New Metabolic Influencer on Oxytocin Release: the Ghrelin

molecules

Article New Metabolic Inﬂuencer on Oxytocin Release: The Ghrelin

Renáta Szabó 1,2,†, Rudolf Ménesi 1,†, Andor H. Molnár 3, Zita Szalai 1, Lejla Daruka 1, Gábor Tóth 4,János Gardi 5 ,Márta Gálﬁ 6, Denise Börzsei 1, Krisztina Kupai 1, Anna Juhász 7,‡, Marianna Radács 6, Ferenc A. László 1, Csaba Varga 1,8 and Anikó Pósa 1,2,*

1 Department of Physiology, Anatomy and Neuroscience, Faculty of Science and Informatics, University of Szeged, 6726 Szeged, Hungary; [email protected] (R.S.); [email protected] (R.M.); [email protected] (Z.S.); [email protected] (L.D.); [email protected] (D.B.); [email protected] (K.K.); [email protected] (F.A.L.); [email protected] (C.V.) 2 Department of Physiology, Anatomy and Neuroscience, Interdisciplinary Excellence Centre, University Of Szeged, Szeged, Hungary 3 Institute of Physical Education and Sport Sciences, Gyula Juhász Faculty of Education, University of Szeged, 6725 Szeged, Hungary; [email protected] 4 Department of Medical Chemistry, Faculty of Medicine, University of Szeged, 6720 Szeged, Hungary; [email protected] 5 First Department of Internal Medicine, Faculty of Medicine, University of Szeged, 6720 Szeged, Hungary; [email protected] 6 Department of Environmental Biology and Education, Gyula Juhász Faculty of Education, Institute of Applied Science, University of Szeged, 6725 Szeged, Hungary; galﬁ@jgypk.u-szeged.hu (M.G.); [email protected] (M.R.) 7 Department of Psychiatry, Faculty of Medicine, University of Szeged, 6725 Szeged, Hungary 8 HR-Pharma Ltd., 6726 Szeged, Hungary * Correspondence: [email protected]; Tel.: +36-62-544884; Fax: +36-62-544291 † Renáta Szabó and Rudolf Ménesi contributed equally to this paper as ﬁrst authors. ‡ The author was deceased.

Academic Editor: Béla Juhász Received: 28 January 2019; Accepted: 13 February 2019; Published: 18 February 2019

Abstract: Background: The hypothalamic–pituitary axis by secreting neuropeptides plays a key role in metabolic homeostasis. In light of the metabolic regulation, oxytocin is a potential neuropeptide for therapies against obesity and related disorders. The aim of our study is to measure ghrelin-induced oxytocin secretion in rats and to detect the changes after administration of ghrelin antagonist. Methods: Ghrelin was administrated centrally (intracerebroventricular, i.c.v., 1.0, 10.0, and 100.0 pmol) or systemically (intravenous, i.v., 1.0, and 10.0 nmol). [D-Lys3]-GHRP-6 ghrelin antagonist was injected 15 min before ghrelin injection in a dose of 10.0 pmol i.c.v. and 10.0 nmol i.v. Results: Either i.c.v. or i.v. administration of ghrelin dose-dependently increased the plasma oxytocin concentration. Following pretreatment with the ghrelin antagonist [D-Lys3]-GHRP-6, the high plasma oxytocin level induced by ghrelin was signiﬁcantly reduced. Conclusion: The results indicate that the release of oxytocin is inﬂuenced directly by the ghrelin system. Examination of the mechanism of ghrelin-induced oxytocin secretion is a new horizon for potential therapeutic options.

Keywords: ghrelin; ghrelin antagonist; oxytocin; neuropeptide regulation

1. Introduction The increasing prevalence of energy metabolism disorders and diseases are both a public health issue and clinical problem. It has been established that the energy homeostasis plays a role in

Molecules 2019, 24, 735; doi:10.3390/molecules24040735 www.mdpi.com/journal/molecules Molecules 2019, 24, 735 2 of 8 controlling the synthesis and secretion of various hormones, thus any disturbance in these contributes to the impairment of the metabolic regulation. The central nervous system (CNS), especially the hypothalamus and pituitary, are important to promote the action of the metabolic regulatory mediators and influence, e.g., the appetite [1]. GhrelinMOLECULES is a 2019 28-amino, 24, X FOR acid-containing PEER REVIEW endogenous ligand for the growth hormone secretagogue2 OF 8 receptor 1a (GHS-R1a) linking the CNS with peripheral tissues that regulate food intake and energy contributes to the impairment of the metabolic regulation. The central nervous system (CNS), homeostasis. The expression of ghrelin has been demonstrated in various tissues, such as the stomach, especially the hypothalamus and pituitary, are important to promote the action of the metabolic kidney, pituitaryregulatory gland mediators and, and hypothalamus influence, e.g., [the2]. appetite Previous [1]. studies suggest that ghrelin could contribute to metabolicGhrelin disturbances. is a 28-amino Low acid-containing plasma ghrelin endogenous is associated ligand with for insulinthe growth resistance, hormone hypertension,secretagogue and increasereceptor the risk 1a of (GHS-R1a) type 2 diabetes linking the [3] CNS and metabolicwith peripher syndromeal tissues that [4]. regulate food intake and energy In ourhomeostasis. previous study,The expression we reported of ghrelin that vasopressinhas been demonstrated and oxytocin in secretionvarious tissues, are increased such as followingthe stomach, kidney, pituitary gland and, hypothalamus [2]. Previous studies suggest that ghrelin could the intracerebroventricular (i.c.v.) or intravenous (i.v.) administration of ghrelin in cell cultures of contribute to metabolic disturbances. Low plasma ghrelin is associated with insulin resistance, neurohypophysealhypertension, tissue. and increase The resultsthe risk indicateof type 2 diabetes that vasopressin [3] and metabolic and oxytocinsyndrome release[4]. are influenced directly by theIn ghrelinour previous system, study, and we the reported effects that of ghrelin vasopressin on vasopressin and oxytocin and secretion oxytocin are increased secretion from the neurohypophysealfollowing the intracerebroventricular tissue in rats can occur (i.c.v.) at or the intravenous level of the(i.v.) posterior administration pituitary of ghrelin [5]. in cell In lightcultures of the of neurohypophyseal importance of hypothalamic–pituitary tissue. The results indicate axis that and vasopressin its mediators and oxytocin in metabolic release regulation, are oxytocininfluenced has a pivotal directly role by in the the ghrelin energy system, homeostasis. and the effects Activation of ghrelin of centralon vasopressin and peripheral and oxytocin oxytocin secretion from the neurohypophyseal tissue in rats can occur at the level of the posterior pituitary [5]. receptors also regulates energy expenditure [6]. Oxytocin promotes glucose uptake and stimulates In light of the importance of hypothalamic–pituitary axis and its mediators in metabolic insulin secretionregulation, [ 7oxytocin]. Zhang has et a pivotal al. found role thatin the oxytocin energy homeostasis. improved Activation the lipid of profile central and by loweringperipheral serum low densityoxytocin lipoprotein receptors andalso cholesterolregulates energy levels expenditure [8]. Summarizing [6]. Oxytoc thein promotes results itglucose can be uptake concluded and that hypothalamic–pituitarystimulates insulin pathwayssecretion [7]. and Zhang its neuropeptideset al. found that areoxytocin closely improved linked tothe metaboliclipid profile regulation. by Hence, thelowering examination serum low and density identification lipoprotein of and the cholesterol neuroendocrine levels [8]. processes Summarizing arenecessary the results toit can understand be the exactconcluded effects of that neuropeptides, hypothalamic–pituitary which promote pathways further and studiesits neuropeptides for metabolic are closely changes. linked to metabolic regulation. Hence, the examination and identification of the neuroendocrine processes are In ournecessary previous to understand work we the proved exact thateffects ghrelin of neuropeptides, administration which enhancespromote further oxytocin studies secretion for in neurohypophysealmetabolic changes. cell cultures. Based on our in vitro results, the aim of our current study is to examine theIn effects our previous of centrally work we and proved systemically that ghrelin administrated administration ghrelin enhances on oxytocin oxytocin secretion release in in rats. Furthermore,neurohypophyseal the effect ofcell ghrelin cultures. receptor Based on (GHS-R1a) our in vitro antagonistresults, the aim [D-Lys of our3]-GHRP-6 current study has is been to also measuredexamine on plasma the effects oxytocin of centrally secretion. and systemically administrated ghrelin on oxytocin release in rats. Furthermore, the effect of ghrelin receptor (GHS-R1a) antagonist [D-Lys3]-GHRP-6 has been also 2. Resultsmeasured on plasma oxytocin secretion.

2.1. Effects2. Results of Centrally (i.c.v.) and Systemically (i.v.) Administrated Ghrelin on Oxytocin Secretion Following2.1. Effects the of Centrally i.c.v. administration (i.c.v.) and Systemically of 1 pmol(i.v.) Administrated ghrelin, the Ghrelin plasma on Oxytocin oxytocin Secretion level signiﬁcantly increased. AfterFollowing administration the i.c.v. administration of a higher ghrelin of 1 pmol dose ghrelin, (10 pmol the plasma and 100 oxytocin pmol), level the oxytocinsignificantly secretion was furtherincreased. enhanced, After butadministration no signiﬁcant of a difference higher ghre waslin dose observed (10 pmol between and 100 the pmol), effects the of 10oxytocin or 100 pmol doses. Datasecretion are presented was furtherin enhanced, Figure1 but. no significant difference was observed between the effects of 10 or 100 pmol doses. Data are presented in Figure 1.

Figure 1. Dose–response effects of centrally (i.c.v.) administrated ghrelin on plasma oxytocin release Figure 1. Dose–response effects of centrally (i.c.v.) administrated ghrelin on plasma oxytocin release (expressed(expressed as pg/ml). as pg/ml). Results Results shown shown as as means ± S.E.M.,± S.E.M., n = 10n. Statistical= 10. Statistical significance: signiﬁcance: * p < 0.05 relative * p < 0.05 relative toto the control control group group..

MOLECULES 2019, 24, X FOR PEER REVIEW 3 OF 8

MoleculesAs 2019shown, 24, 735in Figure 2, i.v. injections of ghrelin significantly enhanced the plasma oxytocin3 of 8 MOLECULES 2019, 24, X FOR PEER REVIEW 3 OF 8 values. Both 1 and 10 nmol doses of ghrelin resulted same elevation in oxytocin level. As shown in Figure 2, i.v. injections of ghrelin significantly enhanced the plasma oxytocin As shown in Figure2, i.v. injections of ghrelin signiﬁcantly enhanced the plasma oxytocin values. values. Both 1 and 10 nmol doses of ghrelin resulted same elevation in oxytocin level. Both 1 and 10 nmol doses of ghrelin resulted same elevation in oxytocin level.

Figure 2. Effects of systemically (i.v.) administered ghrelin on oxytocin secretion (expressed as pg/ml).

Results shown as means ± S.E.M., n = 10. Statistical significance: * p < 0.05 relative to the control group. Figure 2. Effects of systemically (i.v.) administered ghrelin on oxytocin secretion (expressed as pg/ml). Figure 2. Effects of systemically (i.v.) administered ghrelin on oxytocin secretion (expressed as pg/ml). 2.2. EffectsResults of shown Centrally as means (i.c.v.)± andS.E.M., Systemicallyn = 10. Statistical (i.v.) Administrated significance: * pGhrelin< 0.05 relativeAntagonist to the on control Oxytocin group. Results shown as means ± S.E.M., n = 10. Statistical significance: * p < 0.05 relative to the control group. Secretion2.2. Effects of Centrally (i.c.v.) and Systemically (i.v.) Administrated Ghrelin Antagonist on Oxytocin Secretion 2.2. Effects of Centrally (i.c.v.) and Systemically (i.v.) AdministratedD 3 Ghrelin Antagonist on Oxytocin TheThe i.c.v.i.c.v. administeredadministered ghrelin ghrelin antagonist antagonist [D-Lys [ -Lys3]-GHRP-6]-GHRP-6 did notdid induce not induce any significant any significant changes Secretion changesin oxytocin in concentrationoxytocin concentration compared compared to the control to group.the control However, group. the However, higher oxytocin the higher levels oxytocin induced levelsby ghrelinThe induced i.c.v. were administeredby significantly ghrelin were decreased,ghrelin significantly antagonist though decr the[eased,D-Lys plasma 3]-GHRP-6though oxytocin the did plasma concentration not induceoxytocin remainedany concentration significant above remainedchangesthe control in above level.oxytocin the Data control concentration are presented level. Data compared in are Figure presented3 to. the incontrol Figure group. 3. However, the higher oxytocin levels induced by ghrelin were significantly decreased, though the plasma oxytocin concentration remained above the control level. Data are presented in Figure 3.

Figure 3. Effects of centrally (i.c.v.) administered ghrelin antagonist [D-Lys3]–GHRP-6 on the Figure 3. Effects of centrally (i.c.v.) administered ghrelin antagonist [D-Lys3]–GHRP-6 on the plasma plasma oxytocin concentration (expressed as pg/mL). Results shown as means ± S.E.M., n = 10. oxytocinStatistical concentration signiﬁcance: (expressed * p < 0.05 relative as pg/mL). to the Results control shown group as and means # p < 0.05± S.E.M., relative n = to 10. the Statistical 10 pmol significance:Figureghrelin-treated 3. Effects * p group. of< 0.05centrally relative (i.c.v.) to the administered control group ghre andlin antagonist # p < 0.05 [D-Lys3]–GHRP-6relative to the 10 pmolon the ghrelin- plasma treatedoxytocin group. concentration (expressed as pg/mL). Results shown as means ± S.E.M., n = 10. Statistical significance:After the i.v. * administrationp < 0.05 relative ofto thethe ghrelincontrol group antagonist, and # changesp < 0.05 relative in the oxytocin to the 10 concentration pmol ghrelin- were not observed.Aftertreated the group. Thei.v. highadministration plasma oxytocin of the levelsghrelin induced antagonist, by ghrelin changes were in fully the blocked,oxytocinand concentration the control werelevel wasnot observed. observed. The Data high are plasma presented oxytocin in Figure levels4. induced by ghrelin were fully blocked, and the controlAfter level the was i.v. observed. administration Data are of presentedthe ghrelin in anFiguretagonist, 4. changes in the oxytocin concentration were not observed. The high plasma oxytocin levels induced by ghrelin were fully blocked, and the control level was observed. Data are presented in Figure 4.

Molecules 2019, 24, 735 4 of 8 MOLECULES 2019, 24, X FOR PEER REVIEW 4 OF 8

Figure 4. Effects of systemically (i.v.) administered ghrelin antagonist on the plasma oxytocin level Figure 4. Effects of systemically (i.v.) administered ghrelin antagonist on the plasma oxytocin level (expressed as pg/mL). Results shown as means ± S.E.M., n = 10. Statistical significance: * p < 0.05 (expressed as pg/mL). Results shown as means ± S.E.M., n = 10. Statistical significance: * p < 0.05 relative to the control group and # p < 0.05 relative to the 10 pmol ghrelin-treated group. relative to the control group and # p < 0.05 relative to the 10 pmol ghrelin-treated group. 2.3. Behavioral Changes at the End of the Experimental Period 2.3. Behavioral Changes at the End of the Experimental Period Available data suggest that ghrelin possesses a dual role in stress and related behavioral disordersAvailable [9] as data well assuggest it can changethat ghrelin feeding possesses behavior a [10dual]. During role in this stress 30-min-experiment, and related behavioral we did notdisorders observe [9] behavioral as well as changesit can change or a significant feeding beha changevior in[10]. the During consumption this 30-min-experiment, of the rats. we did not observe behavioral changes or a significant change in the consumption of the rats. 3. Discussion 3. Discussion A growing body of evidence indicates that metabolic disorders are significantly linked to the dysregulationA growing of body the CNS. of evidence Considering indicates the importance that metabolic of hypothalamic–pituitary disorders are significantly axis linked in metabolic to the regulation,dysregulation the of objective the CNS. of ourConsidering current study the importance was to investigate of hypothalamic–pituitary the basic regulatory axis pathways in metabolic at the siteregulation, of hypothalamus/pituitary the objective of our current [11]. Numerous study was studiesto investigate examine the and basic prove regulatory the effects pathways of oxytocin at the assite a of potential hypothalamus/pituitary therapeutic option [11]. [12 Numerous]. Using an stud animalies examine model, Camerinoand prove reported the effects that of oxytocinoxytocin oras oxytocina potential receptor therapeutic knockout option mice [12]. gained Using weight an animal combined model, with Camerino impaired reported glucose homeostasisthat oxytocin [13 or]. Experimentsoxytocin receptor in rodents knockout and preclinicalmice gained studies weight show combined that exogenous with impaired oxytocin glucose administration homeostasis reduces [13]. caloricExperiments consumption in rodents and and produces preclinical weight studies loss [show14–16 that]. We exogenous used an alternative oxytocin administration way by administrating reduces (i.c.vcaloric or consumption i.v.) of ghrelin and to increaseproduces oxytocin weight loss signaling. [14–16]. We used an alternative way by administrating (i.c.vAs or i.v.) regards of ghrelin the mechanism to increase of oxytocin ghrelin, signaling. we presume that ghrelin stimulates oxytocin release by actingAs directly regards on the oxytocin-producing mechanism of ghrelin, neurons we presume in the hypothalamic that ghrelin stimulates paraventricular oxytocin or supraopticrelease by nucleiacting [directly17,18]. Previous on oxytocin-producing data suggest that i.c.v.neurons administration in the hypothalamic of ghrelin increases paraventricular c-Fos immunoreactivity, or supraoptic whichnuclei shows[17,18]. that Previous ghrelin modulates data suggest neuronal that activity i.c.v. in administration the hypothalamic of paraventricular ghrelin increases nucleus c-Fos [19]. Consequently,immunoreactivity, there which is afunctional shows that link ghrelin between modu ghrelinlates andneuronal oxytocin activity producing in the cells/ hypothalamic oxytocin neuropeptide.paraventricular Olszewski nucleus [1 et9]. al. Consequently, demonstrated there that is oxytocin a functional serves link as between a negative ghrelin feedback and regulatoroxytocin inproducing feeding-related cells/ oxytocin mechanisms neuropeptide. driven by Olszewski ghrelin. Interplayet al. demonstrated of ghrelin that and oxytocin oxytocin serves may limitas a ghrelin-inducednegative feedback excessive regulator level in feeding-related of food intake, mechanisms whereas interactionsdriven by ghrelin. with orexigenicInterplay of peptides ghrelin (e.g.,and oxytocin orexin and may neuropeptide limit ghrelin-induced Y) promote excessive ghrelin-induced level of food food intake, intake [whereas10]. Based interactions on our earlier with observationsorexigenic peptides related to(e.g., neurohypohyseal orexin and neuropepti oxytocinde secretion, Y) promote the stimulatory ghrelin-induced effect betweenfood intake oxytocin [10]. andBased ghrelin on our seems earlier to observations be similar by related i.c.v. to and neur i.v.ohypohyseal administration oxytocin of ghrelin. secretion, The the mode stimulatory of the oxytocin-increasingeffect between oxytocin action and of systemicallyghrelin seems administered to be similar ghrelin by i.c.v. has and not beeni.v. administration fully clarified. Circulatingof ghrelin. ghrelinThe mode cannot of the penetrate oxytocin-increasing the blood–brain action barrier of syst andemically activate administered the hypothalamic ghrelin magnocellular has not been fully cells sinceclarified. the ghrelinCirculating molecule ghrelin is toocannot large penetrate to pass through the blood–brain the blood–brain barrier and barrier. activate It seems the hypothalamic that oxytocin ismagnocellular released directly cells fromsince the ghrelin neurohypophysis. molecule is too We large earlier to reportedpass through that the dispersed blood–brain cell cultures barrier. ofIt neurohypophysisseems that oxytocin isolated is released from directly adult rats from are the capable neurohypophysis. of synthesizing We andearlie releasingr reported oxytocin that dispersed [20,21]. Ourcell cultures observations of neurohypophysis on neurohypophyseal isolated culturesfrom adult proved rats are the capable significant of synthesizing role of pituicytes and releasing in the ghrelin-inducedoxytocin [20,21]. changes Our observations in oxytocin excretion.on neurohypop To verifyhyseal that ghrelincultures influences proved the oxytocin significant secretion, role weof carriedpituicytes out in examinations the ghrelin-induced with a specific changes ghrelin in oxytocin receptor excretion. antagonist. To The verify synthetic that ghrelin antagonistinfluences oxytocin secretion, we carried out examinations with a specific ghrelin receptor antagonist. The synthetic ghrelin antagonist [D-Lys3]-GHRP-6 (His-DTrp-DLys-Trp-DPhe-Lys-NH2) is widely

Molecules 2019, 24, 735 5 of 8

3 [D-Lys ]-GHRP-6 (His-DTrp-DLys-Trp-DPhe-Lys-NH2) is widely applied under various experimental conditions [22]. We observed that the higher plasma oxytocin level induced by ghrelin was significantly decreased by the ghrelin antagonist administered before the ghrelin injection. It is interesting that ghrelin antagonists could also moderate the neurohypophyseal hormone release-increasing effect of ghrelin only if the antagonists are administered before ghrelin treatment [5]; if ghrelin administration precedes application of the antagonists, the ghrelin receptor antagonists prove ineffective: the increase in oxytocin production is not changed at all. In earlier studies [20,21] we observed the same phenomenon in connection with dopamine or serotonin regulation. Our present findings prove that the plasma oxytocin level is increased following ghrelin administration, and that the GHS-R1a ghrelin antagonist can block the enhancement of oxytocin secretion induced by i.c.v. or i.v. administered ghrelin. Both neurohypophyseal and plasma oxytocin secretion by ghrelin is a pivotal step in improving the understanding of oxytocin physiology and potential application. The importance of ghrelin/oxytocin neuropeptides at hypothalamus/pituitary sites promote new horizon for metabolic disorders. Although the basic mechanism of ghrelin-induced oxytocin secretion is verified, further studies are necessary to investigate the potential therapeutic option of oxytocin in special models.

4. Materials and Methods

4.1. Experimental Protocol The experiments were performed on 3- to 4-month-old male Wistar rats, ranging in weight from 180 to 250 g (bred in our animal house; breeding stock from the Laboratory Animal Insitute, Gödöll˝o, Hungary). The animal care and research protocols were in accordance with the guidelines of our university. In our experiment, all efforts were made to minimize the number of animals as well as the animal suffering. Ghrelin was administered i.v. into the lateral tail vein or i.c.v. into the right lateral cerebral ventricle. For i.c.v. administration, the anesthetized rats were cannulated 7 days before the experiment. The animals were ﬁxed in a stereotaxic instrument, a hole was drilled into the right parietal bone, and a cannula (Plastics One, Raonake, VA, USA) was inserted into the right lateral ventricle 0.7 mm anterior and 1.0 mm lateral to the bregma, according to the stereotaxic atlas. The length of the cannula was 4.0 mm, starting from the cranial bone. The cannula was ﬁxed to the surface of the bone with rapidly hardening dental cement. For i.v. administration, the ghrelin dose was 1.0 or 10.0 nmol in 200 µL of saline. For the i.c.v. injection of ghrelin, the dose was 1.0, 10.0, or 100.0 pmol in 10 µL of saline in 1 min, via a Hamilton syringe. [D-Lys3]-GHRP-6 was administered i.c.v. in a dose of 10.0 pmol in 10 µL of saline in 1 min, 15 min before ghrelin administration. For i.v. administration of the ghrelin antagonist, the dose was 10.0 nmol in 200 µL of saline in 1 min, 15 min before ghrelin injection. In the control rats, 10 µL of physiological NaCl solution was administered i.c.v., and 200 µL of NaCl solution was administered i.v. Thirty min following the administrations of ghrelin or ghrelin antagonist, the rats were decapitated and the blood was collected in cooled polystyrene tubes. The position of the i.c.v. cannula was checked by the injection of 1% methylene blue (Reanal, Budapest, Hungary) following coronal section of the brain. If the position of the cannula was not correct, these cases (6.5%) were excluded from the assessment of the results. The experimental protocol of the study is presented in Figure5. Molecules 2019, 24, 735 6 of 8 MOLECULES 2019, 24, X FOR PEER REVIEW 6 OF 8

FigureFigure 5. The5. experimentalThe experimental protocol protocol of the study. of i.c.v.the =study. intracerebroventricular/central i.c.v. = intracerebroventricular/central administration; i.v.administration; = intravenous/systemic i.v. = intravenous/systemic administration. administration. 4.2. Plasma Oxytocin Determination 4.2. Plasma Oxytocin Determination The oxytocin levels were measured by the RIA technique [5]. Synthetic oxytocin served as The oxytocin levels were measured by the RIA technique [5]. Synthetic oxytocin served as reference preparation for radiolabelling and specific oxytocin antibody was used (Bachem, CA, USA). reference preparation for radiolabelling and specific oxytocin antibody was used (Bachem, CA, USA). Reverse-phase chromatography was used to purify the labelled hormone. The standard curves covered Reverse-phase chromatography was used to purify the labelled hormone. The standard curves the range from 1.0 to 128 pg per assay tube. Oxytocin was extracted from supernatants with an Amprep covered the range from 1.0 to 128 pg per assay tube. Oxytocin was extracted from supernatants with C8 minicolumn (Code RPN 1902, Amersham, Buckinghamshire, UK), with a recovery of more than an Amprep C8 minicolumn (Code RPN 1902, Amersham, Buckinghamshire, UK), with a recovery of 95%. The dry residue was redissolved in 125 µL of assay buffer and 50-µL aliquots were used in more than 95%. The dry residue was redissolved in 125 µL of assay buffer and 50-µL aliquots were duplicate for the RIA. The sensitivity of the assay for oxytocin was 1 pg/tube. Oxytocin values are used in duplicate for the RIA. The sensitivity of the assay for oxytocin was 1 pg/tube. Oxytocin values reported in pg/mg protein. The intra- and interassay coefficients of variation proved to be 13.3% and are reported in pg/mg protein. The intra- and interassay coefficients of variation proved to be 13.3% 16.3%, respectively. and 16.3%, respectively. The following compounds were used; rat ghrelin (synthetized in the Department of Medical The following compounds were used; rat ghrelin (synthetized in the Department of Medical Chemistry, University of Szeged, Szeged, Hungary), [D-Lys3]-GHRP-6 (Tocris, Bristol, UK), oxytocin Chemistry, University of Szeged, Szeged, Hungary), [D-Lys3]-GHRP-6 (Tocris, Bristol, UK), oxytocin (oxytocin; Richter, Budapest, Hungary), and oxytocin antibody (Bachem, Torrance, CA, USA). (oxytocin; Richter, Budapest, Hungary), and oxytocin antibody (Bachem, Torrance, CA, USA). 4.3. Protein Measurement 4.3. Protein Measurement A modified Lowry method was used for the determination of total protein [20,23]. A modified Lowry method was used for the determination of total protein [20,23]. 4.4. Statistical Analysis 4.4. Statistical Analysis The data are expressed as means ± S.E.M. of the results for the total number of rats per experimentalThe data are group expressed (GraphPad as means Prism ± S.E.M. 4). Statistical of the results analysis for was the performedtotal number by of using rats theper Tukey–Kramerexperimental multiplegroup (GraphPad comparison Prism test. p4).-values Statistical less than analysis 0.05 were was consideredperformed significantly by using the different. Tukey– Kramer multiple comparison test. p-values less than 0.05 were considered significantly different.

AuthorAuthor Contributions: contribution: Conceptualization, R.S., R.M., F.A.L., andand A.P.;A.P.; DataData curation,curation, R.S.R.S. and and R.M.; R.M.; Formal Formal analysis,analysis, R.S. R.S. and and A.P., A.P., Investigation, Investigation, R.S., R.S., R.M., R.M., A.H.M., A.H.M., Z.S., Z.S., L.D., L.D., M.G., M.G., M.R., M.R., F.A.L.F.A.L. and and A.P., A.P., Methodology, Methodology, A.H.M.,A.H.M., L.D., L.D., G.T., G.T., J.G., J.G., M.G.,M.G., D.B.,D.B., A.J.,A.J., C.V.C.V. andand M.R.,M.R., Resources,Resources, C.V.,C.V., A.P.,A.P., Supervision,Supervision, C.V.C.V. and and A.P; A.P; Visualization, D.B. and K.K.; Writing-original draft, R.M. and F.A.L.; Writing-review& editing, R.S. R.S. and R.M. contributed equally to this paper as first authors.

Molecules 2019, 24, 735 7 of 8

Visualization, D.B. and K.K.; Writing-original draft, R.M. and F.A.L.; Writing-review & editing, R.S. R.S. and R.M. contributed equally to this paper as first authors. Funding: The study was supported by GINOP-2.3.2-15-2016-00062 and the Ministry of Human Capacities, Hungary grant 20391-3/2018/FEKUSTRAT is acknowledged. Furthermore, this work has been supported by the European Union, cofinanced by the European Social Fund EFOP-3.6.2-16-2017-00009. Conflicts of Interest: The authors declare no conflicts of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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