Download Hypothalamic Nesfatin-1 Stimulates Sympathetic Nerve

Diabetes Volume 64, November 2015
3725
Mamoru Tanida,1 Hitoshi Gotoh,2 Naoki Yamamoto,3 Mofei Wang,1 Yuhichi Kuda,1
Yasutaka Kurata,1 Masatomo Mori,4 and Toshishige Shibamoto1
Hypothalamic Nesfatin-1 Stimulates
Sympathetic Nerve Activity via
Hypothalamic ERK Signaling
Diabetes 2015;64:3725–3736 | DOI: 10.2337/db15-0282
Nesfatin-1 is an 82-amino acid neuropeptide produced in
the hypothalamus that acts on the brain to suppress
1Department of Physiology II, Kanazawa Medical University, Uchinada, Ishikawa,
Japan
2Department of Biology, Kyoto Prefectural University of Medicine, Kyoto, Japan
3College of Pharmacology, Hokuriku University, Kanazawa, Ishikawa, Japan
4Kitakanto Molecular Novel Research Institute for Obesity and Metabolism, Midori
City, Gunma, Japan
Corresponding author: Mamoru Tanida, [email protected].
appetite (1–6), increase energy expenditure (7), and induce cardiovascular changes, leading to body weight reduction (1), blood pressure (BP) elevation (8–10), and
increased insulin sensitivity (11) in animals. Intracerebroventricular (ICV) administration of nesfatin-1 reduces
food intake and body weight gain and elevates BP, heart
rate (HR) (8), and peripheral glucose uptake (11). Thus,
central nesfatin-1 may regulate the function of peripheral
organs through neural activity to maintain homeostasis
and regulate a number of physiological processes.
Regarding the neural mechanism of physiological regulation by nesfatin-1, our previous study demonstrated
that sympathetic nervous supply to the kidneys in anesthetized rats could be stimulated by the ICV injection of
nesfatin-1 (10), suggesting that the sympathetic nervous
system mediates the action of nesfatin-1. Recently, it has
been reported that increased sympathetic stimulation of
white adipose tissue (WAT) and the liver resulted in lipolysis (12) and glucogenesis (13), respectively, with the intracerebral administration of leptin, a feeding regulator
and sympathetic activator. Thus, central nesfatin-1 may
modulate sympathetic nerve outflow to WAT and the
liver and regulate lipid and glucose metabolism; however,
there are no studies reporting the effect of ICV nesfatin-1
on the neural activity of sympathetic nerves to WAT and
the liver.
The hypothalamus performs a crucial role in coordinating the autonomic control of abdominal organs by
the sympathetic and parasympathetic nerves. Nesfatin-1
expression has been demonstrated in several hypothalamic nuclei including the paraventricular nucleus (PVN),
supraoptic nucleus, arcuate nucleus (ARC), and lateral
Received 27 February 2015 and accepted 20 July 2015.
This article contains Supplementary Data online at http://diabetes
.diabetesjournals.org/lookup/suppl/doi:10.2337/db15-0282/-/DC1.
© 2015 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
METABOLISM
Nesfatin-1 acts on the hypothalamus and regulates the
autonomic nervous system. However, the hypothalamic
mechanisms of nesfatin-1 on the autonomic nervous
system are not well understood. In this study, we found that
intracerebroventricular (ICV) administration of nesfatin-1
increased the extracellular signal–regulated kinase (ERK)
activity in rats. Furthermore, the activity of sympathetic
nerves, in the kidneys, liver, and white adipose tissue
(WAT), and blood pressure was stimulated by the ICV
injection of nesfatin-1, and these effects were abolished
owing to pharmacological inhibition of ERK. Renal
sympathoexcitatory and hypertensive effects were also
observed with nesfatin-1 microinjection into the paraventricular hypothalamic nucleus (PVN). Moreover, nesfatin-1
increased the number of phospho (p)-ERK1/2–positive
neurons in the PVN and coexpression of the protein in
neurons expressing corticotropin-releasing hormone (CRH).
Pharmacological blockade of CRH signaling inhibited
renal sympathetic and hypertensive responses to nesfatin-1.
Finally, sympathetic stimulation of WAT and increased
p-ERK1/2 levels in response to nesfatin-1 were preserved
in obese animals such as rats that were fed a high-fat
diet and leptin receptor-deficient Zucker fatty rats.
These findings indicate that nesfatin-1 regulates the autonomic nervous system through ERK signaling in PVNCRH neurons to maintain cardiovascular function and
that the antiobesity effect of nesfatin-1 is mediated by
hypothalamic ERK-dependent sympathoexcitation in
obese animals.
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hypothalamic area (14,15). Moreover, oxytocin neurons in
the PVN play an important role in mediating feeding reduction induced by nesfatin-1 (2). These reports suggest
that neural transmission in the PVN mediates the hypothalamic action of nesfatin-1 on feeding regulation.
Hypothalamic intracellular signaling, including extracellular signal–regulated kinase (ERK), phosphoinositol-3
kinase (PI3K), and AMPK, plays important roles in anorexia and sympathetic stimulation following the central
administration of leptin (12,13,16–18). Although a receptor specific to nesfatin-1 has not yet been identified, the
different signaling systems mediated by nesfatin-1 have
been clarified. Nesfatin-1 stimulates the phosphorylation
of mitogen-activated protein kinase (MAPK) (19), CREBP
(20), and AMPK (11). However, the precise signaling
mechanisms underlying nesfatin-1–induced activation of
the sympathetic nervous tones in the brain remain to be
determined. Thus, we examined the effect of nesfatin-1
on hypothalamic intracellular signaling and, through pharmacological inhibition studies, examined the role of the intracellular pathway in mediating the effect of nesfatin-1
on sympathetic nerve stimulation of abdominal organs,
cardiovascular function, and feeding behavior.
RESEARCH DESIGN AND METHODS
Animals
Male Wistar rats (weighing 250–270 g) and Zucker fatty
rats (weighing 340–385 g) were used in these studies.
Animals were housed in a room maintained at 24 6 1°C
and illuminated for 12 h (8:00 A.M. to 8:00 P.M.). Rats had
free access to food and water, and were allowed to adapt
to the environment for at least 1 week before experimentation. Dietary obesity was induced in rats via feeding
with a 60% high-fat diet (HFD) (HFD-60; Oriental Yeast
Co., Ltd., Tokyo, Japan) for 10 weeks. All animal care and
handling procedures were approved by the Animal Research Committees of Kanazawa Medical University.
Brain Cannulation
Rats were equipped with ICV, lateral cerebroventricular,
and ARC cannulae using a stereotaxic apparatus, as
described previously (12,13). The PVN of rats was cannulated unilaterally using a 25-gauge guide cannula with
coordinates (215 mm anteroposterior, +0.5 mm mediolateral, and 27.5 mm dorsoventral with 0°) according to
the atlas of Paxinos and Watson (21). To verify the accuracy of PVN and ARC injections, the brain was sectioned,
and brain slices were counterstained with cresyl violet
solution to visualize the injection site.
Recording of Sympathetic Nerve Activity
Seven to 10 days after recovery from brain cannulation,
anesthesia was induced in rats via intraperitoneal injections
of a urethane (750 mg/kg) and a-chloralose (75 mg/kg)
mixture. Autonomic nerve activity measurements were performed as described previously (12,13). Renal sympathetic
nerve activity (RSNA), WAT sympathetic nerve activity (WATSNA) projecting to the adipose tissue of the epididymis,
Diabetes Volume 64, November 2015
and liver sympathetic nerve activity (Liv-SNA) in rats
were recorded in separate animals. The BP signal was
also sampled using PowerLab and was stored on a hard
disk for offline analysis calculating mean arterial pressure (MAP) and HR.
The baseline measurements of SNAs were made 5–10 min
prior to the ICV injection of vehicle (artificial cerebrospinal
fluid [aCSF], 10 mL) or nesfatin-1 (200 pmol/10 mL aCSF).
The other groups of rats received unilateral nesfatin-1
(50 pmol/0.5 mL aCSF) microinjection into the PVN or
ARC. In experiments using pharmacological inhibitors,
animals first received ICV U0126 (7 mg), LY294002 (5 mg),
SB203580 (0.5 mg), astressin 2B (30 mg), H4928 (9 nmol), or
vehicle (DMSO; 5 mL) followed 15 min later by ICV nesfatin-1
or vehicle (aCSF).
Feeding Experiment
The body weight and food intake of individually caged
rats were measured before and after treatment. Seven to
10 days after ICV surgery, overnight-fasted rats (n = 7
animals per group) were assigned to receive an ICV injection of vehicle (DMSO; 5 mL) or U0126 (7 mg) followed
15 min later by an ICV injection of vehicle (aCSF; 10 mL)
or nesfatin-1 (200 pmol) at 8:00 P.M. Body weight and food
intake were measured 4 and 12 h after ICV nesfatin-1 or
vehicle administration. In Zucker fatty or HFD rats (n = 5
or 6 animals per group), after overnight fasting, food intake was measured 4 h after ICV injection of vehicle or
nesfatin-1.
Immunohistochemistry and In Situ Hybridization
All rats were fasted overnight. Thirty minutes after the
lateral cerebroventricular injection of nesfatin-1 (400
pmol) or vehicle (aCSF; 10 mL), rats were anesthetized
and perfused intracardially with saline followed by 4%
paraformaldehyde in 0.1 mol/L PBS; lateral cerebroventricular injection of nesfatin-1 increased RSNA in anesthetized rats (Supplementary Fig. 1). Brains were
removed, postfixed at 4°C overnight, and cryoprotected
in 30% sucrose for 2 nights. The brains were sliced, and
then each section was treated with a 0.15% H2O2 solution
and incubated in a 0.1% BSA solution for 1 h. Thereafter,
the immunohistochemical responses of phospho (p)-ERK
in the PVN and ARC were measured as follows: sections
were incubated with primary antibody solutions of specific polyclonal rabbit antibody against p-ERK (in 1:200
dilution; Cell Signaling Technology) as long as 48 h at 4°C.
After washing, sections were incubated at 4°C overnight
with biotinylated anti-rabbit IgG secondary antibody (in
1:500 dilution; Sigma-Aldrich). After washing sections,
immunoreactivity was visualized using a Vectastain ABC
Kit (Vector Laboratories) and 3,39-diaminobenzidine (Dojindo
Molecular Technologies, Inc.) as the chromogen. Images of
the slices were examined under a microscope, and the number of p-ERK–immunoreactive cells in the PVN or ARC was
counted using ImageJ software.
For double staining of the hypothalamic slices, using
fluorescent immunohistochemistry, brain sections were
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incubated with a primary antibody at the following dilutions:
rabbit anti–p-ERK1/2, 1:200 (Cell Signaling Technology);
and mouse anti-oxytocin, 1:600 (MAB5296; Chemicon). Then,
sections were incubated with Alexa Fluor 488–labeled
and Alexa Fluor 543–labeled secondary antibody and
DAPI and were observed using a fluorescence microscope
(DP70 and DP71; Olympus) or a confocal microscope.
Using dual in situ hybridization and immunohistochemistry, we examined the types of neurons in the PVN
that colocalized with the p-ERK1/2–positive cells following
nesfatin-1 injection. Fixed brain sections were incubated
with proteinase K and acetylated with 0.1 mol/L triethanolamine containing 0.25% acetic anhydride. Digoxigeninlabeled probes were applied onto sections and incubated
at 65°C overnight. Slides were washed with 13 sodium
chloride–sodium citrate containing 50% formamide twice
followed by maleic acid buffer containing 0.1% Tween 20.
Sections were incubated with sheep anti–digoxigeninalkaline phosphatase (Roche, Zürich, Switzerland) at 4°C
overnight. Signals were detected via incubation with an
NBT/BCIP solution (Roche). For immunohistochemical
staining after in situ hybridization, sections were treated
with heat by microwaving for 5 min in 10 mmol/L citrate
buffer (pH 6.0) and cooled to room temperature before
incubation with anti–p-ERK1/2 antibody. The signals of
p-ERK were visualized using a peroxidase reaction with
3,39-diaminobenzidine as the substrate. The following
cDNAs were PCR amplified, cloned into p3T (MoBiTec,
Göttingen, Germany) vector, and used for digoxigeninlabeled probe synthesis: corticotropin-releasing hormone
(CRH; NM_031019; nt_203–758), arginine vasopressin
(AVP; NM_016992; nt_27–513), and thyrotropin-releasing
hormone (TRH; NM_013046; nt_136–903). Sections were
observed under a microscope.
Western Blotting
Rats were fasted overnight before the ICV injection of
vehicle or nesfatin-1 (100 or 200 pmol/10 mL). Thirty
minutes after ICV injections, animals were killed by decapitation, and the mediobasal hypothalamus was quickly
removed and homogenized on ice. A total protein assay
and Western blotting with primary antibodies (p-ERK1/2,
p-Akt, p-AMPK, p-p38, p-CREBP, total ERK1/2, total Akt,
total AMPK, total p38, and total CREBP) were performed
as described in our previous studies (12,13).
Measurement of CRH Content
One hundred twenty minutes after ICV injections (vehicle
or nesfatin-1), animals were killed by decapitation, brains
were quickly removed, and the PVN area was dissected in
the frozen hypothalamic sections and homogenized on
ice. The CRH level in the PVN was measured with an
ELISA Kit (YK131; Yanaihara Co., Shizuoka, Japan).
Data Analysis
All data were expressed as the mean 6 SEM. When comparing the responses of nerve activity and cardiovascular
parameters between groups, ANOVA with the Bonferroni
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post hoc test was used. When comparing the Western
blotting and immunohistochemistry data between vehicle
and nesfatin-1, the Student t test was used. P , 0.05 was
considered statistically significant.
RESULTS
Hypothalamic Nesfatin-1 Increases ERK1/2 Activity in
Rats
To determine the signaling pathways crucial for nesfatin-1
action in the hypothalamus, we examined the effect of the
ICV administration of nesfatin-1 on hypothalamic intracellular signaling factors in vivo.
ICV administration of nesfatin-1 increased the levels of
p-ERK1/2 in a dose- and time-dependent manner (Fig. 1A
and Supplementary Fig. 2), with a significant difference
observed 30 min after injection with nesfatin-1 or vehicle
(Fig. 1B). On the contrary, the levels of p38, which is also
involved in MAPK signaling, as well as those of p-Akt,
p-AMPK, and p-CREBP were unaltered 30 min after ICV
injection of nesfatin-1 (Fig. 1C–E).
Pharmacological Blockade of ERK Abrogates
Sympathetic Nerve Stimulation in Response to
Nesfatin-1
We used a pharmacological approach to examine the
effect of ERK inhibition on sympathetic activation via ICV
nesfatin-1 administration. We found that the renal sympathetic response to ICV nesfatin-1 administration was
attenuated by ICV preinjection with U0126 (an ERK
inhibitor) but not LY20996 (a PI3K inhibitor) or
SB203580 (a p38 inhibitor; Fig. 2A and B). On the basis
of our previous report (10) demonstrating that the ICV
administration of nesfatin-1 increased MAP and HR, we
examined the role of hypothalamic ERK signaling in cardiovascular regulation by nesfatin-1. ICV pretreatment with
U0126 abrogated the hypertensive and HR-elevating effect
of nesfatin-1 (Supplementary Fig. 3A and B). In addition,
ICV injection of nesfatin-1 stimulated regional SNA in
WAT (Fig. 2C) and liver (Fig. 2E) of anesthetized rats.
Increased sympathetic nerve outflows in WAT and the
liver in response to nesfatin-1 was blocked by pretreatment with U0126, but not by pretreatment with
LY20996 or SB203580 (Fig. 2D and F). To reveal the
effect of anesthetics on cardiovascular and sympathetic
responses to nesfatin-1, MAP was measured in conscious
rats, and this parameter was elevated 60 min after the ICV
injection of nesfatin-1 (before injection 112 6 4 mmHg,
postinjection 128 6 5 mmHg, change 16 6 4 mmHg, P , 0.05).
Nesfatin-1/NucB2 is expressed in a number of hypothalamic nuclei (14,15), and endogenous nesfatin-1 may
affect sympathetic neurotransmission. To examine this
hypothesis, we investigated the effect of neutralizing antibodies against nesfatin-1 on ICV nesfatin-1–induced
sympathoexcitation and found that pretreatment with
neutralizing antibodies attenuated renal sympathetic activation by ICV nesfatin-1 (Fig. 2B), whereas neutralizing
antibodies administered before the vehicle injection did
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Figure 1—Effect of ICV injection of nesfatin-1 on the phosphorylation levels of ERK, Akt, p38, AMPK, and CREBP in the hypothalamus of
rats. A: Time-course data of the Western blotting of ERK, Akt, p38, AMPK, and CREBP after ICV injection of vehicle or nesfatin-1. The bar
graphs show levels of hypothalamic p-ERK1/2 (B), p-Akt (C), p-p38 (D), p-AMPK (E), and p-CREBP (F) 30 min after the injection of vehicle
or nesfatin-1. Data are shown as the mean 6 SEM. n = 5 rats per group in this experiment. *Significant difference between vehicle group
and nesfatin-1 group was shown (P < 0.05).
not affect RSNA (Fig. 2B). These results suggest that administered nesfatin-1 acting on hypothalamic neurons,
but not on endogenous nesfatin-1, is responsible for stimulation of the sympathetic nervous system.
Crucial Role of Hypothalamic PVN ERK Signaling in
Nesfatin-1–Induced Sympathetic Activation
We further examined ERK activation induced by nesfatin-1
in hypothalamic nuclei using immunohistochemical analysis.
Lateral cerebroventricular injection of nesfatin-1 increased
the number of ERK1/2-positive cells in both the PVN and
ARC (Fig. 3A–C). We further demonstrated that lateral cerebroventricular injection of nesfatin-1 significantly increased
RSNA (Supplementary Fig. 1A and B), indicating that lateral
cerebroventricular administration also acted upon the hypothalamus to induce sympathetic activation.
To address the site of nesfatin-1 activity in the
hypothalamus that is responsible for stimulating SNA,
we investigated the effect of nesfatin-1 microinjection
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Figure 2—The possible role of the hypothalamic ERK in mediating sympathetic nerve responses to nesfatin-1 (Nes) injection. Representative neurograms of RSNA (A), WAT-SNA (C), and Liv-SNA (E) show the effect of ERK inhibitor (U1026 [U]) on regional sympathetic
responses to nesfatin-1 or vehicle (V). Bar graphs show the effects of ICV pretreatment (ICV1) with inhibitors of ERK1/2 (U0126), PI3K
(LY294002 [LY]), and p38 (SB203580 [SB]) on nesfatin-1–induced acceleration of RSNA (B), WAT-SNA (D), and Liv-SNA (F). ICV1 also
expresses preinjection of vehicle and the neutralizing antibody of nesfatin-1 (Anti), and ICV2 depicts the injection of vehicle or nesfatin-1
15 min after ICV1 injection. Data are shown as the mean 6 SEM. n = 5–9 rats per group in this experiment. *Significant difference (P < 0.05)
vs. vehicle plus vehicle group.
into the ARC or PVN on RSNA. RSNA was stimulated by
nesfatin-1 microinjection into the PVN, but not ARC, in
anesthetized rats (Fig. 3D). Using these data, the injection
sites within the ARC and PVN were demonstrated histologically (Fig. 3E). In addition, we confirmed that nesfatin-1
microinjection into the PVN elevated MAP and HR (Supplementary Fig. 3C and D).
Nesfatin-1 Stimulates ERK Signaling in the CRH
Neurons of PVN
A number of neuron types are found within the PVN.
Oxytocin neurons in the PVN are involved in the
development of anorexia, and they mediate the weightreducing effects of hypothalamic nesfatin-1 (2). Thus, we
examined ERK1/2 activation in the oxytocin neurons of
the PVN following nesfatin-1 injection using immunohistochemical double staining. We examined the PVN along
the rostrocaudal axis and found almost no oxytocin coexpression in p-ERK1/2–positive cells induced by the lateral
cerebroventricular injection of nesfatin-1 (Fig. 4A). Other
classes of neurons are found within the PVN, namely
CRH, TRH, and AVP neurons. Thus, to examine the
colocalization of p-ERK1/2–immunoreactive neurons and
other classes of neurons in the PVN, we performed double
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Figure 3—Hypothalamic nesfatin-1 regulates sympathetic nerve outflow via ERK signaling in the PVN. A: Immunohistochemical staining of
p-ERK1/2 in the PVN and ARC after lateral cerebroventricular injection of vehicle or nesfatin-1. High-magnification photographs of the
boxed area in the left panels of each group are represented in the right panels of each group. High-magnification photographs: scale bar =
300 mm in left panels; 75 mm in right panels. Bar graphs show the numbers of p-ERK–positive cells in the hypothalamic PVN (B) and ARC
(C) after lateral cerebroventricular injection of vehicle or nesfatin-1 as the mean 6 SEM. n = 5 rats per group. *Significant difference
between vehicle group and nesfatin-1 group is shown (P < 0.05). D: Time-course data of the RSNA response to nesfatin-1 microinjection
into the PVN or ARC are shown as the mean 6 SEM. n = 5–7 rats per group. *P < 0.05 vs. PVN-vehicle group. E: Nissl-staining data of
microinjection into the PVN (top panel) or ARC (bottom panel). Black arrows in the photographs show injection sites in the PVN or ARC.
3V, third ventricle; ME, median eminence; and VMH, ventromedial hypothalamus.
staining with in situ hybridization and immunohistochemistry. Interestingly, almost all neurons with ERK signaling
activation following lateral cerebroventricular nesfatin-1
administration colocalized with CRH neurons but not
with TRH or AVP neurons (Fig. 4B–G).
We next examined the role of ERK1/2 signaling in CRH
neurons of the PVN in the regulation of SNA induced by
central nesfatin-1. Pretreatment with astressin 2B, a CRH
receptor antagonist, but not H4928, an oxytocin receptor
antagonist, abolished renal sympathetic activation in
response to ICV nesfatin-1 administration (Fig. 5A and C).
Pretreatment with neither astressin 2B nor H4928 inhibited
the stimulatory response of WAT-SNA to nesfatin-1 (Fig. 5B
and D). In addition, preinjection of astressin 2B abrogated
hypertension and tachycardia induced by the ICV injection of nesfatin-1 (Fig. 5E and F). These data support the
hypothesis that ERK signaling in CRH neurons of the
PVN is important for the sympathetic and cardiovascular
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Figure 4—Double staining using double immunofluorescence or in situ hybridization and immunohistochemistry in the hypothalamic PVN
after lateral cerebroventricular injection of nesfatin-1. A: Double-immunofluorescence images show p-ERK1/2–immunoreactive cells (green)
and oxytocin-immunoreactive cells (red) separately, and the cells of colocalization are depicted in yellow in the merged photograph. Scale
bars = 100 mm. Dual staining data for in situ hybridization and immunohistochemistry (B–G) show p-ERK1/2–immunoreactive cells (brown)
and CRH- (B and E), TRH- (C and F), or AVP-positive cells (purple) (D and G) separately. High-magnification photographs of the boxed area
in B–D are shown in E–G. Scale bars = 50 mm.
action of central nesfatin-1. In addition, to reveal the
mechanism of ERK signaling regulation by nesfatin-1,
we examined the effects of astressin 2B on the response
of hypothalamic p-ERK1/2 to nesfatin-1 and found that
increased p-ERK1/2 levels in response to nesfatin-1 were
induced in the astressin 2B group (Fig. 5G and H). These
data suggest that nesfatin-1 directly activates ERK1/2
signaling in the CRH neurons of the PVN. Supporting
this idea, we illustrated that increased CRH levels in the
PVN induced by nesfatin-1 were inhibited by ICV preinjection with U0126 (ERK inhibitor; Supplementary Fig. 4).
Possible Role of Hypothalamic ERK in the Anorexic
and Weight-Reducing Actions of Nesfatin-1
To test the hypothesis that hypothalamic ERK is critical
for the feeding action of nesfatin-1, we examined the
effect of pharmacological ERK inhibition (U0126) on
appetite and body weight in response to ICV administration of nesatin-1. ICV injection of nesfatin-1 following
vehicle pretreatment significantly decreased food intake
and body weight at 4 h after the injection (Fig. 6A and B).
This treatment (vehicle plus nesfatin-1) also reduced body
weight gain at 12 h after the injection (Fig. 6D). Decreased
food intake and body weight in response to nesfatin-1
were attenuated by pretreatment with U0126 (Fig. 6A,
B, and D), suggesting that central nesfatin-1 regulates feeding behavior by reducing appetite and body weight and that
these effects are mediated by hypothalamic ERK signaling.
Central Nesfatin-1 Increases WAT-SNA in Obesity
We assessed whether the effect of central nesfatin-1 on
sympathoexcitation is mediated by the leptin receptor. In
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Figure 5—Possible role of hypothalamic CRH neurons and hypothalamic oxytocin neurons in mediating sympathetic and cardiovascular
responses to nesfatin-1 injection. Time-course data of RSNA (A) WAT-SNA (B), MAP (E), and HR (F) and bar graph data of RSNA (C) and
WAT-SNA (D) averaging the last hour of recording after nesfatin-1 (Nes) or vehicle (V) treatment were shown as the effect of CRH receptor
inhibitor (astressin 2B [Ast]) or oxytosin receptor inhibitor (H4928 [H4]) on regional sympathetic and cardiovascular responses to nesfatin-1.
Data are shown as the mean 6 SEM. n = 5–9 rats per group in this experiment. G: Representative data from Western blotting of ERK1/2 at
30 min after ICV injection of vehicle or nesfatin-1 with or without preinjection of astressin 2B. Bar graph data (H) are shown as the mean 6
SEM. n = 6 rats per group in this experiment. A, C, D, and H: *Significant difference (P < 0.05) vs. vehicle plus vehicle group. E and F:
*Significant difference (P < 0.05) between vehicle plus vehicle and vehicle plus nesfatin-1; #significant difference (P < 0.05) between H4928
plus vehicle and H4928 plus nesfatin-1. ICV1, ICV pretreatment; ICV2, ICV injection.
keeping with the previously demonstrated feeding behavior in
response to nesfatin-1, ICV nesfatin-1 administration caused
an increase in WAT-SNA in Zucker fatty rats (Fig. 7A–C),
indicating the sensitivity of sympathetic nerves to the stimulatory effects of nesfatin-1 despite loss of the leptin receptor.
In addition, we examined the effect of ICV administration of
nesfatin-1 on WAT-SNA in rats fed an HFD, an obesity model
with accompanying leptin resistance, and found that HFD-fed
rats also retained WAT-SNA sensitivity to ICV administration
of nesfatin-1 (Fig. 7A–C). Hypothalamic p-ERK1/2 levels at
30 min following the ICV administration of nesfatin-1 were
increased in Zucker fatty rats and HFD rats (Fig. 7D). In
addition, ICV-administered nesfatin-1 significantly decreased
food intake and body weight at 4 h after the injection in
Zucker fatty rats and HFD rats (Fig. 7E). These data demonstrate that central nesfatin-1–mediated stimulation of hypothalamic ERK signaling and WAT-SNA was retained in both
rat models of obesity, suggesting a beneficial action of
nesfatin-1 on obesity through autonomic nervous control.
Meanwhile, there was no significant difference in blood glucose
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Figure 6—Possible role of hypothalamic ERK in mediating the effects of nesfatin-1 (Nes) on food intake and body weight in rats. Bar graphs
show effects of pretreatment (ICV1) with ERK inhibitor (U0126 [U]) on food intake and body weight responses to ICV injection (ICV2)
of vehicle (V) or nesfatin-1. Control group was performed as vehicle injection in both ICV1 and ICV2. Data are shown as the mean 6 SEM.
n = 7 rats per group in this experiment. *Significant difference (P < 0.05) vs. vehicle plus vehicle group.
levels (mg/dL) in response to nesfatin-1 in anesthetized animals between the vehicle group (before injection 148 6 9,
240 min postinjection 136 6 13, change 213 6 13) and
nesfatin-1 group (before injection 162 6 17, 240 min
postinjection 160 6 17, change 22 6 8), and blood
glucose levels (mg/dL) in Zucker fatty rats were also not
affected by nesfatin-1 (before injection 125 6 7, 240 min
postinjection 113 6 12, change 29 6 10). However, in
the HFD group, blood glucose levels were elevated following
nesfatin-1 administration (before injection 153 6 15, 240
min postinjection 214 6 23, change 60 6 21).
DISCUSSION
Central nesfatin-1 acts on the hypothalamus to regulate
physiological processes such as feeding behavior, cardiovascular function, and energy metabolism (1–7). In the
current study, we determined a previously unreported
mechanism of nesfatin-1 activity through the modulation
of hypothalamic intracellular signaling. Our data demonstrate that nesfatin-1 stimulates hypothalamic ERK signaling and suggest that this pathway is involved in the
nesfatin-1–mediated regulation of feeding behavior, SNA,
and cardiovascular function. Furthermore, we elucidated
the detailed mechanism underlying this effect. Central
nesfatin-1 stimulates ERK1/2 phosphorylation in the
CRH neurons of the PVN, resulting in selective sympathoexcitation of the kidneys, but not of WAT, or the elevation of BP. Finally, this study obtained data demonstrating
that hypothalamic nesfatin-1 increased WAT-SNA and ERK
activity and suppressed food intake independently of leptin
receptor signaling, and this activity led to the stimulation of
sympathetic outflow to WAT and the feeding suppression
of obese rats fed an HFD. These findings suggest that ERK
signaling in the CRH neurons of the PVN may have a crucial
role in accelerating renal sympathetic nerve outflow, BP,
and HR. Moreover, hypothalamic ERK signaling appears
to underlie the sympathoexcitatory action of nesfatin-1,
independent of leptin signaling, on energy intake and fat
metabolism.
Recently, in vitro experiments using a neural cell line
demonstrated that nesfatin-1 stimulates the phosphorylation of MAPK (19), CREBP (20), and AMPK (11), playing
important roles in regulating feeding behavior and energy
metabolism in vivo (12,13,16–18,22,23). Our in vivo finding that the ICV injection of nesfatin-1 concentration
dependently increased MAPK activity (phosphorylation
of ERK1/2), but not AMPK, PI3K, or CREBP activity,
corroborates this finding. Then, we demonstrated that
pharmacological inhibition of hypothalamic ERK signaling attenuated RSNA, WAT-SNA, and Liv-SNA in response to nesfatin-1. Interestingly, the hypertensive
and HR-elevating actions of nesfatin-1 were also abrogated by ERK inhibition. Thus, we suggest that hypothalamic nesfatin-1 activity is mediated by ERK signaling,
which modulates autonomic nervous system and cardiovascular function.
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Nesfatin-1 Regulates Sympathetic Nervous System
Diabetes Volume 64, November 2015
Figure 7—ICV injection of nesfatin-1 (Nes) activates sympathetic nerve outflow to the WAT and hypothalamic ERK signaling in the HFD rats
and Zucker fatty rats. Representative neurograms (A), time-course data (B), and bar graph data (C) of nerve discharges show the effect of
ICV nesfatin-1 or vehicle (V) on WAT-SNA in HFD rats and Zucker fatty rats. Data are shown as the mean 6 SEM. n = 5–10 rats per group in
this experiment. D: Representative data of Western blotting of ERK1/2 at 30 min after ICV injection of vehicle or nesfatin-1 in each rat. E: Bar
graphs show effects of vehicle or nesfatin-1 on food intake in HFD rats and Zucker fatty rats. Data are shown as the mean 6 SEM. n = 5 or 6
rats per group in this experiment. *Significant difference (P < 0.05) vs. vehicle group.
The current study clearly demonstrated that hypothalamic nesfatin-1 stimulated sympathetic nerves, including
those innervating the kidneys, WAT, and liver, and the
cardiovascular system, possibly through the upregulation
of ERK activation in the PVN. On the contrary, neuroanatomical studies identified autonomic neural connections
between PVN and abdominal organs, including the
kidneys, WAT, and liver, as injection of the pseudorabies
virus into these organs resulted in virus-positive cells in
the PVN (24–27). Infected neurons were also identified at
other sites within the hypothalamus and extrahypothalamic nuclei (24–27). Nesfatin-1 administration into the
nucleus of the solitary tract, a pseudorabies virus–positive
area, elevated BP and HR (28), suggesting that an extrahypothalamic area may be responsible for central nesfatin-1
activity within the brain stem. Therefore, additional investigations are required to elucidate the role of the PVN and/or
other nuclei in mediating the sympathetic nerve response
evoked by nesfatin-1.
Several types of endogenous peptide-containing neurons, including CRH, TRH, oxytocin, and AVP neurons, have
been localized to the hypothalamic PVN (29–31). Thus, the
current study examined which types of neurons in the
PVN are involved in nesfatin-1–induced sympathoexcitation
diabetes.diabetesjournals.org
through ERK signaling. The results demonstrated that
ERK1/2-positive cells induced by the central administration of nesfatin-1 colocalized with CRH-expressing neurons in PVN but not with neurons expressing TRH, oxytocin,
or AVP, and supporting these data, ICV nesfatin-1 administration increased CRH levels in PVN. In addition, we
determined that hypothalamic nesfatin-1 selectively stimulated SNA in the kidneys, but not in WAT or the cardiovascular system, through CRH neurons. It appears that
ERK signaling in CRH neurons in the PVN might contribute to the regulation of autonomic and cardiovascular
functions by central nesfatin-1 as an underlying mechanism of the hypothalamic action of nesfatin-1, but the
mechanism by which nesfatin-1 activates ERK signaling
in PVN is unknown. Our data indicated that CRH receptor blocking did not affect the increased phosphorylation of ERK1/2 induced by ICV administration of
nesfatin-1, suggesting that stimulated ERK signaling
in CRH neurons in PVN is induced by a direct action
of nesfatin-1, opposed to secondary action of CRF released from CRH neurons. Similarly, an in vitro study
(32) revealed that CRH failed to stimulate ERK. In addition, an increase in CRH levels in PVN in response to
nesfatin-1 was attenuated by preinjection of an ERK
inhibitor, supporting our aforementioned idea.
Leptin, an appetite suppressor released from WAT, has
been demonstrated to act on the hypothalamus through
a similar mechanism as nesfatin-1 in stimulating SNA in
rats (12,13,16); however, the anorexic effects of hypothalamic nesfatin-1 are not mediated by the same mechanism
observed with activation of the leptin receptor (2). In the
current study, the effects of nesfatin-1 on WAT-SNA were
also preserved in Zucker fatty rats lacking the leptin receptor. Interestingly, rats fed an HFD, causing obesity and
leptin resistance, also had increased WAT-SNA in response to nesfatin-1. In addition, both HFD and Zucker
fatty rats had intact hypothalamic ERK signaling sensitivity and anorexic responses to central nesfatin-1. These
results suggest that central nesfatin-1 suppresses appetite and activates SNA in rats, independent of leptin
signaling. Thus, nesfatin-1 may have antiobesity activity
in obese animals through a neural pathway mediated by
hypothalamic ERK signaling, leading to sympathetic
stimulation of WAT and a consequent increase in energy
metabolism.
The current study had a number of limitations that
should be addressed. First, our study could not determine
whether nesfatin-1 action on the CRH neurons in PVN is
associated with hypothalamic proopiomelanocortin neurons because previous studies of neural circuits mediating
the hypertensive effect of hypothalamic nesfatin-1 in the
hypothalamus indicated that proopiomelanocortin neurons in the ARC are the primary neurons activated by
nesfatin-1 before signaling to CRH neurons in the PVN as
secondary neurons (9). CRH neurons in the PVN are stimulated by nesfatin-1 via two distinct mechanisms; nonetheless, our data demonstrated that nesfatin-1 injection into
Tanida and Associates
3735
the PVN, but not into the ARC, caused renal sympathetic
nerve activation and BP elevation. This suggests that the
direct action of nesfatin-1 on CRH neurons in PVN is
important in the regulation of SNA and cardiovascular
function. Second, the physiological relevance of nesfatin1–induced sympathoexcitation appears to be tissue specific and dependent on the innervation of the organ. For
instance, in our study, increased neural activity in the
kidneys and WAT induced by nesfatin-1 resulted in BP
elevation and metabolic acceleration resulting in body
weight reduction, respectively. On the contrary, previous
studies (13) on the physiological significance of sympathetic innervation of the liver demonstrated hepatic autonomic control of glucose metabolism, as stimulation of
liver sympathetic nerves resulted in hyperglycemia. However, our data illustrating that ICV nesfatin-1–induced
increases in Liv-SNA did not affect blood glucose levels
are inconsistent with those of previous studies of hepatic
autonomic innervation. Because increased parasympathetic stimulation of the liver suppresses glucose production (13), central nesfatin-1 may also increase hepatic
parasympathetic activity, resulting in unchanged blood
glucose levels. Of course, we will need to investigate this
hypothesis in the future.
In conclusion, we demonstrated that ERK signaling in
CRH neurons of the hypothalamic PVN plays a crucial role
in the regulation of SNA in the kidneys and cardiovascular
function by central nesfatin-1. In addition, we described
an ERK-mediated effect of nesfatin-1 on the activation of
WAT-SNA in Zucker fatty rats and a diet-induced rat
model of obesity, suggesting that nesfatin-1, independent
of leptin activity, has beneficial effects in improving
obesity through hypothalamic ERK-SNA signaling.
Funding. This study was supported by grants (to M.T.) from the Ministry of
Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for
Young Scientists 21689008 and 26870672), the Promoted Research from
Kanazawa Medical University (S2014-2), and the Takeda Science Foundation.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
Author Contributions. M.T. conceived and designed the experiments,
performed the experiments, analyzed the data, contributed reagents/materials/
analysis tools, and wrote the article. H.G. performed the experiments and wrote
the article. N.Y. performed the experiments. M.W. and Y. Kud. analyzed the data.
Y. Kur., M.M., and T.S. contributed reagents/materials/analysis tools. M.T. is the
guarantor of this work and, as such, had full access to all the data in the study
and takes responsibility for the integrity of the data and the accuracy of the data
analysis.
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