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Oxytocin and Stimulate by the Ghrelin-Producing Cell Line, MGN3-1 in Vitro

Hiroshi Iwakura, Hiroyuki Ariyasu, Hiroshi Hosoda, Go Yamada, Kiminori Hosoda, Kazuwa Nakao, Kenji Kangawa, and Takashi Akamizu

Ghrelin Research Project (H.I., H.A., T.A.), Translational Research Center, and Department of Medicine and Clinical Science, , and (G.Y., K.H., K.N.), Kyoto University Hospital, Kyoto Downloaded from https://academic.oup.com/endo/article/152/7/2619/2457151 by guest on 30 September 2021 University Graduate School of Medicine, Kyoto 606-8507, Japan; and National Cerebral and Cardiovascular Center Research Institute (H.H., K.K.), Osaka 565-8565, Japan

To understand the physiological role of ghrelin, it is crucial to study both the actions of ghrelin and the regulation of ghrelin secretion. Although ghrelin actions have been extensively revealed, the direct factors regulating ghrelin secretion by ghrelin-producing cells (X/A-like cells), however, is not fully understood. In this study, we examined the effects of and neurotransmit- ters on in vitro ghrelin secretion by the recently developed ghrelin-producing cell line MGN3-1. and significantly stimulated ghrelin secretion by MGN3-1 cells. Because MGN3-1 cells express only oxytocin mRNA, not mRNA, oxytocin is the likely regulator, with the effect of vasopressin mediated by a cross-reaction. We also discovered that dopamine stimulates ghrelin secretion from MGN3-1 cells in a similar manner to the previously known ghrelin stimulators, epinephrine and . MGN3-1 cells expressed mRNA en- coding dopamine receptors D1a and D2. The dopamine receptor D1 fenoldopam stimu- lated ghrelin secretion, whereas the D2, D3 agonist did not. Furthermore, the D1 SKF83566 attenuated the stimulatory effect of dopamine. These results indi- cate that the stimulatory effect of dopamine on ghrelin secretion is mediated by the D1a receptor. In conclusion, we identified two direct regulators of ghrelin, oxytocin and dopamine. These find- ings will provide new direction for further studies seeking to further understand the regulation of ghrelin secretion, which will in turn lead to greater understanding of the physiological role of ghrelin. (Endocrinology 152: 2619–2625, 2011)

hrelin is a -derived 28- peptide sults of in vivo studies suggest that plasma ghrelin levels Ghormone with a unique modification of acylation, are regulated by acute and chronic energy status (8–10), first described by Kojima et al. in 1999 (1). To understand the individual factors regulating ghrelin secretion by ghre- better the physiological function of ghrelin, it is crucial to lin-producing cells (X/A-like cells) remains unclear due to study both ghrelin action and the regulation of ghrelin the lack of an appropriate in vitro assay system. secretion. The actions of ghrelin have been vigorously in- Recently we established a ghrelin-producing cell line, vestigated by multiple groups, revealing a wide variety of MGN (mouse ghrelinoma) 3-1 cells from a gastric ghreli- activities, including GH-stimulating (2), orexigenic (3), noma isolated from ghrelin promoter SV40-T antigen fat-storing (4), cardiovascular (5), gastroprokinetic (6), transgenic mice (11, 12). The MGN3-1 cell is the first cell and -suppressing (7) activities. In contrast, the reg- line derived from a gastric ghrelin-producing cell that pre- ulation of ghrelin secretion from ghrelin-producing cells serves the ability to secrete of substantial amounts of ghre- (X/A-like cells) is not fully understood. Although the re- lin under physiological regulation, making this line one of

ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: ANP, Atrial ; AVP, vasopressin; CNP, C-type natriuretic Printed in U.S.A. peptide; C-RIA, anti-COOH-terminal ghrelin (amino acids 13–28) antiserum used to detect Copyright © 2011 by The Endocrine Society ghrelin and desacyl-ghrelin; GABA, ␥-aminobutyric acid; GHRP2, GH-releasing peptide 2; doi: 10.1210/en.2010-1455 Received December 20, 2010. Accepted April 6, 2011. GIP, gastric inhibitory polypeptide; GLP, -like peptide; GOAT, ghrelin O-acyltrans-

First Published Online April 26, 2011 ferase; N-RIA, anti-NH2-terminal ghrelin (amino acids 1–11) antiserum detects ghrelin only; PP, ; VIP, vasoactive intestinal peptide.

Endocrinology, July 2011, 152(7):2619–2625 endo.endojournals.org 2619 2620 Iwakura et al. Oxytocin and Dopamine Stimulate Ghrelin Endocrinology, July 2011, 152(7):2619–2625

gastric inhibitory polypeptide (GIP), calci- A 350 ** ** tonin, oxytocin, vasopressin (AVP), C-type 300 natriuretic peptide (CNP) (Peptide institute, 250 cells) Inc., Osaka, Japan), GH-releasing peptide 2 6 ** 200 ** (GHRP2; Kaken Pharmaceuticals, Co., Ltd, 150 Tokyo, Japan), insulin (Invitrogen, Carlsbad, 100 CA), or (Pepro Tech, Inc., Rocky Hill, Ϫ6 NJ) were added to each well at 10 M.To

N-RIA (fmol/10 50 screen for , , 0 , muscarine, epinephrine, norepi- PP GH VIP GIP AVP

ANP nephrine, dopamine, , , CNP CCK IGF-I Leptin Insulin GLP-1

Gastrin ␥ Control

GHRP2 glutamate, or -aminobutyric acid (GABA; Thyroxin Oxytocin

Ϫ Downloaded from https://academic.oup.com/endo/article/152/7/2619/2457151 by guest on 30 September 2021

Glucagon 4 Sigma Aldrich Japan) were added at 10 M to each well. To determine the stimulatory ad- Ϫ5 B 2000 renergic receptor subtype, 10 M of isopro- 1800 ** terenol, denopamine, , phenyleph- 1600 ** rine, or (Sigma Aldrich Japan) were

cells) 1400 6 used. To determine the stimulatory dopa- 1200 Ϫ5 ** mine receptor subtype, 10 M , 1000 ** 800 fenoldopam, or bromocriptine (Sigma Aldrich 600 Japan) were used. For the antagonistic studies, 400 1 antagonist [d(CH2)5 , C-RIA (fmol/10 200 Tyr(Me)2,Orn8]-oxytocin (Bachem, Buben- 0 dorf Switzerland), ␤1-receptor antagonist PP GH VIP GIP AVP ANP CNP CCK IGF-I (Sigma Aldrich Japan), and dopamine Leptin GLP-1 Insulin Control GHRP2

Secretin D1 receptor antagonist SKF83566 (Tocris Bio- Thyroxin Oxytocin Glucagon Calcitonin science, Ellisville, MO) were used. Neurotensin Somatostatin FIG. 1. The effects of peptide hormones on ghrelin secretion by MGN3-1 cells. A and B, The amount of ghrelin secreted by MGN3-1 cells incubated for4hinDMEM supplemented with Measurements of ghrelin Ϫ 0.5% BSA and 10 6 M GH, GHRP2, IGF-I, insulin, glucagon, somatostatin, PP, gastrin, CCK, concentrations in culture medium

GLP-1, VIP, GIP, secretin, neurotensin, calcitonin, oxytocin, AVP, T4, leptin, ANP, or CNP. **, To measure ghrelin concentrations in P Ͻ 0.01 in comparison with controls (n ϭ 9). culture medium, the collected culture media were centrifuged, and the resulting super- the best research tools to study the regulation of ghrelin natants were immediately applied to Sep- secretion in vitro. In previous studies, we used MGN3-1 Pak C18 cartridges (Waters Corp., Milford, MA) preequili- brated with 0.9% saline. After washing cartridges with saline cells to examine the effects of insulin and somatostatin, and 5% CH CN/0.1% trifluoroacetic acid, bound was which are well established in in vivo studies to suppress 3 eluted with 60% CH3CN/0.1% trifluoroacetic acid. Eluates ghrelin secretion (13–16). In this study, we examined the were lyophilized and subjected to ghrelin RIA. Two types of effects of peptide hormones and nonpeptide neurotrans- ghrelin RIA were performed: C-RIA, in which an anti-COOH- mitters on in vitro ghrelin secretion from MGN3-1 cells. terminal ghrelin (amino acids 13–28) antiserum is used to detect both ghrelin and desacyl-ghrelin, and N-RIA, in which an anti-

NH2-terminal ghrelin (amino acids 1–11) antiserum detects Materials and Methods ghrelin only, as described (17, 18).

Cell culture RT-PCR and quantitative RT-PCR MGN3-1 cells were cultured in DMEM supplemented with Total RNA was extracted using an RNeasy kit (QIAGEN, ␮ 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml Hilden, Germany). Reverse transcription was performed with streptomycin at 37 C in 10% CO2 as described previously (12). a high-capacity cDNA reverse transcription kit (Applied Bio- systems, Foster City, CA). RT-PCR was performed using a Batch incubation study GeneAmp 9700 cycler (Applied Biosystems) with AmpliTaq ϫ 5 MGN3-1 cells were seeded at 7.5 10 cells/well and cultured Gold using appropriate primers (Supplemental Table 1, pub- for 24 h in 12-well plates. After a washing with PBS, cells were lished on The Endocrine Society’s Journals Online web site at incubated at 37 C for4hinDMEM supplemented with 0.5% BSA http://endo.endojournals.org). Real-time quantitative PCR and the indicated reagents before collecting supernatants. To screen for peptide hormones stimulating or suppressing ghrelin secretion, was performed using an ABI PRISM 7500 sequence detection IGF-I, glucagon, somatostatin, pancreatic polypeptide (PP), gluca- system (Applied Biosystems) using appropriate primers and gon-like peptide (GLP)-1, secretin, neurotensin, thyroxin, atrial na- taqman probes or Power SybrGreen (Supplemental Table 1). triuretic peptide (ANP), GH (Sigma Aldrich Japan, Tokyo, Japan), The mRNA expression of each was normalized to the gastrin, (CCK), vasoactive intestinal peptide (VIP), detected levels of 18S rRNA. Endocrinology, July 2011, 152(7):2619–2625 endo.endojournals.org 2621

A BC cating that the stimulatory effect of va- 400 2000 sopressin is likely secondary to a cross- 1800 350 ** reaction to the oxytocin receptor. MGN 3-1 ** 1600 Stomach ##

cells) 300 cells) 6 6 1400 Actually, addition of oxytocin receptor OxyR 250 ## 1200 1 2 8 200 1000 antagonist [d(CH2)5 ,Tyr(Me) ,Orn ]- AVPR1a 150 800 600 oxytocin significantly attenuated the 100 AVPR1b 400 50 stimulatory effect of vasopressin on ghre-

N-RIA (fmol/10 C-RIA (fmol/10 200 AVPR2 0 0 lin secretion (Fig. 2, B and C). Oxytocin- AVP - ++ AVP - + + mediated of ghrelin-secre- (d(CH ) 1,Tyr(Me)2, (d(CH ) 1,Tyr(Me)2, 2 5 --+ 2 5 --+ 8 8 tion was dose dependent (ED50 value for Orn )-Oxytocin Orn )-Oxytocin N-RIA: 51.22 nM; C-RIA: 21.9 nM; Fig. Downloaded from https://academic.oup.com/endo/article/152/7/2619/2457151 by guest on 30 September 2021 D E F G 2, D and E). Although oxytocin induced 350 1600 1.2 a small, but significant, increase in ghre- ** ** 1.4 ** 300 1400 ** 1 1.2 lin O-acyltransferase (GOAT) mRNA 1200 cells) cells) 6 250 6 1 1000 0.8 levels in MGN3-1 cells (Fig. 2F), ghrelin 200 800 0.8 150 0.6 mRNA levels were unchanged (Fig. 2G). 600 0.6 0.4 100 400 0.4 GOATmRNA (AU)

50 200 Ghrelin mRNA (AU) 0.2 0.2 Effects of nonpeptide N-RIA (fmol/10 C-RIA (fmol/10 0 0 0 0 (-) 10-8 10-710-6 (-) 10-8 10-710-6 neurotransmitters on ghrelin Oxytocin (M) Oxytocin (M) secretion Control Control Oxytocin Oxytocin We next examined the effects of non- FIG. 2. The effect of oxytocin on ghrelin secretion by MGN3-1 cells. A, RT-PCR analysis of peptide neurotransmitters on ghrelin oxytocin receptor (Oxy-R) and vasopressin receptors (AVPR) 1a, 1b, and 2 mRNA expression in secretion by MGN3-1 cells. Ghrelin se- MGN3-1 cells. B and C, The amount of ghrelin secreted by MGN3-1 cells incubated for4hin Ϫ Ϫ DMEM supplemented with 0.5% BSA and 10 6 M AVP with or without 10 6 M cretion by MGN3-1 cells was stimu- 1 2 8 Ͻ [d(CH2)5 ,Tyr(Me) ,Orn ]-oxytocin (oxytocin receptor antagonist). **, P 0.01 in comparison lated by the addition of epinephrine, with controls; ##, P Ͻ 0.01 in comparison with AVP (n ϭ 9). D and E, The amount of ghrelin norepinephrine, or dopamine to the secreted by MGN3-1 cells incubated for4hinDMEM supplemented with 0.5% BSA and Ϫ8 Ϫ6 medium (Fig. 3, A and B). No effects on 10 to 10 M oxytocin. **, P Ͻ 0.01 in comparison with controls (Ϫ)(nϭ 9). F and G, Ϫ Ghrelin and GOAT mRNA levels in MGN3-1 cells after a 24-h incubation with 10 6 M ghrelin secretion were seen after the ad- oxytocin. **, P Ͻ 0.01 in comparison to controls (n ϭ 9). AU, Arbitrary unit. dition of acetylcholine, nicotine, mus- carine, histamine, serotonin, gluta- Statistical analysis mate, or GABA to the medium (Fig. 3, A and B). Ghrelin All values were expressed as the means Ϯ SE. The statistical secretion induced by epinephrine increased in a dose-de- significance of the differences in mean values was assessed by ␮ ANOVA with a post hoc test (Turkey’s test) or Student’s t test as pendent manner (ED50 value for N-RIA: 1.31 M; C-RIA: appropriate. Differences with P Ͻ 0.05 were considered significant. 2.36 ␮M; Fig. 4, A and B). MGN3-1 cells expressed mRNA Statistical analysis was performed by Statcel2 (OMS, Saitama, Japan). encoding of ␣1a- and ␤1- receptors (Fig. 4C). The nonselective ␤-agonist isoproterenol and the ␤1-ag- onist denopamine significantly stimulated ghrelin secre- Results tion by MGN3-1 cells (Fig. 4, D and E). The ␤2-agonist Effects of peptide hormones on ghrelin secretion ritodrine also stimulated ghrelin secretion to a lesser extent, First, we examined the effects of various peptide hor- which may have been secondary to cross-reactivity (Fig. 4, D mones on ghrelin secretion by MGN3-1 cells. Oxytocin and E). No effect on ghrelin secretion was found using the ␣ ␣ and vasopressin significantly stimulated ghrelin secretion 1-agonist , the 1a-agonist A61603 or the ␣ ␤ by MGN3-1 cells, whereas insulin and somatostatin sup- 2-agonist clonidine (Fig. 4, D and E). Addition of 1-receptor pressed the secretion as reported previously (12) (Fig. 1, A antagonist atenolol significantly attenuated the stimulatory ef- and B). Addition of any of the other , including fect of epinephrine on ghrelin secretion (Fig. 4, F and G). These GH, GHRP2, IGF-I, glucagon, PP, gastrin, CCK, GLP-1, results indicate that the stimulation of ghrelin secretion by epi- VIP, GIP, secretin, neurotensin, calcitonin, thyroxin, lep- nephrine or norepinephrine is primarily mediated by the ␤1- tin, ANP, or CNP to the medium had no effect on ghrelin receptor. Isoproterenol significantly increased GOAT mRNA secretion (Fig. 1, A and B). levels but not ghrelin mRNA levels (Fig. 4, H and I). MGN3-1 cells expressed mRNA encoding the oxytocin The stimulation of ghrelin secretion by dopamine was ␮ receptor but did not express mRNA for any subtypes of also dose dependent (ED50 value for N-RIA: 24.7 M; vasopressin receptors (types 1a, 1b, and 2; Fig. 2A), indi- C-RIA: 40.6 ␮M; Fig. 5, A and B). MGN3-1 cells expressed 2622 Iwakura et al. Oxytocin and Dopamine Stimulate Ghrelin Endocrinology, July 2011, 152(7):2619–2625

AB effect (Fig. 5, D and E). Addition of D1 500 2500 ** 450 ** ** ** receptor antagonist SKF83566 signifi- 400 ** ** 2000

cells) cells) cantly attenuated the stimulatory effect

6 350 6 300 1500 of dopamine on ghrelin secretion (Fig. 250 200 1000 5, F and G). These results indicate that 150 100 500 the stimulatory effect of dopamine on C-RIA (fmol/10 N-RIA (fmol/10 50 ghrelin secretion is mediated by the D1a 0 0 receptor. Apomorphine had no effect GABA GABA on ghrelin or GOAT mRNA levels in Control Control Nicotine Nicotine Serotonin Serotonin Histamine Histamine Glutamate Glutamate Muscarine Muscarine Dopamine Dopamine MGN3-1 cells (Fig. 5, H and I). Epinephrine Epinephrine Acetylcholine Acetylcholine Downloaded from https://academic.oup.com/endo/article/152/7/2619/2457151 by guest on 30 September 2021 Norepinephrine Norepinephrine FIG. 3. The effects of neurotransmitters on ghrelin secretion by MGN3-1 cells. A and B, The amount of ghrelin secreted by MGN3-1 cells incubated for4hinDMEM supplemented with Ϫ Discussion 0.5% BSA and 10 4 M acetylcholine, nicotine, muscarine, epinephrine, norepinephrine, dopamine, histamine, serotonin, glutamate, or GABA. **, P Ͻ 0.01 in comparison with controls (n ϭ 9). Ghrelin-producing cells are located in the stomach. These cells secrete ghrelin by responding to various kinds of in- mRNA encoding dopamine receptors D1a and D2 (Fig. puts, possibly hormones, neurotransmitters, or nutrients. 5C). The nonselective dopamine receptor agonist apomor- From these exogenous signals, the cell can sense the phine and the D1 receptor agonist fenoldopam also sig- outside environment and/or interact with other organs nificantly stimulated ghrelin secretion from MGN3-1 to provide appropriate regulation of ghrelin secretion, cells, whereas the D2, D3 agonist bromocriptine had no

D A B C 450 400 ** MGN 3-1Stomach ** 350 a1a cells) 450 3000 6 300 400 ** ** 2500 a1b 250 350

cells) cells) 200 6 300 * 6 2000 a1d 150 250 ** 1500 a2a 100 200 50 a2b N-RIA (fmol/10 150 1000 0 100 a2c 500 50 N-RIA (fmol/10 C-RIA (fmol/10 b1 0 0 Control -7 -6 -5 -7 -6 -5 b2 A61603 Ritodrine

(-) 10 10 10 (-) 10 10 10 Clonidine b3

Epinephrine (M) Epinephrine (M) Denopamine Isoproterenol Phenylephrine E FGH I 2500 600 2500 1.4 1.6 ** 1.4 ** 500 ** ** 1.2 * 2000 ** 2000 1.2 cells) cells) cells) 6 6 1 6 ## 400 ## 1 1500 1500 0.8 300 0.8 0.6 1000 1000 0.6 200 0.4 0.4 500 500 100 GOAT mRNA (AU) Ghrelin mRNA (AU) 0.2 0.2 N-RIA (fmol/10 C-RIA (fmol/10 C-RIA (fmol/10 0 0 0 0 0 Epinephrine - + + Epinephrine - + + Atenolol - - + Atenolol - - + Control Control Control A61603 Ritodrine Clonidine Isoproterenol Isoproterenol Denopamine Isoproterenol Phenylephrine FIG. 4. The effects of epinephrine on ghrelin secretion by MGN3-1 cells. A and B, The amount of ghrelin secreted by MGN3-1 cells incubated for Ϫ Ϫ 4 h in DMEM supplemented with 0.5% BSA and 10 7 to 10 5 M epinephrine. **, P Ͻ 0.05, **, P Ͻ 0.01 in comparison with controls (Ϫ)(nϭ 9). C, RT-PCR analysis of adrenergic receptors-␣1a, -␣1b, -␣1d, -␣2a-c, and -␤1–3 mRNA expression in MGN3-1 cells. D and E, The amount of ghrelin Ϫ secreted by MGN3-1 cells incubated for4hinDMEM supplemented with 0.5% BSA and 10 5 M isoproterenol (␤-agonist), denopamine (␤1- agonist), ritodrine (␤2-agonist), phenylephrine (␣1-agonist), clonidine (␣2-agonist), or A61603 (␣1a-agonist). **, P Ͻ 0.01 in comparison with Ϫ controls (n ϭ 9). F and G, The amount of ghrelin secreted by MGN3-1 cells incubated for4hinDMEM supplemented with 0.5% BSA and 10 5 M Ϫ epinephrine with or without 10 4 M atenolol (␤1-antagonist). **, P Ͻ 0.01 in comparison with controls; ##, P Ͻ 0.01 in comparison with Ϫ epinephrine (n ϭ 9). H and I, Ghrelin and GOAT mRNA levels in MGN3-1 cells after a 24-h incubation with 10 5 M isoproterenol. *, P Ͻ 0.05 in comparison with controls (n ϭ 9). AU, Arbitrary unit. Endocrinology, July 2011, 152(7):2619–2625 endo.endojournals.org 2623

A B C We found that oxytocin signific- 900 3000 antly stimulates ghrelin secretion from 800 ** 2500 ** MGN 3-1Stomach MGN3-1 cells. Oxytocin, a nonapep- 700 cells) cells) DRD1a 6 6 600 2000 tide with a bond, is secreted 500 * DRD2 1500 from the posterior in a 400 * DRD3 300 1000 neuroendocrine manner and is involved 200 500 DRD4 in milk ejection and uterine contrac- 100 C-RIA (fmol/10 N-RIA (fmol/10 0 0 DRD5 tion. Oxytocin also acts as a neu- (-) 10-6 10-5 10-4 (-) 10-6 10-5 10-4 Dopamine (M) Dopamine (M) rotransmitter, specifically as a negative D E F G regulator of food intake to oxytocin- Downloaded from https://academic.oup.com/endo/article/152/7/2619/2457151 by guest on 30 September 2021 450 1600 400 2000 receptive in the paraventricu- ** ** ** ** 400 ** 1400 ** 350 1800 lar nucleus of the (19). 350 1600 ## cells) cells) cells) 1200 cells) 300 6 6 6 6 1400 Only two previous reports have exam- 300 1000 250 250 ## 1200 800 200 1000 ined the effect of oxytocin on plasma 200 600 150 800 150 600 ghrelin levels. Vila et al. (20)described a 400 100 100 400 50 200 50 reduction in basal and lipopolysaccha- C-RIA (fmol/10 C-RIA (fmol/10 N-RIA (fmol/10 N-RIA (fmol/10 200 0 0 0 0 ride-induced ghrelin levels in healthy Dopamine - + + Dopamine - + + men after systemic administration of SKF83566 - - + SKF83566 - - + Control Control oxytocin. Shibata et al. (21) reported Fenoldopam Fenoldopam that inhibition of the suckling-induced Apomorphine Apomorphine Bromocriptine Bromocriptine increase in plasma oxytocin levels by a H I oxytocin antagonist did not alter 1.2 1.2 plasma ghrelin levels in lactating rats. 1 1 0.8 0.8 Although the investigators concluded 0.6 0.6 that oxytocin has no effects on ghrelin 0.4 0.4 secretion, our findings are not in accor- dance with that report. The reason for 0.2 GOAT mRNA (AU) 0.2 Ghrelin mRNA (AU) 0 0 this discrepancy is not clear but may result from indirect effects of additional

Control Control mediators in vivo. Further studies will

pomorphine pomorphine be needed to explore the regulation of A A FIG. 5. The effect of dopamine on ghrelin secretion by MGN3-1 cells. A and B, The amount ghrelin secretion by oxytocin in vivo. of ghrelin secreted by MGN3-1 cells incubated for4hinDMEM supplemented with 0.5% We also found that the nonpeptide Ϫ Ϫ BSA and 10 6 to 10 4 M of dopamine. *, P Ͻ 0.05, **, P Ͻ 0.01 in comparison with controls neurotransmitters epinephrine and nor- (Ϫ)(nϭ 9). C, RT-PCR analysis of dopamine receptor (DR) D1a and D2–5 mRNA expression in MGN3-1 cells. D and E, The amount of ghrelin secreted by MGN3-1 cells incubated for4hin epinephrine strongly stimulate ghrelin se- Ϫ DMEM supplemented with 0.5% BSA and 10 5 M apomorphine (nonselective dopamine cretion by MGN3-1 cells. Ghrelin secre- agonist), fenoldopam (D1 agonist), or bromocriptine (D2, D3 agonist). **, P Ͻ 0.01 in tion has been suggested to be regulated comparison with controls (n ϭ 9). F and G, The amount of ghrelin secreted by MGN3-1 cells Ϫ by the sympathetic nervous system. incubated for4hinDMEM supplemented with 0.5% BSA and 10 4 M dopamine with or Ϫ without 10 4 M SKF83566 (D1 antagonist). **, P Ͻ 0.01 in comparison with controls; ##, Mundinger et al. (22) noted that in- P Ͻ 0.01 in comparison with dopamine (n ϭ 9). H and I, Ghrelin and GOAT mRNA levels in creased portal ghrelin levels in rats after Ϫ5 MGN3-1 cells after a 24-h incubation with 10 M apomorphine (n ϭ 9). AU, Arbitrary unit. electrical sympathetic nerve stimula- tion or iv administration. Ho- which in turn influences various homeostatic systems, soda and Kangawa (23) reported that the administration including energy or growth control. We of adrenergic increased plasma ghrelin levels in sought to understand better the molecular mechanisms rat. Recently Zhao et al. (24) reported that ghrelin secre- governing ghrelin secretion by cells, which may further contribute to understanding the physiological role of tion from the pancreatic ghrelinoma cell line PG-1 and the ghrelin. In previous studies, we have developed a ghre- stomach ghrelinoma cell line SG-1 could be stimulated by ␤ lin-secreting cell line MGN3-1 as a research tool to 1-adrenergic receptors. Our observation demonstrating study the regulation of ghrelin secretion in vitro (12). In increased ghrelin secretion after epinephrine and norepi- this study, we examined the effects of the various pep- nephrine administration is consistent with these results, tide hormones and neurotransmitters on ghrelin secre- supporting the idea that sympathetic nervous system is an tion using MGN3-1 cells. important regulator of ghrelin secretion. 2624 Iwakura et al. Oxytocin and Dopamine Stimulate Ghrelin Endocrinology, July 2011, 152(7):2619–2625

In addition to epinephrine and norepinephrine, dopa- 2. Takaya K, Ariyasu H, Kanamoto N, Iwakura H, Yoshimoto A, mine also significantly stimulated ghrelin secretion from Harada M, Mori K, Komatsu Y, Usui T, Shimatsu A, Ogawa Y, Hosoda K, Akamizu T, Kojima M, Kangawa K, Nakao K 2000 MGN3-1 cells via the D1A receptor. As far as we know, Ghrelin strongly stimulates growth release in humans. this is the first report of ghrelin secretion stimulation by J Clin Endocrinol Metab 85:4908–4911 dopamine. Dopamine is a catecholamine, acting as a neu- 3. Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kan- gawa K, Matsukura S 2001 A role for ghrelin in the central regula- rotransmitter in the certain areas in motor control or tion of feeding. Nature 409:194–198 reward behaviors. A substantial amount of dopamine is 4. Tscho¨ p M, Smiley DL, Heiman ML 2000 Ghrelin induces adiposity also produced in the (25), in which it in rodents. Nature 407:908–913 5. 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These findings hormone-insulin-like growth factor I axis and glucose intolerance. will provide new direction for further studies seeking to Am J Physiol Endocrinol Metab 297:E802–E811 12. Iwakura H, Li Y, Ariyasu H, Hosoda H, Kanamoto N, Bando M, understand better the regulation of ghrelin secretion and Yamada G, Hosoda K, Nakao K, Kangawa K, Akamizu T 2010 the overall physiological role of ghrelin in organism ho- Establishment of a novel ghrelin-producing cell line. Endocrinology meostasis and energy regulation. 151:2940–2945 13. Saad MF, Bernaba B, Hwu CM, Jinagouda S, Fahmi S, Kogosov E, Boyadjian R 2002 Insulin regulates plasma ghrelin concentration. J Clin Endocrinol Metab 87:3997–4000 Acknowledgments 14. Norrelund H, Hansen TK, Orskov H, Hosoda H, Kojima M, Kan- gawa K, Weeke J, Moller N, Christiansen JS, Jorgensen JO 2002 We thank Ms. Chieko Ishimoto and Ms. Chinami Shiraiwa for Ghrelin immunoreactivity in human plasma is suppressed by soma- tostatin. Clin Endocrinol (Oxf) 57:539–546 their excellent technical assistance. 15. Murdolo G, Lucidi P, Di Loreto C, Parlanti N, De Cicco A, Fatone C, Fanelli CG, Bolli GB, Santeusanio F, De Feo P 2003 Insulin is Address all correspondence and requests for reprints to: Hiroshi required for prandial ghrelin suppression in humans. Diabetes 52: Iwakura, M.D., Ph.D., 54 Shogoin Kawahara-cho, Sakyo-ku, 2923–2927 Kyoto 606-8507, Japan. E-mail:[email protected]. 16. Shimada M, Date Y, Mondal MS, Toshinai K, Shimbara T, Fuku- This work was supported by grants from the Ministry of naga K, Murakami N, Miyazato M, Kangawa K, Yoshimatsu H, Matsuo H, Nakazato M 2003 Somatostatin suppresses ghrelin se- Education, Culture, Sports, Science, and Technology of Japan cretion from the rat stomach. Biochem Biophys Res Commun 302: and the Ministry of Health, Labor, and Welfare of Japan. 520–525 Disclosure Summary: All authors have nothing to declare. 17. 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