The investigator was blinded to the treatment.
Gastric Emptying (GE)
GE of a nonnutrient viscous solution was determined by the
phenol red/methyl cellulose method as previously described (27).
Rats were food but not water deprived for 8 h before the dark
phase. Chronically cannulated conscious rats were injected with
nesfatin-1 icv (0.05 or 0.5 ␮g/rat) or into the 4v (0.05 ␮g/rat) at
the beginning of the dark phase, and water bottles were removed.
Likewise, noncannulated rats were injected with nesfatin-1 ic
(0.5 ␮g/rat) under short isoflurane anesthesia. At 150 min after
icv or 30 min after 4v or ic injection, conscious rats received an
orogastric gavage of 1.5 ml viscous solution, and GE was mon-
itored 20 min later. In another study, 8-h fasted rats received two
consecutive icv injections of vehicle or astressin2-B (30 ␮g/rat)
followed by vehicle or nesfatin-1 (0.05 ␮g/rat), and the 20-min
GE was assessed at 170 min after injection. The time of GE
monitoring was selected based on the peak hourly inhibition of
food intake upon icv, 4v, and ic injection of nesfatin-1 estab-
lished in the studies described above.
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endo.endojournals.org 4913
Fos and nesfatin-1 immunohistochemistry
Freely fed rats were injected ip (300 ␮l) with CCK-8S (3 ␮g/
kg, n ⫽ 3) or vehicle (saline, n ⫽ 3; group size was based on a
power analysis) during the dark phase. Ninety minutes later, rats
were anesthetized with sodium pentobarbital (70 mg/kg, ip,
Nembutal; Abbott Laboratories, Chicago, IL). Transcardial per-
fusion and brain processing were performed as described be-
fore (28). Brain sections were incubated in rabbit anti-c-Fos
(1:10,000; Oncogene, Cambridge, MA) followed by the avidin-
biotin-peroxidase complex method and visualized with 3,3⬘-dia-
minobenzidine tetrachloride and nickel ammonium sulfate.
Sections were thereafter incubated in rabbit anti-nesfatin-1 (1:
10,000, catalog no. H-003-22; Phoenix Pharmaceuticals) fol-
lowed by the avidin-biotin-peroxidase complex method and
3,3⬘-diaminobenzidine tetrachloride. Cells with dark blue nu-
clear staining were Fos positive, and cells with strong brown
cytoplasmic staining were nesfatin-1 immunoreactive (ir). The
anti-nesfatin-1 antibody used, raised in rabbits against rat nes-
fatin-1 (1-82 aa), shows rat nesfatin-1 as a 9.7-kDa band on the
Western blot (20). Specificity of the staining as previously stip-
ulated (29) was assessed by preabsorption of the antibody (1 ml,
1:10,000) with rat nesfatin-1 (10 ␮g; Phoenix Pharmaceuticals).
After 24 h incubation, the solution was centrifuged and the su-
pernatant used for immunostaining. Fos-ir and nesfatin-1-ir cells
were counted unilaterally as previously described (28) in the
anterior parvocellular part of the PVN (apPVN, ⫺1.08 to ⫺1.32
mm from bregma, four sections) and NTS at the level of the area
postrema (⫺13.68 to ⫺14.28 mm from bregma, eight sections)
(25). The average number of single- or double-labeled Fos-ir and
nesfatin-1-ir cells per section for each animal was calculated. The
investigator was blinded to the treatment.
In another study, rats freely fed, 24-h fasted, or 24-h fasted
followed by refeeding for 2 h (n ⫽ 3 per group) were anesthetized
at the beginning of the dark phase, transcardially perfused, and
brains processed for Fos and nesfatin-1 as described above. The
number of Fos-ir and nesfatin-1-ir cells was counted in the su-
praoptic nucleus (SON, ⫺0.6 to ⫺1.56 mm from bregma, 12
sections), PVN (⫺1.08 to ⫺2.0 mm from bregma, 12 sections),
and NTS (⫺13.32 to ⫺14.6 mm from bregma, 13 sections). The
average number of Fos-ir and nesfatin-1-positive cells per section
per brain nucleus was assessed.
Nesfatin-1 plasma levels
Rats (n ⫽ 5) were chronically equipped with a jugular vein
catheter and allowed to recover for 4 d after surgery until the
body weight started to increase again. Blood was withdrawn
serially from conscious lightly hand-restrained rats first under ad
libitum feeding during the dark phase (between 2000 and
2100 h), 12 h later during the light phase (between 0800 and
0900 h), and then after a 24-h fast (between 0800 and 0900 h)
followed by a 12-h refeeding period (between 2000 and 2100 h).
Blood was collected in tubes containing EDTA (7.5%, 10 ␮l/
0.5ml blood; Sigma Chemical Co., St. Louis, MO) and apro-
tinin (0.6 U trypsin inhibitor/0.5 ml blood; ICN Pharmaceu-
ticals, Costa Mesa, CA). Plasma nesfatin-1 was measured by
RIA (rat nesfatin-1, detection range 125–16,000 pg/ml; Phoe-
nix Pharmaceuticals).
Statistical analysis
Data are expressed as mean ⫾ SEM and analyzed by one way
ANOVA followed by Tukey post hoc test. The RIA data were
 4914 Stengel et al. Nesfatin-1: Central Actions to Reduce Food Intake
evaluated by paired t test. Differences between groups were con-
sidered significant when P ⬍ 0.05.
Results
Intracerebroventricular nesfatin-1 inhibits dark-
phase feeding and GE without altering blood
glucose in rats chronically implanted with a
cannula into the lateral brain ventricle
In freely fed chronically icv cannulated rats, nesfatin-1
(0.05 ␮g/rat ⫽ 5 pmol) injected icv at the beginning of the
dark phase significantly reduced cumulative food intake
from the third to the sixth hour after injection compared
with vehicle-injected controls (P ⬍ 0.01; Fig. 1A) without
altering blood glucose (supplemental Table 1, published
as supplemental data on The Endocrine Society’s Journals
Online web site at http://endo.endojournals.org) or induc-
ing behavioral changes relative to controls (supplemental
Table 2). The hourly food intake after nesfatin-1 showed
an 87% reduction compared with vehicle-treated animals
FIG. 1. Nesfatin-1 injected into the cerebrospinal fluid at different
levels of the brain decreases dark-phase food intake in freely fed rats.
A and B, Nesfatin-1 or vehicle was injected at the beginning of the
dark phase in ad libitum fed rats chronically implanted with cannula
either into the lateral brain ventricle, icv (A), or the 4v (B); C, injection
ic was performed under short anesthesia in noncannulated rats.
Cumulative food intake was monitored for 6 h after injection. Each bar
represents the mean ⫾ SEM. *, P ⬍ 0.05; **, P ⬍ 0.01 vs. vehicle.
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Endocrinology, November 2009, 150(11):4911– 4919
FIG. 2. Nesfatin-1 injected icv dose-dependently inhibits the dark-
phase GE of a nonnutrient solution in fasted rats: lack of reversal by icv
CRF2 antagonist. A, Chronically icv implanted rats fasted for 8 h were
injected icv with vehicle or nesfatin-1 at the beginning of the dark
phase and 150 min later gavaged; GE was monitored 20 min later. *,
P ⬍ 0.01; **, P ⬍ 0.001 vs. vehicle. B, Astressin2-B (A2-B, 30 ␮g/rat) or
vehicle (V) was injected icv before nesfatin-1 (0.05 ␮g/rat) or vehicle. *,
P ⬍ 0.05 vs. vehicle/vehicle. Each column represents the mean ⫾ SEM
of number of rats indicated at the bottom.
at the 2- to 3-h period after injection (P ⬍ 0.001). Al-
though the 24-h cumulative food intake was not signifi-
cantly changed (nesfatin-1 vs. vehicle, 17.4 ⫾ 1.4 vs.
20.4 ⫾ 0.7 g/300 g body weight, P ⬎ 0.05), the body
weight was reduced at 24 h after nesfatin-1 injection by
1.8 ⫾ 0.5% compared with vehicle (P ⬍ 0.001). The
shorter fragment, Ac-nesfatin-11– 44-NH2 (0.025 or 0.075
␮g/rat ⫽ 5 or 15 pmol, icv), by contrast, did not influence
the 3-h dark phase feeding response (supplemental Fig. 1).
Nesfatin-1 (0.05 or 0.5 ␮g/rat, icv) also dose-depen-
dently reduced the 20-min GE of a nonnutrient viscous
solution to 47.9 ⫾ 5.6% (P ⫽ 0.008) and 30.8 ⫾ 5.8%
(P ⬍ 0.001), respectively, compared with 73.5 ⫾ 4.4% in
the vehicle group as measured during the dark phase at
150 –170 min after icv injection (Fig. 2A).
Nesfatin-1 (0.5 ␮g/rat) injected icv during the light
phase in overnight fasted rats did not reduce the refeeding
response compared with vehicle (P ⬎ 0.05) when food was
given either immediately (supplemental Fig. 2A) or 2 h
after injection (supplemental Fig. 2B).
CRF2 receptor antagonist injected into the lateral
brain ventricle (icv) selectively blocks icv nesfatin-
1-induced reduction of dark-phase food intake but
not GE inhibition
Nesfatin-1 (0.05 ␮g/rat, icv) reduced the 3- and 6-h
cumulative food intake by 53.7 and 51.8% in icv vehicle-
pretreated rats compared with vehicle alone (Fig. 3A).
Nesfatin-1 inhibitory action was blocked by astressin-B
 Endocrinology, November 2009, 150(11):4911– 4919 endo.endojournals.org 4915

injection and a dose-related inhibition of cumulative food

intake over 6 h after injection (Fig. 1C and supplemental
Table 3) without influencing GE monitored for the 30- to
50-min period after injection (nesfatin-1 0.5 ␮g/rat:
51.6 ⫾ 3.4%, n ⫽ 8 vs. vehicle 59.2 ⫾ 3.8%, n ⫽ 4; P ⬎
0.05). In addition, ic (0.5 ␮g/rat) nesfatin-1-induced sup-
pression of dark-phase food intake was not altered by
astressin-B or astressin2-B (30 ␮g/rat) (Fig. 3B).
Nesfatin-1 injected ip (2 ␮g/rat) had no effect on the
dark-phase food intake (supplemental Fig. 3) as well as
when injected into the cisterna magna during the light
phase in overnight fasted rats (supplemental Fig. 4).

CCK-8S injected peripherally activates

nesfatin-1 immunopositive neurons in the PVN
and NTS
CCK-8S (3 ␮g/kg, ip) significantly increased the number
of Fos-positive neurons in the apPVN (P ⬍ 0.01; Fig. 4, B and

FIG. 3. A, CRF receptor antagonists injected icv prevent icv nesfatin-1-

induced reduction of dark-phase food intake in freely fed rats.
Chronically icv implanted rats were injected icv with 30 ␮g/rat
astressin-B, astressin2-B, astressin2-B analog devoid of CRF2 binding
affinity (inactive peptide), or vehicle before icv nesfatin-1 (0.05 ␮g/rat)
or vehicle; cumulative food intake was monitored for 6 h. *, P ⬍ 0.02;
**, P ⬍ 0.001 vs. vehicle/vehicle. B, CRF receptor antagonists injected
ic do not prevent ic nesfatin-1-induced reduction of dark-phase food
intake. Rats under brief anesthesia were injected ic with 30 ␮g/rat
astressin-B, astressin2-B, or vehicle before ic injection of nesfatin-1 (0.5
␮g/rat) or vehicle, and cumulative food intake monitored for 6 h. *,
P ⬍ 0.05; **, P ⬍ 0.005 vs. vehicle/vehicle. Each bar in A and B
represents the mean ⫾ SEM of number of rats indicated at the bottom.
bw, Body weight.

and astressin2-B (30 ␮g/rat, icv) whereas the astressin2-B

analog devoid of CRF antagonist activity (30 ␮g/rat, icv)
had no effect (Fig. 3A). By contrast, astressin2-B (30 ␮g/
rat, icv) did not modify the delayed GE induced by icv
nesfatin-1 (0.05 ␮g/rat, Fig. 2B). The CRF antagonists
injected icv alone under the same conditions modified nei-
ther basal food intake nor basal GE (Figs. 3A and 2B).

Nesfatin-1 injected into the 4v or cisterna magna

inhibits dark-phase food intake through CRF2-
independent mechanisms while not altering GE
Nesfatin-1 injected into the 4v (0.05 ␮g/rat) in freely fed
conscious rats chronically implanted with a cannula sig-
nificantly decreased dark-phase cumulative food intake
during the first hour (29%, P ⬍ 0.05) and up to 5 h (41%,
P ⬍ 0.05) after injection (Fig. 1B) while not reducing GE
(74.3 ⫾ 5.3 vs. vehicle 75.1 ⫾ 6.8%, n ⫽ 4 per group; P ⬎
0.05) as measured in the dark period during the 30 –50 min
after injection. Likewise, when nesfatin-1 (0.05 or 0.5 ␮g/
rat) was injected under brief anesthesia into the cisterna
magna of noncannulated freely fed rats, there was a re-
duction of dark-phase food intake by 59 – 60% at 1 h after
FIG. 4. CCK injected ip induces Fos expression in nesfatin-1-ir neurons
of the apPVN and NTS. Nonfasted rats were injected ip with vehicle (A
and E) or CCK-8S (3 ␮g/kg) (B and F) and at the beginning of the dark
phase euthanized 90 min later. CCK increased the number of Fos-
positive neurons (black) in the apPVN (B and C) and NTS (D and F). The
higher magnification shows that a proportion of these activated
neurons colocalized with nesfatin-1 (brown) (B and F). Scale bars (A
and E) 100 ␮m; scale bars in insets of B and F, 25 ␮m. Each bar
represents the mean ⫾ SEM of three rats. *, P ⬍ 0.05; **, P ⬍ 0.01 vs.
ip vehicle (C and D). AP, Area postrema; CC, central canal; DMV,
dorsal motor nucleus of the vagus nerve; 3V, third ventricle.

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 4916 Stengel et al. Nesfatin-1: Central Actions to Reduce Food Intake
C) and the NTS (P ⬍ 0.01; Fig. 4, D and F) compared with
ip vehicle (Fig. 4, A and C–E). Double labeling revealed that
after CCK-8S, 43% of all nesfatin-1-ir cells in the apPVN and
24% in the NTS were activated as shown by double labeling
of Fos and nesfatin-1 immunoreactivity (Fig. 4, C and D).
After preabsorption of the anti-nesfatin-1 antibody, no im-
munostaining could be detected (data not shown).
Changes in Fos expression in nesfatin-1-ir neurons
in the hypothalamus and NTS and plasma
nesfatin-1 levels in response to fasting and
refeeding
Fasting for 24 h did not significantly change the low Fos
expression in the SON, PVN, and NTS (fewer than five neu-
rons per section) compared with ad libitum fed rats (P ⬎
0.05). A 2-h refeeding period after 24 h food deprivation
significantly increased the number of Fos-positive neurons in
the SON (P ⬍ 0.001, supplemental Fig. 5, C and D) and the
NTS (P ⬍ 0.001, supplemental Fig. 6, C and D), whereas
there was no change in the PVN (data not shown). In the
SON, 99% of all activated cells induced by refeeding
after a fast were nesfatin-1-ir (supplemental Fig. 5, C
and D), whereas no significant difference in the number
of double-labeled nesfatin-1/Fos-immunopositive neu-
rons was detected in the NTS (supplemental Fig. 6).
Plasma levels of nesfatin-1 under ad libitum feeding con-
ditions showed no significant difference between dark and
light phase (2000 h 537 ⫾ 32 vs. 0800 h 548 ⫾ 42 pg/ml, P ⬎
0.05, n ⫽ 5; Fig. 5). Fasting for 24 h significantly decreased
plasma nesfatin-1 levels compared with ad libitum feeding
conditions (448 ⫾ 57 vs. 548 ⫾ 42 pg/ml, P ⬍ 0.05), whereas
12 h refeeding restored circulating nesfatin-1 levels similar to
ad libitum feeding (Fig. 5).
FIG. 5. Fasting for 24 h reduces plasma levels of nesfatin-1 compared
with fed rats. Serial blood collections from a chronically implanted
intrajugular catheter were performed at the onset of the dark phase
(2000 h) and 12 h later (0800 h) in rats fed ad libitum and then after a
24-h fast and after a 12-h refeeding period. Data are expressed as
mean ⫾ SEM (n ⫽ 5). *, P ⬍ 0.05 vs. ad libitum and 12-h refeeding,
respectively.
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Endocrinology, November 2009, 150(11):4911– 4919
Discussion
NUCB2 or nesfatin-1 (5 or 25 pmol) injected into the third
brain ventricle was initially reported to inhibit nocturnal
feeding in Wistar rats, whereas nesfatin-2 and -3 had no
effect (1). In the present study, we extend these observa-
tions by showing that the low dose of nesfatin-1 (0.05
␮g/rat ⫽ 5 pmol) injected into the lateral brain ventricle
before the onset of the dark period significantly reduced
the 3- to 6-h cumulative food intake by 45% in freely fed
Sprague Dawley rats without altering blood glucose or
inducing unusual changes in behavior. Although nesfa-
tin-1 did not modify the 24-h cumulative food intake,
body weight at 24 h after injection was significantly
decreased indicative of increased energy expenditure.
Whether nesfatin-1 induces changes in respiratory ex-
change ratio, body temperature, and uncoupling protein
levels in brown adipose tissue requires further investiga-
tions. Next, we assessed whether a shorter fragment of
nesfatin-1 may carry some biological activity. We hypoth-
esized that in the absence of double basic residues, the
Asp43-Pro45 bond may be more labile than any other
bond. We synthesized the acetylated and amidated nes-
fatin-11– 44 to mimic the amide bond of the extended
molecule at both the N and C termini and therefore
eliminating the possible charge effect. However, Ac-nes-
fatin-11– 44-NH2, injected icv at 5 or 15 pmol, failed to
influence the dark-phase food intake in freely fed rats,
showing the specificity of nesfatin-1 action and indicating
that the full-length peptide is required for biological ac-
tivity. However, a recent study showed that a middle frag-
ment of nesfatin-1 consisting of amino acids 24 –53 re-
duced food intake after peripheral injection in mice
whereas the N-terminal and C-terminal fragments were
without effect (19). Yet to be established, as pointed out by
the authors, is whether this middle fragment represents an
endogenous form of nesfatin-1 (19).
Present data and existing evidence supports that icv
nesfatin-1-induced reduction of nocturnal feeding in-
volves the activation of hypothalamic CRF2 receptors.
First, there is similarity between nesfatin-1 and CRF2 ago-
nists, urocortins, in the temporal pattern of food reduc-
tion. We found that icv nesfatin-1 inhibited nocturnal
feeding in freely fed rats with a 3-h delay without influ-
encing the refeeding response to a fast in the light phase.
Likewise, several reports indicate that urocortin 2 or 3
injected icv or microinjected into the PVN or ventromedial
hypothalamic nucleus reduced nocturnal food intake with
a 2- to 3-h delayed onset in freely fed rats through acti-
vation of hypothalamic CRF2 receptors while not altering
the food intake in response to a fast (30 –32). Second,
astressin-B, which blocks both CRF receptors, and the
 Endocrinology, November 2009, 150(11):4911– 4919
selective CRF2 antagonist astressin2-B (9) injected icv
completely prevented the nesfatin-1-induced inhibition of
nocturnal feeding. Injected alone, the CRF antagonists
had no effect as previously reported (33, 34). Nonspecific
potential peptide-peptide interaction was ruled out by
showing the unchanged icv nesfatin-1 anorexic action in
the presence of an astressin analog that bears structural
similarity with astressin2-B but is devoid of CRF1 and
CRF2 receptor affinity (⬎100 nM) (9). Taken together,
these data are consistent with the hypothalamic CRF2 sig-
naling system being part of underlying mechanisms of nes-
fatin-1-induced reduction of dark-phase food intake. In
addition, we demonstrated that low doses of nesfatin-1
injected at the hindbrain level into the 4v (0.05 ␮g/rat)
through a chronic cannula or into the cisterna magna (0.5
␮g/rat) under light anesthesia inhibited the first hour of
dark-phase food intake by 29 and 60%, respectively, with
a cumulative food intake reduction still maintained for 5 h
after injection. However, ic astressin-B and astressin2-B
did not influence ic nesfatin-1-induced decrease in food
intake. It is unlikely that the lack of CRF antagonist effect
injected ic relates to the 10-fold higher dose of nesfatin-1
given ic vs. icv because astressin-B and astressin2-B were
injected ic at a 10-fold higher dose than the maximally
effective dose to block ic CRF inhibitory action on gastric
motor function (27). CRF2-independent inhibition of the
dark-phase food intake along with the short vs. delayed
onset of the inhibitory effect induced by ic or 4v vs. icv
nesfatin-1, respectively, provide compelling indication
that icv and hindbrain (4v and cisterna magna) injections
represent distinct sites of nesfatin-1 action. The hindbrain
action site upon 4v and ic injections is also supported by
the demonstration that compounds injected into the 4v are
not seen rostrally to the caudal brainstem (35). The abun-
dant distribution of nesfatin-1 in several hypothalamic as
well as medullary nuclei such as the NTS and dorsal motor
nucleus of the vagus (1–3, 36) (present study) linked with
food intake regulation (37) also gives neuroanatomical
support for differential action sites of nesfatin-1. Distinct
forebrain and hindbrain sites of action were established
previously for several peptides (melanocortin, oxytocin,
and bombesin) based on comparison between injection
into the lateral cerebroventricle vs. 4v (38 – 40). Intrapa-
renchymal microinjection studies will be required to iden-
tify specific hypothalamic and brainstem nuclei responsive
to nesfatin-1.
The present study also demonstrates that the anorexic
effect of icv or ic nesfatin-1 was specific to the physiolog-
ical feeding occurring in the dark phase while not influ-
encing the feeding response to a fast during the light phase.
The lack of effect is not related to the delayed onset of icv
nesfatin-1 inhibitory action, which may have occurred at
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endo.endojournals.org 4917
a time when the drive to eat is low after the initial binge
eating. When food was given to fasted rats at 2 h after icv
nesfatin-1 injection, the peptide still did not decrease food
intake. Moreover, the ic injection of nesfatin-1, which
showed a rapid food intake-suppressing effect during the
dark phase, did not influence food intake of overnight
fasted rats during the light phase. This segregation of nes-
fatin-1 effects under ad libitum feeding during the dark
phase vs. fasted conditions during the light phase may be
linked with the interaction of nesfatin-1 with specific brain
neuropeptide circuitries recruited during nighttime feed-
ing as shown in the abrupt rise or fall of several neuropep-
tide genes at the onset of the dark phase before and during
the feeding period (41– 43).
Most of the centrally acting peptides reducing food in-
take also influence the process of digestion (44). Here we
show for the first time that icv nesfatin-1 dose-depen-
dently suppresses GE of a nonnutrient viscous solution. In
addition, we demonstrate distinct mechanisms for icv nes-
fatin-1-induced inhibition of dark-phase food intake and
gastric motor function because icv astressin2-B blocked
the reduction of food intake without influencing the de-
layed GE. Moreover, we show that nesfatin-1 injected ic or
into the 4v did not influence GE, supporting a forebrain
site of action for icv nesfatin-1 to regulate gastric propul-
sive motor function. These data also indicate that delayed
GE is unlikely to contribute to icv, ic, or 4v nesfatin-1-
induced food intake reduction. Nesfatin-1 is colocalized
with a large proportion of oxytocin neurons in the PVN (2,
16, 36) and also modulates the activity of PVN oxytocin
neurons (6). In a previous study, we showed a prominent
expression of nesfatin-1-ir in the magnocellular part of the
PVN (3), and more than 70% of those neurons have been
shown to be oxytocin-ir (16). Other neuroanatomical and
functional studies established that oxytocinergic neurons
of the PVN project to gastric subregions of the NTS (45)
and inhibit GE in rats (46).
The present studies also revealed the activation of 43% of
nesfatin-1 immunoreactivity neurons in the apPVN, a region
where many neurons also contain CRF (47), and 24% of
nesfatin-1-ir neurons in the NTS induced by ip injection of
CCK-8S at a dose known to reduce food intake (15) in ad
libitum fed rats in the dark phase under conditions where
nesfatin-1 decreased food intake. These data suggest a pos-
sible implication of nesfatin-1 in the mediation of gut hor-
mone-related reduction of food intake. Moreover, 2 h refeed-
ing after 24 h food deprivation, a condition associated with
satiety, induced Fos expression in SON and NTS neurons.
The activation of neurons in the SON during refeeding after
48 h fasting has been reported before (36). In the SON, a
region recently implicated in food intake regulation
(48), the majority (99%) of activated neurons were nes-
 4918 Stengel et al. Nesfatin-1: Central Actions to Reduce Food Intake
fatin-1-ir, whereas in the NTS other than nesfatin-1-
immunopositive neurons were activated by refeeding.
In addition to the brain, our recent report characterized
the expression of NUCB2/nesfatin-1 in the stomach, most
prominently within ghrelin cells of the rat gastric oxyntic
mucosa (20). In particular, we demonstrated a significant
down-regulation of NUCB2 in rat gastric small endocrine
cells after 24 h fasting (20). In the present study, we
showed that nesfatin-1 plasma levels decreased signifi-
cantly after 24 h fasting and returned to baseline after
refeeding, providing the first evidence of circulating
NUCB2/nesfatin-1 and its modulation by changes in feed-
ing status. However, circulating NUCB2/nesfatin-1 levels
did not change between dark phase and light phase under
ad libitum feeding conditions, suggesting the absence of
circadian changes. Collectively, these data suggest that
fasting inhibits both the synthesis and release of gastric
nesfatin-1/NUCB2. The present findings add to a recent
report showing that ip injection of nesfatin-1 at approx-
imately 60 ␮g/mouse decreases nocturnal food intake in
mice (19), which would suggest peripheral signaling in
addition to the central signaling by nesfatin-1 to suppress
food intake. However, we found that nesfatin-1 injected ip
at a dose 40-fold higher than the icv effective dose did not
alter the nocturnal food consumption in rats, indicative
that the anorexic effect of nesfatin-1 is more readily ob-
served upon brain than ip injection. Likewise in mice, nes-
fatin-1 injected ip at low doses (2 and 12 ␮g/mouse) did
not influence food intake (19). Whether the lack of effect
of nesfatin-1 upon ip injection at low doses in rodents
reflects poor pharmacokinetics to reach the site of action
under these conditions compared with direct systemic
level changes will need to be ascertained.
In conclusion, we demonstrate that 5 pmol nesfatin-1
injected icv in freely fed rats induce a delayed inhibition of
dark-phase feeding that involves the activation of CRF2
receptors. We also established a responsiveness of 4v or
cisterna magna to nesfatin-1, resulting in a rapid onset
reduction of dark-phase food intake that is CRF2 inde-
pendent. These data point to distinct forebrain and hind-
brain sites of nesfatin-1 consistent with the presence of
nesfatin-1-immunopositive cells in both hypothalamic
and hindbrain nuclei regulating ingestive behavior. Nes-
fatin-1 icv action on food intake is not linked with changes
in blood glucose levels, locomotor activity, or grooming
behavior. In addition, we established a novel central ac-
tion of nesfatin-1 to influence visceral function as shown
by the inhibition of GE selectively occurring upon icv in-
jection and mediated by mechanisms distinct from those
influencing food intake because they are CRF2 indepen-
dent. A role of hypothalamic nesfatin-1 in satiety signaling
induced by exogenous or endogenous gut peptides re-
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Endocrinology, November 2009, 150(11):4911– 4919
leased by eating is suggested by the activation of nesfatin-
1-ir neurons primarily within the apPVN by ip CCK and
SON after 2 h refeeding after a fast. Our observations of
a functional relationship between nesfatin-1 and the CRF2
signaling system after lateral brain ventricle injection to
inhibit feeding may have implications in the understand-
ing of stress-related eating disorders.
Acknowledgments
We thank Mrs. Honghui Liang for her excellent technical
support, and we thank Ms. Eugenia Hu for reviewing the
manuscript.
Address all correspondence and requests for reprints to:
Yvette Taché, Ph.D., Center for Neurovisceral Sciences and
Women’s Health CURE, Building 115, Room 117, Veterans Ad-
ministration Greater Los Angeles Healthcare System, 11301
Wilshire Boulevard, Los Angeles, California 90073. E-mail:
ytache@mednet.ucla.edu.
This work was supported by German Research Foundation
Grants STE 1765/1-1 (A.S.), GO 1718/1-1 (M.G.), Veterans Ad-
ministration Research Career Scientist Award, Veterans Admin-
istration Merit Award, NIHDK 33061, Center Grant DK-41301
(Animal Core) (Y.T.), and DK PO1-26741 (J.R.).
Disclosure Summary: A.S., M.G., L.W., P.K., H.M.,
N.W.G.L., and Y.T. have nothing to disclose. J.R. is Founder of
Sentia Medical Sciences, Inc. No conflicts of interest exist.
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