Ghrelin in the lateral parabrachial nucleus influences the excitability of glucosensing neurons, increases food intake and body weight

in Endocrine Connections
View More View Less
  • 1 Special Medicine Department, College of Basic Medicine, Qingdao University, Qingdao, China
  • 2 Hyperbaric Oxygen Therapy Department, Yuhuangding Hospital Affiliated to Qingdao University, Yantai, China
  • 3 Physiology Department, College of Basic Medicine, Qingdao University, Qingdao, China
  • 4 Intensive Care Unit Department, Affiliated Hospital of Qingdao University, Qingdao, China
  • 5 Institute of Acupuncture, Shandong University of Traditional Chinese Medicine, Jinan, China
  • 6 Medical Microbiology Department, College of Basic Medicine, Qingdao University, Qingdao, China

Correspondence should be addressed to B Wang or J Dong: wangbin532@126.com or dongjing6@hotmail.com

*(C Zhang and J Yuan contributed equally to this work)

Ghrelin plays a pivotal role in the regulation of food intake, body weight and energy metabolism. However, these effects of ghrelin in the lateral parabrachial nucleus (LPBN) are unexplored. C57BL/6J mice and GHSR−/− mice were implanted with cannula above the right LPBN and ghrelin was microinjected via the cannula to investigate effect of ghrelin in the LPBN. In vivo electrophysiological technique was used to record LPBN glucose-sensitive neurons to explore potential udnderlying mechanisms. Microinjection of ghrelin in LPBN significantly increased food intake in the first 3 h, while such effect was blocked by [D-Lys3]-GHRP-6 and abolished in GHSR−/− mice. LPBN ghrelin microinjection also significantly increased the firing rate of glucose-excited (GE) neurons and decreased the firing rate of glucose-inhibited (GI) neurons. Additionally, LPBN ghrelin microinjection also significantly increased c-fos expression. Chronic ghrelin administration in the LPBN resulted in significantly increased body weight gain. Meanwhile, no significant changes were observed in both mRNA and protein expression levels of UCP-1 in BAT. These results demonstrated that microinjection of ghrelin in LPBN could increase food intake through the interaction with growth hormone secretagogue receptor (GHSR) in C57BL/6J mice, and its chronic administration could also increase body weight gain. These effects might be associated with altered firing rate in the GE and GI neurons.

Abstract

Ghrelin plays a pivotal role in the regulation of food intake, body weight and energy metabolism. However, these effects of ghrelin in the lateral parabrachial nucleus (LPBN) are unexplored. C57BL/6J mice and GHSR−/− mice were implanted with cannula above the right LPBN and ghrelin was microinjected via the cannula to investigate effect of ghrelin in the LPBN. In vivo electrophysiological technique was used to record LPBN glucose-sensitive neurons to explore potential udnderlying mechanisms. Microinjection of ghrelin in LPBN significantly increased food intake in the first 3 h, while such effect was blocked by [D-Lys3]-GHRP-6 and abolished in GHSR−/− mice. LPBN ghrelin microinjection also significantly increased the firing rate of glucose-excited (GE) neurons and decreased the firing rate of glucose-inhibited (GI) neurons. Additionally, LPBN ghrelin microinjection also significantly increased c-fos expression. Chronic ghrelin administration in the LPBN resulted in significantly increased body weight gain. Meanwhile, no significant changes were observed in both mRNA and protein expression levels of UCP-1 in BAT. These results demonstrated that microinjection of ghrelin in LPBN could increase food intake through the interaction with growth hormone secretagogue receptor (GHSR) in C57BL/6J mice, and its chronic administration could also increase body weight gain. These effects might be associated with altered firing rate in the GE and GI neurons.

Introduction

Ghrelin is a 28 amino acids acylated peptide, which was first discovered as an endogenous natural ligand for the growth hormone secretagogue receptor(GHSR) 1α by Kojima et al. in 1999 (1). Ghrelin was first described to stimulate the release of growth hormone from the pituitary (1, 2, 3). Then it was reported to be also involved in the regulation of feeding (4), body weight (5), and energy metabolism (6). A key role had been established for ghrelin in energy metabolism regulation. Ghrelin acts on its receptor GHSR, a G-protein-coupled receptor in diverse central and peripheral areas to exert multiple effects (7). In peripheral, circulatory ghrelin is primarily produced by X-A cells in stomach (8) and its main orexigenic effects are exerted in the brain. In central, ghrelin-producing neurons are located in the hypothalamus (1, 9, 10), while GHSR were expressed dispersedly and widely in forebrain, hypothalamus and hindbrain (11, 12), indicating that ghrelin might act as a neuroregulatory transmitter in various brain regions. Previous studies have shown that ghrelin injection into hypothalamic nuclei such as arcuate nucleus (ARC), paraventricular nucleus (PVN) and lateral hypothalamic nucleus (LH) could induce polyphagia and body weight gain (13). These effects were also observed following ghrelin injection into the fourth ventricle and dorsal vagal complex (DVC) in brainstem (14). Additionally, GHSR mRNA has been detected in the lateral parabrachial nucleus (LPBN) (11, 15). However, whether LPBN ghrelin-signaling mediates the regulation of energy balance remains unknown.

LPBN is a brain structure located in the dorsolateral pons that surrounds the superior cerebellar peduncle (16), and the latter has become an increasingly recognized region involved in the control of food intake and energy balance. Abundant direct nerve projections are present between LPBN and hypothalamus or brainstem. Moreover, many feeding relevant peptides including PYY (17), leptin (18) and glucagon-like peptide-1 (GLP-1) (19) are involved in the control of food intake in the LPBN. Besides, our recent research showed that administration of anorexia peptide nesfatin-1 in rats’ LPBN could modulate food intake, body weight and enhance uncoupling protein1 (UCP-1) expression in brown adipose tissue (BAT) (20). However, the acute and chronic effects and mechanisms of ghrelin in the LPBN on feeding behavior and energy expenditure are yet to be elucidated.

Glucose is crucial to the central control of energy homeostasis (21). The neuronal glucosensation was first defined by Mayer in 1953 as that certain neurons could regulate food intake by transducing glucose level fluctuations into neural signals (22). Glucose-sensitive (GS) neurons possess the ability to alter their firing rates in response to fluctuating ambient glucose concentrations (23) as well as participating in food intake regulation (24, 25). GS neurons are classified as glucose-excited (GE) neurons and glucose-inhibited (GI) neurons. The GI neurons play especially important roles in the counterregulatory response to hypoglycemia (25, 26). A recent study showed that ghrelin is permissive for the counterregulatory response (27), indicating that ghrelin signaling might be closely related to the physiological function of GS neurons. In fact, these specific neurons distribute widely in the central nervous system (CNS), and our previous studies have demonstrated that they were involved in the mechanisms of feeding regulations in PVN, LH and ventromedial nucleus (VMH) (24), as well as DVC in brainstem (25). Recently, LPBN was proposed to serve as a novel glucose-sensitive territory in the brain, in addition to the well-known hypothalamic and hindbrain centers (28, 29), which aroused our interest. Our previous studies proposed that ghrelin could influence the excitability of GS neurons in rats and take part in regulating food intake in hypothalamic nucleus (PVN, VMH, LH) (30). With the addition of our recent finding that GS neurons were associated with the feeding and energy regulation in LPBN (20).

In the current study, we investigated the influence of LPBN ghrelin in the firing rate of GS neurons by the in vivo electrophysiological technique. Then we explored whether LPBN ghrelin could increase the cumulative food intake and body weight and whether GS neurons were involved in the mechanism of ghrelin’s effects mentioned previously.

Materials and methods

Animals

Adult male wild-type (GHSR+/+) and GHSR-knockout (GHSR−/−) mice (on a C57BL/6J background) were housed in standard rodent cages. The original GHSR−/− mice were purchased from Shanghai Research Center For Model Organisms Of China, and the GHSR−/− mice were obtained from crosses between heterozygous and homozygous knockout animals that were backcrossed over 10 generations (31). Animals (ages 8 weeks) were housed in a temperature-controlled (23 ± 2°C) animal room (illumination from 7:00 to 19:00), with free access to standard food and tap water for at least 1 week to adapt to their surroundings. The experimental protocols were approved by the Qingdao University Animal Care and Use Committee and Animal Welfare Committee (No. QYFY WzLL25734) in accordance with the National Institutes of Health guidelines.

Reagents

Ghrelin was purchased from Tocris Bioscience (No. 1463/1). [D-Lys3]-GHRP-6 (GHSR antagonist) was purchased from APEXBIO (No. B5234). Pontamine sky blue (C8679) and glucose (G7528) were purchased from Sigma-Aldrich (for electrophysiology experiment). 0.9% NaCl and glucose were obtained from Qingdao University hospital. The chloral hydrate was purchased from Tianjin Ruijinte chemical company. All other chemicals are of the highest grade obtainable.

Surgery

C57BL/6J mice and GHSR−/− mice were anesthetized with a combination of chloral hydrate (400 mg/kg, i.p.) and ketorolac (1 mg/kg, i.m.) (32) and then positioned in a stereotaxic apparatus (SN-3; Narishige, Tokyo, Japan) for implantation of a 26-gauge chronic guide cannula above the right LPBN without damaging the target area. The placement coordinates for the LPBN were: anteroposterior 5.0 mm, lateral 1.2 mm and ventral 3.3 mm. After surgery, mice were individually housed and allowed to recover for 7 days before further experiments.

Experimental procedures

Experiment 1: Effects of LPBN ghrelin microinjection on nocturnal food intake in C57BL/6J mice and GHSR−/−- mice

To measure the effects of LPBN ghrelin microinjection on nocturnal food intake in C57BL/6J mice, mice (n = 13) were randomly assigned into two groups: NS (0.5 μL normal saline, n = 7) or ghrelin (0.5 μL, 300 pmol/mouse, n = 6) (33). On the day of experiment, mice were fasted for 2 h, from 16:00 to 18:00. At 18:00, mice were administrated NS or ghrelin with a finer injector via a microsyringe extended below the guide cannula. Then, 12 h food intake after refeeding was measured using an electronic scale (TE412-L; 110 Sartorius, Gottingen Germany). Schematic diagram of catheterization and method for determination of injection site were both as described in our previous study (20).

To investigate whether these effects of LPBN ghrelin on nocturnal food intake were mediated through GHSR, C57BL/6J mice (n = 22) were randomly assigned into four groups: NS (0.5 μL normal saline, n = 5) or ghrelin (0.5 μL, 300 pmol/mouse, n = 7) or [D-Lys3]-GHRP-6 (GHSR antagonist, 0.5 μL, 10 nmol/mouse, n = 5) or co-injection of ghrelin and [D-Lys3]-GHRP-6 (0.5 μL, n = 5). GHSR−/− mice (n = 7) were randomly assigned into two groups: NS (0.5 μL normal saline, n = 3) or ghrelin (0.5 μL, 300 pmol/mouse, n = 4). The experimental treatment is the same as described previously, 3 h food intake after refeeding was measured.

Experiment 2: Effects of LPBN ghrelin microinjection on long-term body weight gain in C57BL/6J mice

To investigate the effects of chronic LPBN ghrelin administration on long-term body weight gain, two groups–C57BL/6J mice (NS (0.5 μL normal saline, n = 7) as control group and ghrelin (0.5 μL, 300 pmol/mouse/day, n = 6)) as treatment group were injected with NS or ghrelin (13). Mice were fasted for 2 h before daily monitor of their body weight before the injection. Ghrelin injections were administered at 18:00 daily from day 1 and lasted for 7 days. On the 8th day’s morning, animals were killed.

Experiment 3: Effects of ghrelin microinjection on glucosesensitive neurons in the LPBN

C57BL/6J mice (n = 44) were anesthetized with urethane (1 g/kg body weight, i.p.) and positioned on a stereotaxic apparatus. The placement coordinates for the LPBN were: anteroposterior 5.0–5.2 mm, lateral 1.0–1.2 mm and ventral 3.3–3.4 mm.

Four-barrel glass microelectrodes (total tip diameter 3–10 Am, resistance 5–20 MV) were used for electrophysiological recording and micro-pressure injection (20, 30): 5 mM glucose solution , 0.9% NaCl , 1.5 × 10−8 M ghrelin and 0.5 M sodium acetate in 2% pontamine sky blue for recording. During the electrophysiological recording, the first three microelectrodes were connected to a four-channel pressure injector (PM2000B; Micro Data Instrument, Inc., USA) to inject drugs by gas pressure (30). Drugs were directly ejected on the surface of firing cells with short pulse gas pressure (1500 ms, 5.0–15.0 psi) (34). The recorded electrical signals were amplified and displayed on a Memory Oscilloscpe (VC-11, Nihon Kohden) as described in our previous study (35). The acquired analog signals were fed into a signal analyzer and then relayed to computer for analysis with Histo software. Although the firing frequencies of neurons vary, the sampling threshold was set to twice the background noise level, signals below which were considered as background noises and therefore not recorded to improve the accuracy of the measurements.

Table 1

Numbers of LPBN neurons responsive to glucose (5 mM) and ghrelin (1.5 × 10−8 M).

LPBN neurons (n = 44)Ghrelin responsiveGhrelin nonresponsive
ExcitedInhibited
GE neurons (n = 18; 40.9%)10 (55.6%) 4 (22.2%)4 (22.2%)
GI neurons (n = 17; 38.6%) 3 (17.6%)11(64.7%)3 (17.6%)
Glucose-insensitive neurons (n = 9; 20.4%) 3 (33.3%) 1 (11.1%)5 (55.6%)

GE neurons, glucose-excited neurons; GI neurons, glucose-inhibited neurons.

After recording the firing frequencies of neurons in the LPBN, we injected 5 mM glucose solution to identify whether they were glucose-sensitive neurons. After the firing frequencies returned to baseline, 0.9% NaCl was given as a control. Next, the 1.5 × 10−8 M ghrelin was administered to investigate the effects of ghrelin on glucose-sensitive neurons in the LPBN. At the end of each experiment, pontamine sky blue was injected through the electrode to confirm positions of the recording electrode.

To distinguish between GE and GI neurons, the spontaneous firing frequency before and after injection of 5 mM glucose solution were recorded and analyzed. Neurons with an elevation of firing rate greater than 20% were identified as GE neurons, while those with a reduction of firing rate greater than 20% were identified as GI neurons. Cells with firing rate changes less than 20% were classified as glucose-insensitive neurons.

After the spontaneous firing frequency of identified GS neurons had returned to the baseline, ghrelin was injected. If more than 20% changes in spontaneous firing frequency were observed (increased or decreased), the neurons were recorded and analyzed.

Experiment 4: Effects of chronic LPBN ghrelin administration on UCP-1 expression in brown adipose tissue

In order to assess the UCP-1 expression at both mRNA and protein levels, BATs from interscapular region were collected soon after the mice were killed in experiment 2.

Western blot

The BATs were homogenized in RIPA buffer (P0013B, Beyotime, Beijing, China) supplemented with 1:100 protease inhibitors (P1005, Beyotime, Beijing, China) and centrifuged at 14,000 g, 10 min. Protein concentrations were adjusted with BCA assay (P0012, Beyotime, Beijing, China), denatured in SDS sampling buffer (95°C, 5 min) and then subjected to 12% SDS-PAGE. Protein samples were then electrotransferred to a polyvinylidene fluoride membrane (Millipore Corp.) for 2 h. After blocking with 5% fetal bovine serum (FBS) for 2 h at room temperature, membranes were incubated with primary antibodies: UCP-1 (ab10983, 1:2000; Abcam); β-actin (#4967, 1:4000 CST, Danvers, MA, USA) at 4°C overnight and with secondary antibodies (goat anti-rabbit IgG H&L (HRP (ab6721, Abcam, 1:2000)) at room temperature for 1 h. Bands were visualized with Immobilon Western chemiluminescent substrate (Millipore, cat. no. WBKLS0100, 200 µL) and UVP 810 gel-imager (UVP, Upland, CA, USA), and the intensities were analyzed using Image J software (NIH, USA).

Real-time PCR

TRIzol reagents (135306; Ambion) were used to extract RNA from BATs. We used 4× gDNA Wiper Mix to rinse genomic DNA, and used 5× HisScript II qRT SuperMix II form RNA reverse-transcription. Relative mRNA levels were determined using the SYBR Green RT-PCR Kit (Q311; Vazyme Biotech Co., Ltd., Nanjing, China) and the Realplex Real Time PCR Thermocycle Instrument (Realplex 4; Eppendorf, Westbury, NY, USA). Primer sequences we used in this study were as follows: UCP-1: 5′-A C T G C C A C A C C T C C A G T C A T T-3′, 5′-C T T T G C C T C A C T C A G G A T T G G-3′; β-actin: 5′-A G G C C C A G A G C A A G A G A G G T A-3’, 5’-G G G G T G T T G A A G G T C T C A A A C A-3′ (32). Real-time PCR data were analyzed using the 2–∆∆Ct method. Ribosomal L32 mRNA levels were used as the internal control.

Experiment 5: Effects of LPBN ghrelin microinjection on c-fos expression in LPBN.

C57BL/6J mice (n = 6) were killed 2 h after injection of NS (n = 3) or ghrelin (n = 3) into the LPBN. Then brains were carefully dissected for subsequent immunohistochemistry.

Immunohistochemistry

Brains samples were fixed with 4% paraformaldehyde for 24 h and were transferred into 30% sucrose at 4°C for 12 h. Samples were then cryo-sectioned into 15 μm sections by microtome (CM1860; Leica) and dried at 60°C in an incubator (DHG-9101; SANFA, Yangzhou, China) for 4 h. Sections were then hydrated in 0.01 M PBS for 15 min, antigen retrieval was performed following manufacturer’s instructions (P0083, Beyotime, Beijing, China). Sections were further washed with 0.01 M PBS for three times, blocked with 0.1% FBS for 1 h at 37°C, and then incubated with a c-fos primary antibody (rabbit antimouse, ab190289, 1:1000; Abcam) for 2 h at 37°C and HRP (horse radish peroxidase) secondary antibody (PV-6001, 1:200; Zsbio, Tianjin, China) for 20 min at 37°C. Between incubation by antibody and after incubation by secondary antibody, sections were washed as described previously. A DAB kit (ZLI-9018; Zsbio) was used to develop the staining. Morphology was assessed using a light microscope (CX31; OLYMPUS). According to the mouse brain in stereotaxic coordinates (36), LPBN is located in the dorsolateral pons that surrounds the superior cerebellar peduncle (scp), which we considered as the neuroanatomical landmark to delimit LPBN in this work. By using ImagePro Plus 6 (Media Cybernetics, Inc. USA), the number of c-fos immunopositive neurons within LPBN were counted.

Statistical analysis

Data are expressed as means ± s.e.m. Statistical analyses were performed using a commercially available statistical package (SPSS 17.0). Normal distribution was confirmed with Shapiro–Wilk test, and homogeneity of variances was determined with Levene’s test. We applied Student’s t-test to analyze statistical differences between two groups, to compare firing rates before and after drug treatment, and one-way ANOVA for multiple groups. Data were analyzed using repeated measures analyses of variance where appropriate. Degree of freedom and the statistic's value were also reported. In all cases, P < 0.05 was considered to be significant.

Results

Effects of ghrelin injection in the LPBN on nocturnal feeding in mice

Comparing with the 0.9% NaCl group, ghrelin (300 pmol) injection into the LPBN significantly increased the cumulative food intake at 1st, 2nd and 3rd h (1st h 0.81 ± 0.07 vs 0.31 ± 0.09, P < 0.05; 2nd h 1.10 ± 0.09 vs 0.64 ± 0.05, P < 0.05; 3rd h 1.40 ± 0.13 vs 1.04 ± 0.09, P < 0.05; Fig. 1A).

Figure 1
Figure 1

Effects of ghrelin injection in LPBN on nocturnal feeding in mice. (A) Ghrelin increased food intake in 1, 2, 3 h after injection of ghrelin in LPBN. (B) [D-Lys3]-GHRP-6 blocked the effects of LPBN ghrelin on food intake in 2, 3 h after injection of ghrelin in LPBN. (C) Ghrelin receptor knockout abolished the effects of LPBN ghrelin on food intake in 1, 2, 3 h after injection of ghrelin in LPBN. *P < 0.05 ghrelin vs 0.9% NaCl, #P < 0.05 GHRP-6+ghrelin vs GHRP-6.

Citation: Endocrine Connections 9, 12; 10.1530/EC-20-0285

The orexigenic effect of ghrelin was blocked by [D-Lys3]-GHRP-6 when comparing co-injection group with ghrelin only group (1st h 0.67 ± 0.10 vs 0.43 ± 0.05, P < 0.05; 2nd h 0.98 ± 0.12 vs 1.03 ± 0.10, P > 0.05; 3rd h 1.82 ± 0.15 vs 1.57 ± 0.12, P > 0.05; Fig. 1B). And it was further abolished in GHSR−/− mice when comparing GHSR−/− mice injected with ghrelin to GHSR−/− mice injected with NaCl (1st h 0.48 ± 0.17 vs 0.23 ± 0.05, P > 0.05; 2nd h 0.94 ± 0.20 vs 0.70 ± 0.14, P > 0.05; 3rd h 1.37 ± 0.34 vs 1.24 ± 0.08, P > 0.05; Fig. 1C).

Effects of ghrelin injection in LPBN on chronic body weight gain in mice

For mice treated with 300 pmol ghrelin daily for 7 days, their body weight gain was significantly higher relative to mice treated with 0.9% NaCl injection starting from the 4th day (4th day 1.45 ± 0.15 vs 0.82 ± 0.22, P < 0.05; 5th day 1.97 ± 0.26 vs 1.17 ± 0.25, P < 0.05; 6th day 2.19 ± 0.28 vs 1.12 ± 0.20, P < 0.05; Fig. 2).

Figure 2
Figure 2

Effects of ghrelin injection in LPBN on long-time body weight gain in mice. After the injection of ghrelin, body weight gain was increased from the 4th day. *P < 0.05 ghrelin vs 0.9% NaCl.

Citation: Endocrine Connections 9, 12; 10.1530/EC-20-0285

Effects of ghrelin on the LPBN glucosesensitive neurons in vivo

Forty-four GS neurons were investigated in the LPBN, among which 40.9% were GE neurons and 38.6% were GI neurons (Table 1). The general average spike frequency of GE neurons in the absence and presence of glucose were 3.74 and 8.16 Hz, respectively, and the data of GI neurons were 4.40 and 1.88 Hz. Treatment with ghrelin (1.5 × 10−8 M) significantly increased the firing rates of GE neurons (3.19 ± 0.64 to 6.56 ± 1.21 Hz, P < 0.05; Fig. 3A) and decreased the firing rates of GI neurons relative to the control group (3.66 ± 0.77 to 1.51 ± 0.34 Hz, P < 0.05; Fig. 3B).

Figure 3
Figure 3

Effects of ghrelin on LPBN glucose-sensitive neurons in vivo. (A) Ghrelin of LPBN affects the activity of glucose-excited neurons. (B) Ghrelin of LPBN affects the activity of glucose-inhibited neurons. The first arrow indicates the addition of 5 mM glucose, the second arrow indicates the 0.9% NaCl-treated control, and the third arrow indicates ghrelin (1.5 × 10−8 M) application. (a) Baseline before glucose application. (b) Firing rate after glucose application. (c) Firing rate recovered to baseline before 0.9% NaCl application. (d) Firing rate after 0.9% NaCl application and the baseline before ghrelin application. (e) Firing rate after ghrelin application.

Citation: Endocrine Connections 9, 12; 10.1530/EC-20-0285

Effects of long-term LPBN ghrelin on UCP-1 expression in BAT

After the administration of 300 pmol ghrelin in LPBN for 7 days, no remarkable differences were observed in the BAT expression of UCP-1 at either protein level (1.97 ± 0.24 vs 2.18 ± 0.32, P > 0.05; Fig. 4A) or mRNA level (2.12 ± 0.44 vs 1.00 ± 0.69, P > 0.05; Fig. 4B) relative to those of 0.9% NaCl injection.

Figure 4
Figure 4

Effects of long-term LPBN ghrelin on UCP-1 expression in BAT UCP-1 protein expression in BAT had no change in ghrelin group (n = 7) compared to 0.9% NaCl group (n = 6) (A), UCP-1 mRNA expression in BAT had no change in ghrelin group (n = 5) compared to 0.9% NaCl group (n = 4) (B) (P > 0.05).

Citation: Endocrine Connections 9, 12; 10.1530/EC-20-0285

Effects of LPBN ghrelin on c-fos expression in LPBN

Comparing with the 0.9% NaCl injection group, injection of 300 pmol ghrelin significantly increased c-fos expression in the LPBN (179.64 ± 10.76 vs 129.88 ± 14.67, P < 0.05; Fig. 5).

Figure 5
Figure 5

Effects of LPBN ghrelin on c-fos expression in LPBN. Microinjection of ghrelin in LPBN increased c-fos expression in LPBN, ghrelin group (n = 3), 0.9% NaCl group (n = 3). *P < 0.05 ghrelin vs 0.9% NaCl. scp, superior cerebellar peduncle; MPBN, medial parabrachial nucleus.

Citation: Endocrine Connections 9, 12; 10.1530/EC-20-0285

Discussion

The underlying mechanism for regulation of feeding and body weight is crucial in understanding obesity pathogenesis. Since 2000, when ghrelin was found to promote food intake and body weight gain in rodents (37), numerous studies have corroborated these findings by administering ghrelin to rodents either peripherally or centrally (13, 37, 38).

Microinjection minimized drug diffusion in brain, thus facilitates the investigation of the drug effects on a specific nucleus. In the current study, ghrelin injection in LPBN significantly increased nocturnal cumulative food intake in the first 3 h, which was blocked by [D-Lys3]-GHRP-6 and abolished in GHSR−/− mice. Therefore, our results demonstrated that ghrelin acted through GHSR to play orexigenic effect in the LPBN, which was consistent with the previously observed effects in the forebrain (13). An interesting phenomenon was presented in Fig. 1C that GHSR−/− mice tend to have a small size food intake compared with the wide type C57 mice though the difference was not significant. This may resulted from the truth that GHSR was knocked out, which prevented endogenous ghrelin from orexigenic effect. However, it is worth noting that the sensation of forebrain and pons to ghrelin was different. Wren et al. (13) used various doses of ghrelin, and reported that the lowest dose of ghrelin to significantly stimulate food intake was 30 pmol, but they indeed resorted to 300 pmol to confirm whether nuclei were really nonresponsive or just requires a higher dose. In our preliminary experiments, we took the 300 pmol to confirm whether LPBN should be considered nonresponsive or just needs stronger stimulation. Interestingly, as reported, PVN, a nucleus that projects LPBN is among the nucleus in hypothalamus that only responded to 300 pmol but not 30 pmol ghrelin, suggesting that ghrelin signaling in LPBN probably is associated with PVN.

Moreover, the present study demonstrated that LPBN ghrelin injection increased the number of Fos-positive neurons in the LPBN for the first time, corroborated that ghrelin could increase food intake and body weight through GHSR in the LPBN. To the best of our knowledge, some of the GHSR-expressing sites, such as the Arc, have direct access to ghrelin circulating in the blood stream, as peripheral ghrelin and ghrelin mimetic administration induce c-fos expression there (39, 40, 41). However, the GHSR expression in CNS is fairly widespread. One possible explanation for GHSR expression in those sites without immediate access to the blood–brain barrier comes from the observations of ghrelin-producing central neurons (1, 42, 43). Those neurons were observed to produce ghrelin centrally, which would be readily available for those GHSR-expressing nuclei without access to circulating ghrelin. Our experiments provided evidences of central ghrelin’s effect in energy metabolism regulation as a neurotransmitter. However, the source(s) and physiological significance of central nerves system ghrelin remain to be determined.

In the current study, we focused on a group of specialized neurons in LPBN called GS neurons. Brain glucose regulation is crucial for the maintenance of cell activities, neurotransmitter synthesis, and nerve synapses (44, 45), in which the GS neurons play major roles. Significant increase in the firing rate of GE neurons and decrease in the firing rate of GI neurons were observed following administration of ghrelin in LPBN, along with increased food intake. These findings suggest that GS neurons in the LPBN might be involved in regulation of food intake. Our findings revealed that ghrelin’s effect is at least partially associated with GS neurons in LPBN. Our finding expanded the knowledge about the neuronal mechanism of LPBN mediated food intake regulation as well as the area/function of ghrelin. While the route of administration used in electrophysiology experiment achieved ghrelin treatment on single targeted neuron and measured responses, further evidences, such as utilization of knockout mice as control and/or investigate specific current with in vitro patch clamp technique are necessary before a conclusion may be reached regarding whether such effect is a direct effect or indirect through the release of other peptides. Further investigations are planned.

Maintaining energy balance is about reaching the balance between energy intake and expenditure. The changes in body weight result from changes in such balance (46). In the current study, chronic administration of ghrelin in the LPBN did not result in remarkable differences in either protein level or mRNA level of UCP-1 in brown adipose tissue, implying that the ghrelin treatment for 7 days was not enough to translate into a robust expression change of UCP-1 in BAT. Notably, the mean mRNA levels seemed to be elevated in ghrelin-treated samples (not statistically significant). This effect will be further pursued. Studies have raised that ghrelin administration increased body weight gain through promoting a positive energy balance which led to high food intake and low energy expenditure (4, 38). Additionally, ghrelin was found to promote adipose accumulation, reduce storage of lipids and then reduce energy expenditure (47, 48). The next step could be studying its effect on fat mass.

Conclusion

Ghrelin in the LPBN could increase nocturnal cumulative food intake through the interaction with GHSR in C57BL/6J mice, and influence the firing rate of GS neurons in the LPBN. Long term ghrelin administration increased the body weight gain. It was also found to promote c-fos expression in the LPBN.

Declaration of interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

Funding

This work was supported by grants (to Dr Jing Dong) from the National Natural Science Foundation of China (No. 31872791) and Science Foundation of Shandong Province of China (No. ZR201807070189).

Author contribution statement

J D, Z J, C Z and J Y conceived and designed the experiments. C Z, X C, J Y, Q L, M L, L W, R W, K Z and X L C performed the experiments. C Z and J Y analyzed the data. C Z, J Y and J D wrote the paper. J D, C Z, J Y and B W edited the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors would like to thank the help of Ying-chang Pang team in Qingdao University, Shandong, China during conceiving the experiment.

References

  • 1

    Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H & Kangawa K Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999 402 6566 6 0. (https://doi.org/10.1038/45230)

    • Search Google Scholar
    • Export Citation
  • 2

    Akman MS, Girard M, O’Brien LF, Ho AK & Chik CL Mechanisms of action of a second generation growth hormone-releasing peptide (Ala-His-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2) in rat anterior pituitary cells. Endocrinology 1993 132 128612 91. (https://doi.org/10.1210/endo.132.3.8095015)

    • Search Google Scholar
    • Export Citation
  • 3

    Cheng K, Chan WW, Barreto A, Convey EM & Smith RG The synergistic effects of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on growth hormone (GH)-releasing factor-stimulated GH release and intracellular adenosine 3′,5′-monophosphate accumulation in rat primary pituitary cell culture. Endocrinology 1989 124 2791279 8. (https://doi.org/10.1210/endo-124-6-2791)

    • Search Google Scholar
    • Export Citation
  • 4

    Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K & Matsukura S A role for ghrelin in the central regulation of feeding. Nature 2001 409 19419 8. (https://doi.org/10.1038/35051587)

    • Search Google Scholar
    • Export Citation
  • 5

    Mihalache L, Gherasim A, Nita O, Ungureanu MC, Padureanu SS, Gavril RS & Arhire LI Effects of ghrelin in energy balance and body weight homeostasis. Hormones 2016 15 186196. (https://doi.org/10.14310/horm.2002.1672)

    • Search Google Scholar
    • Export Citation
  • 6

    Ledderose C, Kreth S & Beiras-Fernandez A Ghrelin, a novel peptide hormone in the regulation of energy balance and cardiovascular function. Recent Patents on Endocrine, Metabolic and Immune Drug Discovery 2011 5 16. (https://doi.org/10.2174/187221411794351897)

    • Search Google Scholar
    • Export Citation
  • 7

    Thomas MA, Ryu V & Bartness TJ Central ghrelin increases food foraging/hoarding that is blocked by GHSR antagonism and attenuates hypothalamic paraventricular nucleus neuronal activation. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 2016 310 R275R2 85. (https://doi.org/10.1152/ajpregu.00216.2015)

    • Search Google Scholar
    • Export Citation
  • 8

    Liu B, Garcia EA & Korbonits M Genetic studies on the ghrelin, growth hormone secretagogue receptor (GHSR) and ghrelin O-acyl transferase (GOAT) genes. Peptides 2011 32 21912 2 07. (https://doi.org/10.1016/j.peptides.2011.09.006)

    • Search Google Scholar
    • Export Citation
  • 9

    Kageyama H, kitamura Y, Hosono T, Kintaka Y, Seki M, Takenoya F, Hori Y, Nonaka N, Arata S & Shioda S Visualization of ghrelin-producing neurons in the hypothalamic arcuate nucleus using ghrelin-EGFP transgenic mice. Regulatory Peptides 2008 145 1161 21. (https://doi.org/10.1016/j.regpep.2007.09.026)

    • Search Google Scholar
    • Export Citation
  • 10

    Cowley MA, Smith RG, Diano S, Tschop M, pronchuk N, Grove KL, Strasburger CJ, Bidlingmaier M, Esterman M & Heiman ML et al. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003 37 6496 6 1. (https://doi.org/10.1016/s0896-6273(0300063-1)

    • Search Google Scholar
    • Export Citation
  • 11

    Zigman JM, Jones JE, Lee CE, Saper CB & Elmquist JK Expression of ghrelin receptor mRNA in the rat and the mouse brain. Journal of Comparative Neurology 2006 494 5285 48. (https://doi.org/10.1002/cne.20823)

    • Search Google Scholar
    • Export Citation
  • 12

    Guan XM, Yu H, palyha OC, Mckee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, Van Der Ploeg LH & Howard AD Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Research: Molecular Brain Research 1997 48 232 9. (https://doi.org/10.1016/s0169-328x(9700071-5)

    • Search Google Scholar
    • Export Citation
  • 13

    Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA & Ghatei MA et al. Ghrelin causes hyperphagia and obesity in rats. Diabetes 2001 50 2540254 7. (https://doi.org/10.2337/diabetes.50.11.2540)

    • Search Google Scholar
    • Export Citation
  • 14

    Faulconbridge LF, Cummings DE, Kaplan JM & Grill HJ Hyperphagic effects of brainstem ghrelin administration. Diabetes 2003 52 2260226 5. (https://doi.org/10.2337/diabetes.52.9.2260)

    • Search Google Scholar
    • Export Citation
  • 15

    Perello M, Scott MM, Sakata I, Lee CE, Chuang JC, Osborne-Lawrence S, Rovinsky SA, Elmquist JK & Zigman JM Functional implications of limited leptin receptor and ghrelin receptor coexpression in the brain. Journal of Comparative Neurology 2012 520 2812 94. (https://doi.org/10.1002/cne.22690)

    • Search Google Scholar
    • Export Citation
  • 16

    Davern PJ A role for the lateral parabrachial nucleus in cardiovascular function and fluid homeostasis. Frontiers in Physiology 2014 5 436. (https://doi.org/10.3389/fphys.2014.00436)

    • Search Google Scholar
    • Export Citation
  • 17

    Alhadeff AL, Golub D, Hayes MR & Grill HJ Peptide YY signaling in the lateral parabrachial nucleus increases food intake through the Y1 receptor. American Journal of Physiology: Endocrinology and Metabolism 2015 309 E759E7 66. (https://doi.org/10.1152/ajpendo.00346.2015)

    • Search Google Scholar
    • Export Citation
  • 18

    Alhadeff AL, Hayes MR & Grill HJ Leptin receptor signaling in the lateral parabrachial nucleus contributes to the control of food intake. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 2014 307 R1338R13 44. (https://doi.org/10.1152/ajpregu.00329.2014)

    • Search Google Scholar
    • Export Citation
  • 19

    Richard JE, Farkas I, Anesten F, Anderberg RH, Dickson SL, Gribble FM, Reimann F, Jansson JO, Liposits Z & Skibicka KP GLP-1 receptor stimulation of the lateral parabrachial nucleus reduces food intake: neuroanatomical, electrophysiological, and behavioral evidence. Endocrinology 2014 155 435643 67. (https://doi.org/10.1210/en.2014-1248)

    • Search Google Scholar
    • Export Citation
  • 20

    Yuan JH, Chen X, dong J, Zhang D, Song K, Zhang Y, Wu GB, Hu XH, Jiang ZY & Chen P Nesfatin-1 in the lateral parabrachial nucleus inhibits food intake, modulates excitability of glucosensing neurons, and enhances UCP1 expression in brown adipose tissue. Frontiers in Physiology 2017 8 235. (https://doi.org/10.3389/fphys.2017.00235)

    • Search Google Scholar
    • Export Citation
  • 21

    lopez-Gambero AJ, Martinez F, Salazar K, Cifuentes M & Nualart F Brain glucose-sensing mechanism and energy homeostasis. Molecular Neurobiology 2019 56 769796. (https://doi.org/10.1007/s12035-018-1099-4)

    • Search Google Scholar
    • Export Citation
  • 22

    Mayer J Glucostatic mechanism of regulation of food intake. 1953. Obesity Research 1996 4 49349 6. (https://doi.org/10.1002/j.1550-8528.1996.tb00260.x)

    • Search Google Scholar
    • Export Citation
  • 23

    Watts AG & Donovan CM Sweet talk in the brain: glucosensing, neural networks, and hypoglycemic counterregulation. Frontiers in Neuroendocrinology 2010 31 3243. (https://doi.org/10.1016/j.yfrne.2009.10.006)

    • Search Google Scholar
    • Export Citation
  • 24

    Chen X, Dong J & Jiang ZY Nesfatin-1 influences the excitability of glucosensing neurons in the hypothalamic nuclei and inhibits the food intake. Regulatory Peptides 2012 177 212 6. (https://doi.org/10.1016/j.regpep.2012.04.003)

    • Search Google Scholar
    • Export Citation
  • 25

    Dong J, Guan HZ, Jiang ZY & Chen X Nesfatin-1 influences the excitability of glucosensing neurons in the dorsal vagal complex and inhibits food intake. PLoS ONE 2014 9 e98967. (https://doi.org/10.1371/journal.pone.0098967)

    • Search Google Scholar
    • Export Citation
  • 26

    Routh VH, Donovan CM & Ritter S 2. Hypoglycemia detection. Translational Endocrinology and Metabolism 2012 3 47–87. (https://doi.org/10.1210/team.9781936704200.ch2)

    • Search Google Scholar
    • Export Citation
  • 27

    Shankar K, Gupta D, Mani BK, Findley BG, Lord CC, Osborne-Lawrence S, Metzger NP, Pietra C, Liu C & Berglund ED et al. Acyl-ghrelin is permissive for the normal counterregulatory response to insulin-induced hypoglycemia. Diabetes 2020 69 228237. (https://doi.org/10.2337/db19-0438)

    • Search Google Scholar
    • Export Citation
  • 28

    De Araujo IE Expanding the brain glucosensing territory. Cell Metabolism 2014 20 93393 5. (https://doi.org/10.1016/j.cmet.2014.11.013)

  • 29

    Garfield AS, Shah BP, Madara JC, Burke LK, Patterson CM, Flak J, Neve RL, Evans ML, Lowell BB & Myers Jr MG et al. A parabrachial-hypothalamic cholecystokinin neurocircuit controls counterregulatory responses to hypoglycemia. Cell Metabolism 2014 20 10301037. (https://doi.org/10.1016/j.cmet.2014.11.006)

    • Search Google Scholar
    • Export Citation
  • 30

    Chen X, Ge YL, Jiang ZY, Liu CQ, Depoortere I & Peeters TL Effects of ghrelin on hypothalamic glucose responding neurons in rats. Brain Research 2005 1055 13113 6. (https://doi.org/10.1016/j.brainres.2005.06.080)

    • Search Google Scholar
    • Export Citation
  • 31

    Sun Y, Butte NF, Garcia JM & Smith RG Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology 2008 149 8438 50. (https://doi.org/10.1210/en.2007-0271)

    • Search Google Scholar
    • Export Citation
  • 32

    Wang R, Yuan J, Zhang C, Wang L, Liu Y, Song L, Zhong W, Chen X & Dong J Neuropeptide Y-positive neurons in the dorsomedial hypothalamus are involved in the anorexic effect of Angptl8. Frontiers in Molecular Neuroscience 2018 11 451. (https://doi.org/10.3389/fnmol.2018.00451)

    • Search Google Scholar
    • Export Citation
  • 33

    Naleid AM, Grace MK, Cummings DE & Levine AS Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens. Peptides 2005 26 2274227 9. (https://doi.org/10.1016/j.peptides.2005.04.025)

    • Search Google Scholar
    • Export Citation
  • 34

    Jhamandas JH, Lind RW & Renaud LP Angiotensin II may mediate excitatory neurotransmission from the subfornical organ to the hypothalamic supraoptic nucleus: an anatomical and electrophysiological study in the rat. Brain Research 1989 487 5261. (https://doi.org/10.1016/0006-8993(8990939-6)

    • Search Google Scholar
    • Export Citation
  • 35

    Wang WG, Chen X, Jiang H & Jiang ZY Effects of ghrelin on glucose-sensing and gastric distension sensitive neurons in rat dorsal vagal complex. Regulatory Peptides 2008 146 1691 75. (https://doi.org/10.1016/j.regpep.2007.09.007)

    • Search Google Scholar
    • Export Citation
  • 36

    Konsman JPJP The Mouse Brain in Stereotaxic Coordinates, 2nd ed. (Deluxe). Eds G Paxinos & KBJ Franklin. New York: Academic Press, 2003, ISBN 0-12-547637-X. 2001, 28, pp. 827828.

    • Search Google Scholar
    • Export Citation
  • 37

    Tschop M, Smiley DL & Heiman ML Ghrelin induces adiposity in rodents. Nature 2000 407 9089 13. (https://doi.org/10.1038/35038090)

  • 38

    Theander-Carrillo C, Wiedmer P, Cettour-Rose P, Nogueiras R, Perez-Tilve D, Pfluger P, Castaneda TR, Muzzin P, Schurmann A & Szanto I et al. Ghrelin action in the brain controls adipocyte metabolism. Journal of Clinical Investigation 2006 116 198319 93. (https://doi.org/10.1172/JCI25811)

    • Search Google Scholar
    • Export Citation
  • 39

    Hewson AK & Dickson SL Systemic administration of ghrelin induces Fos and Egr-1 proteins in the hypothalamic arcuate nucleus of fasted and fed rats. Journal of Neuroendocrinology 2000 12 1047104 9. (https://doi.org/10.1046/j.1365-2826.2000.00584.x)

    • Search Google Scholar
    • Export Citation
  • 40

    Wang L, Saint-Pierre DH & Taché Y Peripheral ghrelin selectively increases Fos expression in neuropeptide Y – synthesizing neurons in mouse hypothalamic arcuate nucleus. Neuroscience Letters 2002 325 4751. (https://doi.org/10.1016/s0304-3940(0200241-0)

    • Search Google Scholar
    • Export Citation
  • 41

    Traebert M, riediger T, Whitebread S, Scharrer E & Schmid HA Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus. Journal of Neuroendocrinology 2002 14 58058 6. (https://doi.org/10.1046/j.1365-2826.2002.00810.x)

    • Search Google Scholar
    • Export Citation
  • 42

    Horvath TL, Diano S, Sotonyi P, Heiman M & Tschop M Minireview: ghrelin and the regulation of energy balance – a hypothalamic perspective. Endocrinology 2001 142 4163416 9. (https://doi.org/10.1210/endo.142.10.8490)

    • Search Google Scholar
    • Export Citation
  • 43

    Van Der Lely AJ, Tschop M, Heiman ML & Ghigo E Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocrine Reviews 2004 25 4264 57. (https://doi.org/10.1210/er.2002-0029)

    • Search Google Scholar
    • Export Citation
  • 44

    Haberg A, Qu H, Saether O, Unsgard G, Haraldseth O & Sonnewald U Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival. Journal of Cerebral Blood Flow and Metabolism 2001 21 145114 63. (https://doi.org/10.1097/00004647-200112000-00010)

    • Search Google Scholar
    • Export Citation
  • 45

    Howarth C, Gleeson P & Attwell D Updated energy budgets for neural computation in the neocortex and cerebellum. Journal of Cerebral Blood Flow and Metabolism 2012 32 12221232. (https://doi.org/10.1038/jcbfm.2012.35)

    • Search Google Scholar
    • Export Citation
  • 46

    Stevanovic D, Trajkovic V, Muller-Luhlhoff S, Brandt E, Abplanalp W, Bumke-Vogt C, Liehl B, Wiedmer P, Janjetovic K & Starcevic V et al. Ghrelin-induced food intake and adiposity depend on central mTORC1/S6K1 signaling. Molecular and Cellular Endocrinology 2013 381 2802 90. (https://doi.org/10.1016/j.mce.2013.08.009)

    • Search Google Scholar
    • Export Citation
  • 47

    Li Z, Mulholland M & Zhang W Ghrelin O-acyltransferase (GOAT) and energy metabolism. Science China: Life Sciences 2016 59 2812 91. (https://doi.org/10.1007/s11427-015-4973-6)

    • Search Google Scholar
    • Export Citation
  • 48

    Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, Moncrieffe M, Thabet K, Cox HJ & Yancopoulos GD et al. Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. PNAS 2004 101 822782 32. (https://doi.org/10.1073/pnas.0402763101)

    • Search Google Scholar
    • Export Citation

If the inline PDF is not rendering correctly, you can download the PDF file here.

 

     European Society of Endocrinology

     Society for Endocrinology

Sept 2018 onwards Past Year Past 30 Days
Abstract Views 0 0 0
Full Text Views 101 101 84
PDF Downloads 34 34 23
  • View in gallery

    Effects of ghrelin injection in LPBN on nocturnal feeding in mice. (A) Ghrelin increased food intake in 1, 2, 3 h after injection of ghrelin in LPBN. (B) [D-Lys3]-GHRP-6 blocked the effects of LPBN ghrelin on food intake in 2, 3 h after injection of ghrelin in LPBN. (C) Ghrelin receptor knockout abolished the effects of LPBN ghrelin on food intake in 1, 2, 3 h after injection of ghrelin in LPBN. *P < 0.05 ghrelin vs 0.9% NaCl, #P < 0.05 GHRP-6+ghrelin vs GHRP-6.

  • View in gallery

    Effects of ghrelin injection in LPBN on long-time body weight gain in mice. After the injection of ghrelin, body weight gain was increased from the 4th day. *P < 0.05 ghrelin vs 0.9% NaCl.

  • View in gallery

    Effects of ghrelin on LPBN glucose-sensitive neurons in vivo. (A) Ghrelin of LPBN affects the activity of glucose-excited neurons. (B) Ghrelin of LPBN affects the activity of glucose-inhibited neurons. The first arrow indicates the addition of 5 mM glucose, the second arrow indicates the 0.9% NaCl-treated control, and the third arrow indicates ghrelin (1.5 × 10−8 M) application. (a) Baseline before glucose application. (b) Firing rate after glucose application. (c) Firing rate recovered to baseline before 0.9% NaCl application. (d) Firing rate after 0.9% NaCl application and the baseline before ghrelin application. (e) Firing rate after ghrelin application.

  • View in gallery

    Effects of long-term LPBN ghrelin on UCP-1 expression in BAT UCP-1 protein expression in BAT had no change in ghrelin group (n = 7) compared to 0.9% NaCl group (n = 6) (A), UCP-1 mRNA expression in BAT had no change in ghrelin group (n = 5) compared to 0.9% NaCl group (n = 4) (B) (P > 0.05).

  • View in gallery

    Effects of LPBN ghrelin on c-fos expression in LPBN. Microinjection of ghrelin in LPBN increased c-fos expression in LPBN, ghrelin group (n = 3), 0.9% NaCl group (n = 3). *P < 0.05 ghrelin vs 0.9% NaCl. scp, superior cerebellar peduncle; MPBN, medial parabrachial nucleus.

  • 1

    Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H & Kangawa K Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999 402 6566 6 0. (https://doi.org/10.1038/45230)

    • Search Google Scholar
    • Export Citation
  • 2

    Akman MS, Girard M, O’Brien LF, Ho AK & Chik CL Mechanisms of action of a second generation growth hormone-releasing peptide (Ala-His-D-beta Nal-Ala-Trp-D-Phe-Lys-NH2) in rat anterior pituitary cells. Endocrinology 1993 132 128612 91. (https://doi.org/10.1210/endo.132.3.8095015)

    • Search Google Scholar
    • Export Citation
  • 3

    Cheng K, Chan WW, Barreto A, Convey EM & Smith RG The synergistic effects of His-D-Trp-Ala-Trp-D-Phe-Lys-NH2 on growth hormone (GH)-releasing factor-stimulated GH release and intracellular adenosine 3′,5′-monophosphate accumulation in rat primary pituitary cell culture. Endocrinology 1989 124 2791279 8. (https://doi.org/10.1210/endo-124-6-2791)

    • Search Google Scholar
    • Export Citation
  • 4

    Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K & Matsukura S A role for ghrelin in the central regulation of feeding. Nature 2001 409 19419 8. (https://doi.org/10.1038/35051587)

    • Search Google Scholar
    • Export Citation
  • 5

    Mihalache L, Gherasim A, Nita O, Ungureanu MC, Padureanu SS, Gavril RS & Arhire LI Effects of ghrelin in energy balance and body weight homeostasis. Hormones 2016 15 186196. (https://doi.org/10.14310/horm.2002.1672)

    • Search Google Scholar
    • Export Citation
  • 6

    Ledderose C, Kreth S & Beiras-Fernandez A Ghrelin, a novel peptide hormone in the regulation of energy balance and cardiovascular function. Recent Patents on Endocrine, Metabolic and Immune Drug Discovery 2011 5 16. (https://doi.org/10.2174/187221411794351897)

    • Search Google Scholar
    • Export Citation
  • 7

    Thomas MA, Ryu V & Bartness TJ Central ghrelin increases food foraging/hoarding that is blocked by GHSR antagonism and attenuates hypothalamic paraventricular nucleus neuronal activation. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 2016 310 R275R2 85. (https://doi.org/10.1152/ajpregu.00216.2015)

    • Search Google Scholar
    • Export Citation
  • 8

    Liu B, Garcia EA & Korbonits M Genetic studies on the ghrelin, growth hormone secretagogue receptor (GHSR) and ghrelin O-acyl transferase (GOAT) genes. Peptides 2011 32 21912 2 07. (https://doi.org/10.1016/j.peptides.2011.09.006)

    • Search Google Scholar
    • Export Citation
  • 9

    Kageyama H, kitamura Y, Hosono T, Kintaka Y, Seki M, Takenoya F, Hori Y, Nonaka N, Arata S & Shioda S Visualization of ghrelin-producing neurons in the hypothalamic arcuate nucleus using ghrelin-EGFP transgenic mice. Regulatory Peptides 2008 145 1161 21. (https://doi.org/10.1016/j.regpep.2007.09.026)

    • Search Google Scholar
    • Export Citation
  • 10

    Cowley MA, Smith RG, Diano S, Tschop M, pronchuk N, Grove KL, Strasburger CJ, Bidlingmaier M, Esterman M & Heiman ML et al. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003 37 6496 6 1. (https://doi.org/10.1016/s0896-6273(0300063-1)

    • Search Google Scholar
    • Export Citation
  • 11

    Zigman JM, Jones JE, Lee CE, Saper CB & Elmquist JK Expression of ghrelin receptor mRNA in the rat and the mouse brain. Journal of Comparative Neurology 2006 494 5285 48. (https://doi.org/10.1002/cne.20823)

    • Search Google Scholar
    • Export Citation
  • 12

    Guan XM, Yu H, palyha OC, Mckee KK, Feighner SD, Sirinathsinghji DJ, Smith RG, Van Der Ploeg LH & Howard AD Distribution of mRNA encoding the growth hormone secretagogue receptor in brain and peripheral tissues. Brain Research: Molecular Brain Research 1997 48 232 9. (https://doi.org/10.1016/s0169-328x(9700071-5)

    • Search Google Scholar
    • Export Citation
  • 13

    Wren AM, Small CJ, Abbott CR, Dhillo WS, Seal LJ, Cohen MA, Batterham RL, Taheri S, Stanley SA & Ghatei MA et al. Ghrelin causes hyperphagia and obesity in rats. Diabetes 2001 50 2540254 7. (https://doi.org/10.2337/diabetes.50.11.2540)

    • Search Google Scholar
    • Export Citation
  • 14

    Faulconbridge LF, Cummings DE, Kaplan JM & Grill HJ Hyperphagic effects of brainstem ghrelin administration. Diabetes 2003 52 2260226 5. (https://doi.org/10.2337/diabetes.52.9.2260)

    • Search Google Scholar
    • Export Citation
  • 15

    Perello M, Scott MM, Sakata I, Lee CE, Chuang JC, Osborne-Lawrence S, Rovinsky SA, Elmquist JK & Zigman JM Functional implications of limited leptin receptor and ghrelin receptor coexpression in the brain. Journal of Comparative Neurology 2012 520 2812 94. (https://doi.org/10.1002/cne.22690)

    • Search Google Scholar
    • Export Citation
  • 16

    Davern PJ A role for the lateral parabrachial nucleus in cardiovascular function and fluid homeostasis. Frontiers in Physiology 2014 5 436. (https://doi.org/10.3389/fphys.2014.00436)

    • Search Google Scholar
    • Export Citation
  • 17

    Alhadeff AL, Golub D, Hayes MR & Grill HJ Peptide YY signaling in the lateral parabrachial nucleus increases food intake through the Y1 receptor. American Journal of Physiology: Endocrinology and Metabolism 2015 309 E759E7 66. (https://doi.org/10.1152/ajpendo.00346.2015)

    • Search Google Scholar
    • Export Citation
  • 18

    Alhadeff AL, Hayes MR & Grill HJ Leptin receptor signaling in the lateral parabrachial nucleus contributes to the control of food intake. American Journal of Physiology: Regulatory, Integrative and Comparative Physiology 2014 307 R1338R13 44. (https://doi.org/10.1152/ajpregu.00329.2014)

    • Search Google Scholar
    • Export Citation
  • 19

    Richard JE, Farkas I, Anesten F, Anderberg RH, Dickson SL, Gribble FM, Reimann F, Jansson JO, Liposits Z & Skibicka KP GLP-1 receptor stimulation of the lateral parabrachial nucleus reduces food intake: neuroanatomical, electrophysiological, and behavioral evidence. Endocrinology 2014 155 435643 67. (https://doi.org/10.1210/en.2014-1248)

    • Search Google Scholar
    • Export Citation
  • 20

    Yuan JH, Chen X, dong J, Zhang D, Song K, Zhang Y, Wu GB, Hu XH, Jiang ZY & Chen P Nesfatin-1 in the lateral parabrachial nucleus inhibits food intake, modulates excitability of glucosensing neurons, and enhances UCP1 expression in brown adipose tissue. Frontiers in Physiology 2017 8 235. (https://doi.org/10.3389/fphys.2017.00235)

    • Search Google Scholar
    • Export Citation
  • 21

    lopez-Gambero AJ, Martinez F, Salazar K, Cifuentes M & Nualart F Brain glucose-sensing mechanism and energy homeostasis. Molecular Neurobiology 2019 56 769796. (https://doi.org/10.1007/s12035-018-1099-4)

    • Search Google Scholar
    • Export Citation
  • 22

    Mayer J Glucostatic mechanism of regulation of food intake. 1953. Obesity Research 1996 4 49349 6. (https://doi.org/10.1002/j.1550-8528.1996.tb00260.x)

    • Search Google Scholar
    • Export Citation
  • 23

    Watts AG & Donovan CM Sweet talk in the brain: glucosensing, neural networks, and hypoglycemic counterregulation. Frontiers in Neuroendocrinology 2010 31 3243. (https://doi.org/10.1016/j.yfrne.2009.10.006)

    • Search Google Scholar
    • Export Citation
  • 24

    Chen X, Dong J & Jiang ZY Nesfatin-1 influences the excitability of glucosensing neurons in the hypothalamic nuclei and inhibits the food intake. Regulatory Peptides 2012 177 212 6. (https://doi.org/10.1016/j.regpep.2012.04.003)

    • Search Google Scholar
    • Export Citation
  • 25

    Dong J, Guan HZ, Jiang ZY & Chen X Nesfatin-1 influences the excitability of glucosensing neurons in the dorsal vagal complex and inhibits food intake. PLoS ONE 2014 9 e98967. (https://doi.org/10.1371/journal.pone.0098967)

    • Search Google Scholar
    • Export Citation
  • 26

    Routh VH, Donovan CM & Ritter S 2. Hypoglycemia detection. Translational Endocrinology and Metabolism 2012 3 47–87. (https://doi.org/10.1210/team.9781936704200.ch2)

    • Search Google Scholar
    • Export Citation
  • 27

    Shankar K, Gupta D, Mani BK, Findley BG, Lord CC, Osborne-Lawrence S, Metzger NP, Pietra C, Liu C & Berglund ED et al. Acyl-ghrelin is permissive for the normal counterregulatory response to insulin-induced hypoglycemia. Diabetes 2020 69 228237. (https://doi.org/10.2337/db19-0438)

    • Search Google Scholar
    • Export Citation
  • 28

    De Araujo IE Expanding the brain glucosensing territory. Cell Metabolism 2014 20 93393 5. (https://doi.org/10.1016/j.cmet.2014.11.013)

  • 29

    Garfield AS, Shah BP, Madara JC, Burke LK, Patterson CM, Flak J, Neve RL, Evans ML, Lowell BB & Myers Jr MG et al. A parabrachial-hypothalamic cholecystokinin neurocircuit controls counterregulatory responses to hypoglycemia. Cell Metabolism 2014 20 10301037. (https://doi.org/10.1016/j.cmet.2014.11.006)

    • Search Google Scholar
    • Export Citation
  • 30

    Chen X, Ge YL, Jiang ZY, Liu CQ, Depoortere I & Peeters TL Effects of ghrelin on hypothalamic glucose responding neurons in rats. Brain Research 2005 1055 13113 6. (https://doi.org/10.1016/j.brainres.2005.06.080)

    • Search Google Scholar
    • Export Citation
  • 31

    Sun Y, Butte NF, Garcia JM & Smith RG Characterization of adult ghrelin and ghrelin receptor knockout mice under positive and negative energy balance. Endocrinology 2008 149 8438 50. (https://doi.org/10.1210/en.2007-0271)

    • Search Google Scholar
    • Export Citation
  • 32

    Wang R, Yuan J, Zhang C, Wang L, Liu Y, Song L, Zhong W, Chen X & Dong J Neuropeptide Y-positive neurons in the dorsomedial hypothalamus are involved in the anorexic effect of Angptl8. Frontiers in Molecular Neuroscience 2018 11 451. (https://doi.org/10.3389/fnmol.2018.00451)

    • Search Google Scholar
    • Export Citation
  • 33

    Naleid AM, Grace MK, Cummings DE & Levine AS Ghrelin induces feeding in the mesolimbic reward pathway between the ventral tegmental area and the nucleus accumbens. Peptides 2005 26 2274227 9. (https://doi.org/10.1016/j.peptides.2005.04.025)

    • Search Google Scholar
    • Export Citation
  • 34

    Jhamandas JH, Lind RW & Renaud LP Angiotensin II may mediate excitatory neurotransmission from the subfornical organ to the hypothalamic supraoptic nucleus: an anatomical and electrophysiological study in the rat. Brain Research 1989 487 5261. (https://doi.org/10.1016/0006-8993(8990939-6)

    • Search Google Scholar
    • Export Citation
  • 35

    Wang WG, Chen X, Jiang H & Jiang ZY Effects of ghrelin on glucose-sensing and gastric distension sensitive neurons in rat dorsal vagal complex. Regulatory Peptides 2008 146 1691 75. (https://doi.org/10.1016/j.regpep.2007.09.007)

    • Search Google Scholar
    • Export Citation
  • 36

    Konsman JPJP The Mouse Brain in Stereotaxic Coordinates, 2nd ed. (Deluxe). Eds G Paxinos & KBJ Franklin. New York: Academic Press, 2003, ISBN 0-12-547637-X. 2001, 28, pp. 827828.

    • Search Google Scholar
    • Export Citation
  • 37

    Tschop M, Smiley DL & Heiman ML Ghrelin induces adiposity in rodents. Nature 2000 407 9089 13. (https://doi.org/10.1038/35038090)

  • 38

    Theander-Carrillo C, Wiedmer P, Cettour-Rose P, Nogueiras R, Perez-Tilve D, Pfluger P, Castaneda TR, Muzzin P, Schurmann A & Szanto I et al. Ghrelin action in the brain controls adipocyte metabolism. Journal of Clinical Investigation 2006 116 198319 93. (https://doi.org/10.1172/JCI25811)

    • Search Google Scholar
    • Export Citation
  • 39

    Hewson AK & Dickson SL Systemic administration of ghrelin induces Fos and Egr-1 proteins in the hypothalamic arcuate nucleus of fasted and fed rats. Journal of Neuroendocrinology 2000 12 1047104 9. (https://doi.org/10.1046/j.1365-2826.2000.00584.x)

    • Search Google Scholar
    • Export Citation
  • 40

    Wang L, Saint-Pierre DH & Taché Y Peripheral ghrelin selectively increases Fos expression in neuropeptide Y – synthesizing neurons in mouse hypothalamic arcuate nucleus. Neuroscience Letters 2002 325 4751. (https://doi.org/10.1016/s0304-3940(0200241-0)

    • Search Google Scholar
    • Export Citation
  • 41

    Traebert M, riediger T, Whitebread S, Scharrer E & Schmid HA Ghrelin acts on leptin-responsive neurones in the rat arcuate nucleus. Journal of Neuroendocrinology 2002 14 58058 6. (https://doi.org/10.1046/j.1365-2826.2002.00810.x)

    • Search Google Scholar
    • Export Citation
  • 42

    Horvath TL, Diano S, Sotonyi P, Heiman M & Tschop M Minireview: ghrelin and the regulation of energy balance – a hypothalamic perspective. Endocrinology 2001 142 4163416 9. (https://doi.org/10.1210/endo.142.10.8490)

    • Search Google Scholar
    • Export Citation
  • 43

    Van Der Lely AJ, Tschop M, Heiman ML & Ghigo E Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocrine Reviews 2004 25 4264 57. (https://doi.org/10.1210/er.2002-0029)

    • Search Google Scholar
    • Export Citation
  • 44

    Haberg A, Qu H, Saether O, Unsgard G, Haraldseth O & Sonnewald U Differences in neurotransmitter synthesis and intermediary metabolism between glutamatergic and GABAergic neurons during 4 hours of middle cerebral artery occlusion in the rat: the role of astrocytes in neuronal survival. Journal of Cerebral Blood Flow and Metabolism 2001 21 145114 63. (https://doi.org/10.1097/00004647-200112000-00010)

    • Search Google Scholar
    • Export Citation
  • 45

    Howarth C, Gleeson P & Attwell D Updated energy budgets for neural computation in the neocortex and cerebellum. Journal of Cerebral Blood Flow and Metabolism 2012 32 12221232. (https://doi.org/10.1038/jcbfm.2012.35)

    • Search Google Scholar
    • Export Citation
  • 46

    Stevanovic D, Trajkovic V, Muller-Luhlhoff S, Brandt E, Abplanalp W, Bumke-Vogt C, Liehl B, Wiedmer P, Janjetovic K & Starcevic V et al. Ghrelin-induced food intake and adiposity depend on central mTORC1/S6K1 signaling. Molecular and Cellular Endocrinology 2013 381 2802 90. (https://doi.org/10.1016/j.mce.2013.08.009)

    • Search Google Scholar
    • Export Citation
  • 47

    Li Z, Mulholland M & Zhang W Ghrelin O-acyltransferase (GOAT) and energy metabolism. Science China: Life Sciences 2016 59 2812 91. (https://doi.org/10.1007/s11427-015-4973-6)

    • Search Google Scholar
    • Export Citation
  • 48

    Wortley KE, Anderson KD, Garcia K, Murray JD, Malinova L, Liu R, Moncrieffe M, Thabet K, Cox HJ & Yancopoulos GD et al. Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference. PNAS 2004 101 822782 32. (https://doi.org/10.1073/pnas.0402763101)

    • Search Google Scholar
    • Export Citation