GMJ.2020;9:e1767 www.gmj.ir

Received 2019-11-27 Revised 2019-12-22 Accepted 2020-02-03 Influences of Serotonin Hydrochloride on , Ghrelin and KiSS1 Expression

Fariba Mahmoudi 1, Khadijeh Haghighat Gollo 1

1 Faculty of Sciences, University of Mohaghegh Ardabili, Ardabil, Iran

Abstract

Background: Serotonin and stimulates gonadotropin-releasing hormone (GnRH) and luteinizing hormone (LH) release while ghrelin and adiponectin inhibit them. In the present experimental study, the effects of central injection of serotonin were investigated on LH con- centration, KiSS1, adiponectin, and ghrelin genes expression. Materials and Methods: Fifteen Wistar male rats in three groups received saline or serotonin hydrochloride via the third cerebral ventricle. Blood samples were collected via the tail vein. Serum LH concentration and relative expression were evaluated by radioimmunoassay and real-time polymerase chain reaction method, respectively. Results: Serotonin significantly increased the mean serum LH concen- tration and KiSS1 levels compared to the saline group. Serotonin significantly decreased the mean ghrelin and adiponectin genes expression levels compared to the saline group. Conclusion: The serotonergic pathway may have stimulatory effects on hypothalamic kisspeptin synthesis, partly via inhibiting hypothalamic ghrelin and adiponectin neural activity. [GMJ.2020;9:e1767] DOI:10.31661/gmj.v9i0.1767

Keywords: Serotonin; Kisspeptin; Ghrelin; Adiponectin

Introduction dition to the regulation of food intakes, sero- tonin stimulates gonadotropin-releasing hor- everal hypothalamic neuronal circuits act mone (GnRH) and luteinizing hormone (LH) Stogether to control the hypothalamus-pitu- release [5-6]. Kisspeptin is a 54 amino acid itary- gonadal (HPG) axis. Serotonin is a neu- that is coded by of KiSS1 gene rotransmitter that is synthesized from trypto- and shorter products, including kisspeptin-14, phan amino acid by the action of tryptophan 13, and 10 proteolytically cleave of it. All kis- hydroxylase and aromatic amino acid decar- speptins exert their physiological functions boxylase enzymes [1]. In the brain, serotonin via binding to G -coupled , is synthesized mainly in the raphe nucleus GPR54 [7-9]. Kisspeptin and its receptor are of the brainstem and from this site, its neu- widely distributed in the hypothalamic nuclei ral axons project to most parts of the brain, involved in regulating reproduction, including e.g., the hypothalamus [1-2]. Also, in the pe- the arcuate nucleus (ARC) and medial preop- ripheral organs, serotonin and its receptors are tic area (mPOA) [7-9]. GPR54 is extensively expressed in the gut and gonads [3-4]. In ad- expressed on GnRH neurons, and kisspeptin

GMJ  Correspondence to: Fariba Mahmoudi, Faculty of Physiology, Ph. D. Fac- Copyright© 2020, Galen Medical Journal. This is ulty of Sciences, University of Mohaghegh Ardabili, an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International Ardabil, Iran License (http://creativecommons.org/licenses/by/4.0/) Telephone Number: 09144190422 Email:[email protected] Email Address: [email protected] Mahmoudi F, et al. Serotonin Affects KiSS1, Adiponectin, and Ghrelin Serotonin Affects KiSS1, Adiponectin, and Ghrelin Mahmoudi F, et al.

injection is associated with strong stimulation lowing injections via the tail vein. Blood sam- of GnRH/LH release [9-10]. Adiponectin, a ples were centrifuged to 15min at 3000 rpm, 244 amino acid , is synthesized in sev- and the serum stored at –20°C until assayed eral tissues, including the adipose tissue, hy- for LH concentration. Mean serum LH con- pothalamus, and gonads [11]. The physiolog- centration was determined by using the rat LH ical actions of it are mediated by two types of kit and the method of the radioimmunoassay receptors named 1 (Adi- (RIA). Following deep anesthetization by a

poR1) and 2 (AdipoR2), which are extensively mixture of ketamine and xylazine, hypotha- expressed in the lateral and medial hypothal- lamic samples were dissected according to amus gonads. It takes part in the regulation Paxinos & Watson atlas and stored at -80°C. of energy homeostasis, food intakes, cardio- RNA of samples was isolated using the acid vascular protection, and it inhibits GnRH/LH guanidinium thiocyanate-phenol-chloroform secretion [12]. There is a close relationship extraction method according to PureZol man- between metabolic and reproductive disorders ufacturer instruction. The RNA concentration and plasma adiponectin levels [13]. Ghrelin was determined by nanodrop spectrophotom- is a 28 amino acid peptide that is secreted eter, and 1μg of total RNA of each sample by the stomach and central nervous system, was used for reverse transcription with Strand specially hypothalamic nuclei involved in cDNA Synthesis Kit following the manufac- controlling energy balance and reproduction turer’s protocol. The Corbett rotor gene 6000 [14-15]. It stimulates food intakes and growth real-time polymerase chain reaction (PCR) hormone secretion via binding to growth hor- detection system and SYBR Green I kit were mone secretagogues receptor (GHSR-Ia) [14- used for determination the gene expression 15]. It inhibits GnRH/LH hormone secretion) levels. The PCR cycling conditions were as [14-15]. Due to the interference of serotonin, following: first denaturation 95 Cº for 2 min, ghrelin, adiponectin, and kisspeptin in the en- followed by 40 cycles of denaturation at 95 Cº docrine control of LH secretion, in the present for 5 sec, annealing at 54 Cº for 20 sec (ghrelin) study, the effects of serotonin were investigat- and annealing at 60 Cº for 20 sec (adiponectin, ed on serum LH concentration and the relative KiSS1 or GAPDH) and extension at 60 Cº for gene expression of ghrelin, adiponectin, and 25 sec. Specific oligonucleotide sequences for KiSS1 in the hypothalamus of male rats. forward and reverse primers used were: KiSS1 forward 5′-TGATCTCGCTGGCTTCTTG- Materials and Methods GC-3′ and reverse 5′-GGGTTCAGG- GTTCACCACAGG-3′, adiponectin forward: In the present experimental study, male Wis- 5′-AATCCTGCCCAGTCATGAAG-3′ and tar rats were housed in the cages under con- reverse: 5′-CATCTCCTGGGTCACCCT- trolled temperature and light (12h light/ dark TA-3′, ghrelin forward: 5′-AATGCTC- cycle). Animals had free access to food and CCTTCGAT GTTGG-3′ and reverse: water all the time. Following anesthetization 5′-CAGTGGTTACTTGTTAGCTGG-3′ and by a mixture of ketamine and xylazine (ket- GAPDH forward: 5′-AAGTTCAACGGCA- amine 80 mg/kg + xylazine 10 mg/ kg), a 22- CAGTCAAG-3′ and reverse: 5′- CATACT- gauge stainless cannulae was implanted into CAGCACCAGCATCAC-3′. The adiponec- third cerebral ventricle coordinates (AP=- 2.3, tin, ghrelin, KiSS1, and GAPDH amplified ML=0.0, DV=6.5). After surgery, animals products were 214, 98, 132, and 120 base were kept in individual cages for one week. pairs, respectively. The calculation of relative Then fifteen rats in three groups received sa- gene expression levels of the target mRNAs line (3µl) or serotonin hydrochloride (10 or 20 were calculated by the equation 2-ΔΔCT. The µg/3µl). Serotonin hydrochloride (Sigma Al- data were analyzed by SPSS software version drich, USA) was dissolved in distilled water, 16 ( SPSS Inc. USA) One- way ANOVA test and it was injected by a 27- gauge stainless was used to analyze the gene expression and steel injector by using a hamilton micro sy- hormonal data. Post hoc Tukey test was used ringe via third cerebral ventricle at 8:00- 9:00. to compare the significant difference between Blood samples were collected at 60min fol- control and experimental groups. The results

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are presented as mean ± SEM. In all cases, Mean adiponectin gene expression levels de- significance was defined by P≤0.05. creased by 0.17 or 0.56 times following in- jections of 10 or 20µg serotonin compared to Ethical Issue saline. This decrease in 10µg serotonin group All procedures for the maintenance and the was not statistically significant, but in 20µg use of experimental animals were executed serotonin group was statistically significant in with the Guide for the Care and Use of Lab- comparison to the saline group (P≤0.05, Fig- oratory Animals (National Institute of Health ure-2). Injections of 20µg serotonin signifi- Publication No. 80-23, revised 1996. This cantly decreased the mean adiponectin gene research was approved by ethic committee expression levels by 0.39 compared to 10µg of University of Mohaghegh Ardabili (code: serotonin receiving rats (P≤0.05, Figure-2). 94:253). Infusion of 10 or 20µg serotonin significantly decreased the mean ghrelin gene expression Results levels by 0.61 or 0.79 times compared to the saline group, respectively (P≤0.05, Figure-2). Injections of 10 or 20µg serotonin significant- Injections of 20µg serotonin decreased the ly increased the mean serum LH concentra- mean ghrelin gene expression levels by 0.46 tions by 0.31 or 0.65 times compared to saline, compared to 10µg serotonin receiving rats, respectively (P≤0.05, Figure-1). Injections of but this decrease was not statistically signif- 20µg serotonin significantly increased the icant (Figure-2). mean serum LH concentration by 0.25 com- pared to the 10µg serotonin group (P≤0.05, Discussion Figure-1). Injections of 10 or 20µg of sero- tonin significantly increased the mean KiSS1 The results of the present study showed that gene expression levels by 0.42 or 1.17 times the mean serum LH concentration signifi- in comparison to the saline group, respectively cantly increased in serotonin receiving rats in (P≤0.05, Figure-2). Injections of 20µg sero- comparison to the saline group. The present tonin significantly increased the mean KiSS1 data are in accordance with previous studies gene expression levels by 0.52 compared to that established the stimulatory influences 10µg serotonin group (P≤0.05, Figure-2). of the serotonergic pathway on controlling

Figure 1. The effects of serotonin hydrocloride on mean serum LH concentration. *: compared to saline, #: compared to 10µg serotonin (data presented as mean ± SEM, P≤0.05, n=5 in each group).

2 GMJ.2020;9:e1767 GMJ.2020;9:e1767 3 www.gmj.ir www.gmj.ir Mahmoudi F, et al. Serotonin Affects KiSS1, Adiponectin, and Ghrelin Serotonin Affects KiSS1, Adiponectin, and Ghrelin Mahmoudi F, et al.

Figure 2. The effects of serotonin hydrocloride on mean gene expression levels of KiSS1, adiponectin and ghrelin. *: compared to saline, #: compared to 10µg serotonin (data presented as mean ± SEM, P≤0.05, n=5 in each group).

GnRH/LH release [5-6]. It is completely es- hibit the serotonin reuptake [17-19]. It has tablished that the kisspeptin/GPR54 signaling been revealed that these drugs play a crucial pathway is an important stimulatory neuronal role in decreasing plasma ghrelin concentra- pathway upstream GnRH neurons for con- tion and food intakes via activating 5HT2C trolling LH secretion [7-10]. We investigated and 5HT1B receptor subtype of serotonin [2, the effects of third cerebral ventricular injec- 17, 20]. The 5HT2C receptor is expressed tions of serotonin on KiSS1 gene expression in on pro-opiomelanocortin (POMC) neurons vivo. The results demonstrated the stimulato- that are involved in producing alpha-melano- ry effects of serotonin on KiSS1 mRNA levels cyte-stimulating hormone (αMSH), which it compared to saline receiving rats. The present turn is an important stimulatory hormone for data are in agreement with the previous stud- kisspeptin synthesis and GnRH/LH release ies, which demonstrated an interaction be- [21]. Also, it has been shown that serotonin tween serotoninergic and kisspeptin signaling increases the production of αMSH via bind- systems. In zebrafish, a relationship has been ing to 5HT2C [1-2]. The 5HT1B receptor is shown between kisspeptin and serotonergic expressed on (NPY)/agoti- signaling system. So that kisspeptin neurons related peptide(AgRP) neurons of the ARC, modulate the raphe nucleus serotonergic neu- and synthesis of these is suppressed ron activity [16]. To find some mechanisms by binding serotonergic drugs to 5HT1B [1- involved in the regulation of kisspeptin syn- 2]. Previous studies demonstrated a reverse thesis by serotonin, we try to investigate the relationship between αMSH and ghrelin lev- effects of serotonin on gene expression lev- els and a direct relationship between NPY/ els of some hypothalamic , in- AgRP and ghrelin levels [22-23]. So inhib- cluding ghrelin and adiponectin, which act iting NPY/ AgRP or stimulating αMSH syn- upstream of kisspeptin and GnRH neurons. thesis by serotonin may partly be a missing The present results showed that ghrelin gene link for the inhibitory effects of serotonin on expression levels significantly suppressed ghrelin secretion. Also, previous studies have following the central injection of serotonin. shown that ghrelin decreases hypothalamic The results are in line with the effects of con- kisspeptin synthesis [24]. While kisspeptin suming the drugs, which increase serotonin activates αMSH and inactivates neuropeptide secretion such as fenfluramine and m-chlo- Y(NPY) neurons [25]. So down-regulation rophenylpiperazine and the drugs, which in- of ghrelin synthesis following the injection

4 GMJ.2020;9:e1767 www.gmj.ir Serotonin Affects KiSS1, Adiponectin, and Ghrelin Mahmoudi F, et al.

of serotonin might be a possible mechanism results of our lab, inhibition of the synthesis of for stimulatory effects of the serotoninergic hypothalamic adiponectin following injection pathway on hypothalamic KiSS1 gene expres- of serotonin may be partly involved in stim- sion. Also, the present results showed that ulatory effects of the serotoninergic pathway serotonin suppressed the hypothalamic adi- on hypothalamic KiSS1 gene expression and ponectin gene expression levels. The present following increased serum LH concentration. results are in agreement with previous studies that demonstrated the inhibitory effects of se- Conclusion rotonin on adiponectin hormone secretion in male rats [26]. It has been revealed that the The intra-cerebroventricular injections of se- injection of serotonin receptor antagonists, rotonin hydrochloride significantly increased including the 5HT2 receptor, significantly the mean serum LH concentration and hy- increases adiponectin concentration in type 2 pothalamic KiSS1 gene expression levels diabetes, which is accompanied by decreased compared to saline receiving rats. Serotonin plasma levels of adiponectin [27]. Adiponec- hydrochloride significantly decreased the tin receptors are expressed in hypothalamic mean hypothalamic ghrelin and adiponec- nuclei involved in the regulation of repro- tin gene expression levels compared to the duction, and their expression is reported on saline group. The serotonergic pathway may GnRH neuron, GTI-7 cell line, and lactotroph exert stimulatory effects on hypothalamic kis- cells of pituitary [28]. Intracerebroventricular speptin synthesis, partly via inhibiting hypo- injection of adiponectin inhibits GnRH/ LH thalamic ghrelin and adiponectin neural activ- release via activating AMP-activated protein ity. kinase (AMPK) pathway signaling pathway [29]. Also, there is a negative correlation be- Acknowledgment tween adiponectin and kisspeptin signaling pathway so that increased level of adiponectin The authors appreciate to Dr. Homayoun results in a significant decrease of kisspeptin Khazali from Shahid Beheshti University for synthesis [26] or decreased adiponectin levels supplying the instruments. is associated to increased levels of kisspeptin synthesis in polycystic ovary syndrome and Conflict of Interest following increased LH/FSH ratio [30]. Based on previous studies mentioned above and the There is no conflict of interest in this article.

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