Energy homeostasis is is determined by food intake and energy expenditure, which are partly regulated by the cross-talk between central and peripheral hormonal signals. Phoenixin (PNX) is a recently discovered pleiotropic neuropeptide with isoforms of 14 (PNX-14) and 20 (PNX-20) amino acids. It is a potent reproductive peptide in vertebrates, regulating the hypothalamo-pituitary-gonadal axis (HPG). It has been identified as a regulator of food intake during light phase when injected intracerebroventricularly in rats. In addition, plasma levels of PNX also increased after food intake in rats, suggesting that it might have possible roles in energy homeostasis. We hypothesized that gut is a source and site of action of PNX in mice. Immunoreactivity for PNX and its putative receptor, super-conserved receptor expressed in brain (SREB3; also known as the G-protein coupled receptor 173/GPR 173) was found in the stomach and intestine of male C57/BL6 J mice, and in MGN3-1 (mouse stomach endocrine) cells and STC-1 (mouse enteroendocrine) cells. In MGN3-1 cells, PNX-20 significantly upregulated ghrelin (10 nM) and ghrelin-O-acyl transferase (GOAT) mRNAs (1000 nM) at 6 h. In STC-1 cells, it significantly suppressed CCK (100 nM) at 2 h. No effects were found on other intestinal hormones tested (glucagon like peptide-1, glucose dependent insulinotropic polypeptide, and peptide YY). Together, these results indicate that PNX-20 is produced in the gut, and it could act directly on gut cells to regulate metabolic hormones.
Gastrointestinal hormones are major players in the peripheral regulation of energy balance. Among different orexigenic peptides, ghrelin is the only one derived primarily from the stomach or periphery and is released in a pulsatile manner [1,2]. The satiety or anorexigenic signals received by the hypothalamus are termed as meal responsive short-term signals (e.g. cholecystokinin; CCK, glucagon like peptide-1; GLP-1) or long-term signals (e.g. leptin, insulin) that monitors energy stores (Cummings and Overduin, 2007; Monteiro and Batterham, 2017; Wabitsch, 2017). Several lines of research are in progress to identify bioactive peptides that regulate physiological processes. One such newly identified peptide is phoenixin (PNX), which has been identified in 2003 in silico as a C-terminally amidated form of small integral membrane protein 20 (SMIM20) peptide or C4orf52 (chromosome 4; open reading frame 52) [[3], [4], [5]]. Among different isoforms, PNX-14 and PNX-20 are found to be the most prominent ones across species and are highly conserved in vertebrates including human, rodents, bovids, chicken and zebrafish [3,5,6]. G-protein coupled receptor 173 (GPR173), also known as the super conserved receptor expressed in brain (SREB3) is considered to be the putative receptor for this peptide [3,4,7]. Immunohistochemical studies by Enzyme-linked Immuno Assay (ELISA) and RT-qPCR showed that PNX is expressed in tissues including the hypothalamus, heart, kidney, thymus, cerebrum, duodenum, jejunum, ileum, and pancreas of both male and female rats [[3], [4], [5]]. The highest levels of PNX-14-like immunoreactivity in rats have been detected in the hypothalamus, duodenum, jejunum, and ileum respectively [8].
ICV administration of PNX-14 increased food intake in male rats during light phase at 1.7 nmol concentration [9]. This study showed no effect on food intake in the dark phase or during IP administration (dark phase or light phase). This peptide is co-expressed with another novel anorexigenic peptide, nesfatin-1 in the rat hypothalamus [10]. It is also shown that after food intake, the plasma concentration of PNX increases compared to the pre-prandial levels in rats [11]. IP injection of PNX-20 suppressed food intake in male and female zebrafish, and short-term fasting resulted in the downregulation of SMIM20 mRNA expression [12]. PNX-14 and PNX-20 stimulated growth hormone releasing hormone receptor (ghrhr) and growth hormone (gh) expression in pituitary and growth hormone receptor (ghr) expression in liver both in vitro and in vivo in spotted scat [13]. The mechanism of food intake regulation by PNX is not very clear. It is unclear whether PNX acts centrally or peripherally (through vagus nerve or crossing blood brain barrier) and also whether it acts in an autocrine or paracrine manner. GPR173 is also expressed in similar hypothalamic food intake regulating areas of the brain [14,15]. GPR173 was discovered as a receptor that has a G αs alpha subunit coupled to it. This subunit stimulates the adenylate cyclase and finally activates the cAMP dependent pathway. In the presence of PNX-20, the phosphorylation of cAMP response element binding protein (CREB) increases, suggesting an activation of the cAMP/PKA pathway. Overall, it appears that GPR173 and a GPCR-mediated mode of action for PNX-20 exists in mammals. Whether PNX-20 affects important metabolic hormones remain unknown. Based on these information, we hypothesized that mouse gut is a source and site of action of PNX in mice. Our specific objectives were to: a) detect the possible expression of PNX and its putative receptor, SREB3 expression in the gut tissues and cells of mice, and b) determine whether PNX-20 affects the synthesis (at the mRNA level) of metabolic hormones in the gut cells of mice in vitro.
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Mouse stomach ghrelinoma (MGN3-1) cells (a gift from Dr. Hiroshi Iwakura, Kyoto University Medical School, Japan; [16]) were isolated from gastric ghrelin tumors in transgenic mice that were isolated from gastric ghrelin tumors in transgenic mice that expressed the ghrelin promoter linked to Simian virus 40 (SV 40) Tag antigen [17]. This novel ghrelin producing cell line retained three important properties of native ghrelin producing gastric cells that include synthesizing ghrelin in
PNX (Fig. 1A) and SREB3 (Fig. 1B) immunoreactive cells were found to be abundant in the stomach glandular mucosa of male wild type C57/BL6 J mice. In the small intestinal sections from the same mice, the presence of PNX (Fig. 1C, E and G) and SREB3-like (Fig. 1D, F and G) immunoreactivity were relatively more abundant in the mucosa of duodenum (Fig. 1C and D) when compared to the jejunum (Fig. 1E and F) and ileum (Fig. 1G and H). In the small intestine, the immunoreactivity was primarily
PNX is emerging as a regulator of reproduction in mammals [7,15,27,28]. There were a limited number of reports on an orexigenic role for centrally administered PNX-20 on food intake in rats [9,29,30]. Our research provided additional, new information on some novel aspects of PNX-20 in male mice, including its hormone-like actions.
Immunohistochemical studies showed that PNX-14 is expressed in the hypothalamus, duodenum, jejunum and ileum of rats [8]. GPR173/SREB3, the putative receptor that is
The gastrointestinal tract of male mice is a source and site of action of PNX. PNX-20 effects on some of the gut hormones in vitro strongly support a functional role for its putative receptors present in the gut cells. While more information is needed on these aspects, this research provides evidence for PNX-20 as yet another regulator of metabolic hormones. It is possible that PNX-20 indirectly influence physiological processes regulated by these gut hormones. How much of the circulating PNX
Kundanika Mukherjee: conducted the experiments, analyzed the data and prepared the manuscript and revised it. Suraj Unniappan: conceived the idea, provided research funding, assisted in the experiments, helped interpret the data, revised the original manuscript and revised it for resubmission.
This research was supported by funding from the Canadian Institutes of Health Research, Canada Foundation for Innovation, Saskatchewan Health Research Foundation and the University of Saskatchewan Centennial Enhancement Chair in Comparative Endocrinology to SU. KM was supported by trainee stipend from the University of Saskatchewan College of Graduate and Postdoctoral Studies and the Western College of Veterinary Medicine.
