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 PVN-CRH neurons to maintain cardiovascular function and that the antiobesity effect of nesfatin-1 is mediated by hypothalamic ERK-dependent sympathoexcitation in obese animals.
Nesfatin-1 is an 82-amino acid neuropeptide produced in the hypothalamus that acts on the brain to suppress 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 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
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 ± 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.
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 (−15 mm anteroposterior, +0.5 mm mediolateral, and −7.5 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 α-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 (WAT-SNA) projecting to the adipose tissue of the epididymis, 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 μL) or nesfatin-1 (200 pmol/10 μL aCSF). The other groups of rats received unilateral nesfatin-1 (50 pmol/0.5 μL aCSF) microinjection into the PVN or ARC. In experiments using pharmacological inhibitors, animals first received ICV U0126 (7 μg), LY294002 (5 μg), SB203580 (0.5 μg), astressin 2B (30 μg), H4928 (9 nmol), or vehicle (DMSO; 5 μL) followed 15 min later by ICV nesfatin-1 or vehicle (aCSF).
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 μL) or U0126 (7 μg) followed 15 min later by an ICV injection of vehicle (aCSF; 10 μL) 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 μL), 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 3,3′-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 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. Digoxigenin-labeled probes were applied onto sections and incubated at 65°C overnight. Slides were washed with 1× sodium chloride–sodium citrate containing 50% formamide twice followed by maleic acid buffer containing 0.1% Tween 20. Sections were incubated with sheep anti–digoxigenin-alkaline 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 3,3′-diaminobenzidine as the substrate. The following cDNAs were PCR amplified, cloned into p3T (MoBiTec, Göttingen, Germany) vector, and used for digoxigenin-labeled 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.
Rats were fasted overnight before the ICV injection of vehicle or nesfatin-1 (100 or 200 pmol/10 μL). 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).
All data were expressed as the mean ± SEM. When comparing the responses of nerve activity and cardiovascular parameters between groups, ANOVA with the Bonferroni 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.
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 ± 4 mmHg, postinjection 128 ± 5 mmHg, change 16 ± 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 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 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 weight-reducing 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 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 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 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 levels (mg/dL) in response to nesfatin-1 in anesthetized animals between the vehicle group (before injection 148 ± 9, 240 min postinjection 136 ± 13, change −13 ± 13) and nesfatin-1 group (before injection 162 ± 17, 240 min postinjection 160 ± 17, change −2 ± 8), and blood glucose levels (mg/dL) in Zucker fatty rats were also not affected by nesfatin-1 (before injection 125 ± 7, 240 min postinjection 113 ± 12, change −9 ± 10). However, in the HFD group, blood glucose levels were elevated following nesfatin-1 administration (before injection 153 ± 15, 240 min postinjection 214 ± 23, change 60 ± 21).
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.
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 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 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 nesfatin-1–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.