Levels of the neuropeptide phoenixin-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue in rat model of polycystic ovary syndrome

https://doi.org/10.1016/j.bbrc.2020.05.101Get rights and content

Highlights

  • PNX-14 serum levels was significantly higher in PCOS rats.

  • PNX-14/GPR173 expression was changed in hypothalamus, ovary and PAT of PCOS rats.

  • Phosphorylation of kinases ERK1/2, Akt and PKA are correlated with PCOS.

Abstract

Phoenixin (PNX) is a newly discovered peptide produced by proteolytic cleavage of a small integral membrane protein 20 (Smim20), which acts as an important regulator of energy homeostasis and reproduction. Since dysfunction of reproduction is characteristic in polycystic ovarian syndrome (PCOS), the role of PNX in pathogenesis of PCOS needs further investigation. The objective of this study was to determine expression of Smim20, PNX-14 and its receptor GRP173 in the hypothalamus, ovary and periovarian adipose tissue (PAT) of letrozole induced PCOS rats. Phosphorylation of extracellular signal-regulated kinase (ERK1/2), protein kinases A (PKA) and B (Akt) were also estimated. We observed that PCOS rats had high weight gain and a number of ovarian cyst, high levels of testosterone, luteinizing hormone and PNX-14, while low estradiol. Smim20 mRNA expression was higher in the ovary and PAT, while PNX-14 peptide production was higher only in the ovary of PCOS rat. Moreover, in PCOS rats Gpr173 level was lower in PAT but at the protein level increased only in the ovary. Depending on the tissues, kinases phosphorylation were significantly differ in PCOS rats. Our results showed higher levels of PNX-14 in PCOS rats and indicated some novel findings regarding the mechanisms of PCOS pathophysiology.

Introduction

Phoenixin (PNX) is a newly discovered peptide produced mainly in hypothalamus by proteolytic cleavage of a small integral membrane protein 20 (Smim20) [1]. This neuropeptide mainly exists in two active isoforms: phoenixin – 14 (PNX-14) and phoenixin – 20 (PNX-20) [1,2]. PNX-14 is a 14 residue peptide and was found in multiple species including humans, rats, mice, pigs and dogs [1,2]. PNX-20 is a 20 residue peptide, an N-terminal extended PNX-14 and differs in one amino acid between the coding region of human, canine or porcine sequences [1]. Moreover, PNX was detected in various tissues, including the hypothalamus, pituitary, heart, gastrointestinal and pancreatic islets [3]. PNX has been linked to metabolic syndrome, different behaviours, sensory perception, the cardiovascular system as well as food intake [3]. An in vitro study has shown that PNX increased preadipocytes proliferation and differentiation into mature adipocytes via cAMP/Epac – dependent pathways [4]. Moreover, PNX stimulated insulin secretion by cAMP/Epac signaling, while cell growth and insulin expression were mediated via extracellular signal-regulated kinase (ERK1/2)- and protein kinase B (Akt)-pathways, indicating that PNX may play a role in controlling glycaemia by interacting with pancreatic beta cells [5].

In recent years effect of PNX has been observed also in reproduction [1,6,7]. In vitro experiments on anterior pituitary cells showed that PNX stimulated gonadotropin secretion by increasing expression of GnRH receptors [1]. Interestingly, most of the reproductive functions of PNX including stimulation of mRNA expression in GnRH neurons, modulation of reproductive genes [8], delaying the onset of estrous [9] were mediated through the G protein-coupled receptor 173 (GRP173); inhibition of GRP173 by siRNA reduced these effects. A recent study showed that PNX is also an intraovarian factor, which stimulates ovarian follicular cells development via the receptor GRP173 and protein kinase A (PKA) by accelerating proliferation of human granulosa cells (Gc) and inducing estradiol (E2) secretion [10]. Moreover, only one study showed higher plasma levels of PNX-14 in reproductive pathology - polycystic ovarian syndrome (PCOS) patients [11].

PCOS is a very common endocrine disorder in women of reproductive age and characterized by ovulatory dysfunction, polycystic ovaries, increased luteinizing hormone (LH) concentrations, insulin resistance and hyperandrogenic symptoms such as hirsutism, acne and menstrual irregularity [12]. PCOS women are frequently overweight or obese with increased risk of type – 2 diabetes, endometrial cancer, impaired glucose tolerance and cardiovascular disease [13]. However, the exact pathophysiology of PCOS remains largely unclear, therefore novel insights into the mechanism of PCOS are imperative to identify potential diagnostic markers and therapeutic targets for this endocrine disease.

The aim of this study was to identify mRNA and protein expressions of Smim20/PNX-14 and GPR173 receptor in the hypothalamus, ovary and periovarian adipose tissue (PAT) as well as plasma PNX-14 levels in control and PCOS rats. Additionally, the phosphorylation of kinases ERK1/2, PKA, Akt were also examined. In our experiments, we used letrozole induced rat model of PCOS. Letrozole, a non-steroidal aromatase inhibitor, blocks conversion of testosterone (T) into E2 and leads to higher levels of androgen [14] thus reflecting reproductive disorders and metabolic abnormalities of clinical PCOS [15].

Section snippets

Animal and ethics statement

Six-week-old female Wistar rats (weighing 176.9 g ± 14.54 g) were purchased from the Faculty of Pharmacy JU Medical College and Mossakowski Medical Research Centre Polish Academy of Sciences (Poland). Animals were housed in controlled conditions of temperature, humidity and light (LD 12/12) with ad libitum availability of food and water. Rats were allowed to acclimatize for one week before treatment. All procedures performed in studies were approved by the 1st Local Ethical Commission on Animal

Results and discussion

PCOS is a very common endocrine disorder in women of reproductive age and it’s characterized by abnormal gonadotropin secretion and androgen production, resulting in chronic anovulation which leads to infertility. Its etiology and pathophysiology has not yet been fully understood, so in our study, we employed a letrozole induced rat model of PCOS, which exhibits hormonal, reproductive and metabolic signs similar to the women PCOS [14,15]. We observed in PCOS rats a significant increase in body

Funding

This work was supported by the Jagiellonian University, Poland (K/ZDS/00008) and 2017/25/N/NZ4/02760 from the National Science Centre of Poland.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

We sincerely appreciate Michel Khoury, PhD, for reviewing the English grammar.

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