Expression and localization of the neuropeptide phoenixin-14 and its receptor GRP173 in the canine reproductive organs and periovarian adipose tissue.

Phoenixin-14 (PNX-14) is a regulatory neuropeptide encoded by the SMIM20 gene, which has been implicated in the reproductive cycle by modulating the hypothalamic-pituitary-gonadal (HPG) axis. Recently, we showed that PNX-14 is downregulated in bitches with cystic endometrial hyperplasia and pyometra. The objective of this study was to determine the expression of Smim20, PNX-14, and its putative receptor GRP173 in the canine ovary (both healthy and those with ovarian cysts), periovarian adipose tissue (PAT) and in the endometrium during the oestrous cycle. The expression was analysed by RT-qPCR and Western blot. In tissue sections, peptides were localised by immunofluorescent assays, and blood plasma concentrations of PNX-14 were detected by EIA. The results demonstrated increased levels of PNX in bitches in the anestrus groups compared to diestrus animals. The expression of GPR173 increased in PAT during the diestrus phase and endometrial tissue in late diestrus bitches. In the ovary, strong signals of PNX-14 and GPR173 were detected in the luteal and follicular cells. Furthermore, bitches with cystic ovaries were characterised by elevated circulating PNX levels and a significantly higher expression of PNX and GPR173 in gonadal tissues, when compared with healthy animals. Moreover, a positive correlation between PNX and progesterone in the blood of healthy bitches was noted, which changed to a negative correlation in females affected by cystic ovaries. These studies expand the knowledge regarding the expression and localization of the PNX/GRP173 system in canine reproductive organs during physiological and pathological conditions.

The role of phoenixin in the proliferation and migration of ectopic epithelial cells in vitro.

AIM: Endometriosis is one of the most common gynecologic diseases in women of reproductive age. The pathophysiology of endometriosis is still not fully understood. Phoenixin (PNX-14) is a newly discovered neuropeptide that regulates the hypothalamo-pituitary-gonadal (HPG) axis and reproductive functions. Recently, we reported that PNX-14, its precursor protein and receptor were expressed in human endometrium. Moreover, PNX-14 serum levels in endometriosis were reduced. This study aimed to evaluate the in vitro biological functions of physiological PNX-14 concentrations on the ectopic endometrium Z12 cells. METHODS: The proliferation and migration of Z12 cells were assessed using the xCELLigence® RTCA DP system following 72 h of stimulation with 0.05 and 0.2 nM of PNX-14. GPR173 and small integral membrane protein 20 (SMIM20) gene expression was evaluated using quantitative polymerase chain reaction (qPCR) and the protein levels of GPR173 were analyzed using Western blot analysis. RESULTS: PNX-14 at the concentration observed in the serum of patients with endometriosis (0.05 nM) reduced GPR173 and increased SMIM20 expression, while protein levels of GPR173 remained unchanged. Cell proliferation was increased by the 0.02 nM PNX-14- the concentration found in healthy subjects. The 0.2 nM of PNX-14 decreased SMIM20 expression with no change to GPR173 expression and reduced ectopic epithelial cell proliferation during the first 5 h after stimulation. However, at 72 h, the proliferation increased. CONCLUSIONS: This study shows that PNX-14 at endometriosis specific concentration desensitized ectopic epithelium to PNX-14, and increased the expression of SMIM20 to restore the physiological levels of PNX-14.

Levels of spexin and its receptors GALR2 and GALR3 in the hypothalamus and ovary of letrozole-induced polycystic ovary syndrome in rats.

Spexin (SPX) is a newly identified neuropeptide, a natural ligand for the galanin receptors (GALR) 2/3, which is involved in maintaining physiological functions including female reproduction. One of the most common endocrine disorder in reproductive system is polycystic ovary syndrome (PCOS), however the role of SPX in PCOS is still unknown. The objective of this study was to determine the expression of mRNA and peptide levels of SPX and its receptors GALR2/3 in the hypothalamus and ovary (by real time PCR and Western blot) as well as plasma levels of SPX (ELISA) in letrozole - induced PCOS rats. We observed that SPX plasma level does not change in PCOS rats. In the hypothalamus transcript level of Spx and Galr3 were significantly higher in PCOS rats compared to the control, while mRNA of Galr2 and protein expression of GALR2/3 were lower. Moreover, expression of Spx and Galr2/3 mRNA as well as GALR2/3 peptide production were lower in the ovary of PCOS rats. In summary, while our results did not show differences in plasma SPX levels, we observed tissue-dependent significant differences in the SPX/GALR2/3 levels between PCOS and control rats, what indicates possible new mechanisms of PCOS neuroendocrinology.

Canine cystic endometrial hyperplasia and pyometra may downregulate neuropeptide phoenixin and GPR173 receptor expression.

The most common uterine diseases affecting bitches are cystic endometrial hyperplasia (CEH) and pyometra. The neuropeptide phoenixin (PNX) and its receptor (GPR173) are potential key factors involved in the proliferative and inflammatory regulation of the reproductive system in females. This study aimed to evaluate the expression of PNX and GPR173 by qPCR, western blot and immunofluorescence assays in the endometrium of bitches suffering from CEH or pyometra compared to clinically healthy females. Additionally, PNX and progesterone (P4) plasma concentrations were analysed. The results showed a significantly lower expression levels of PNX and GPR173 (mRNA and protein production) in bitches with the CEH or pyometra groups compared to healthy animals. Immunofluorescence staining examination also confirmed a lower concentration of PNX and GPR173 signals in bitches with pathological uteri. Moreover, a lower concentration of PNX blood levels in bitches suffering from pyometra was observed. The PNX concentration was negatively correlated with P4 but only in healthy bitches. These results illustrate that the development of canine uterine disorders may cause a lower expression of PNX and its receptor GPR173.

Phoenixin as a New Target in the Development of Strategies for Endometriosis Diagnosis and Treatment.

Small integral membrane protein 20/phoenixin (SMIM20/PNX) and its receptor GPR173 (G Protein-Coupled Receptor 173) play a role in the regulation of the hypothalamic-pituitary-gonadal axis (HPG). The aim of the study was to determine PNX, FSH, LH, and 17ß-estradiol association in women with endometriosis, and the expression of SMIM20/PNX signaling via GPR173. Serum PNX, FSH, LH, and 17ß-estradiol concentrations were measured by enzyme and electrochemiluminescence immunoassay. SMIM20/PNX and GPR173 expression in the eutopic and ectopic endometrium was assessed by qPCR and immunohistochemistry. Reduced PNX level, increased LH/FSH ratio and elevated 17ß-estradiol concentration were found in patients with endometriosis. No differences in SMIM20 expression were observed between the studied endometria. GPR173 expression was lower in ectopic than in eutopic endometria. SMIM20 expression was mainly restricted to stroma. GPR173 was detected in some eutopic and ectopic stromal cells and in eutopic glandular epithelial cells. Discriminant analysis indicates the diagnostic relevance of PNX and LH/FSH ratio in patients with endometriosis. In women with endometriosis, reduced PNX levels and GPR173 expression may be responsible for HPG axis dysregulation. These new insights may contribute to a better understanding of the pathophysiology of endometriosis and provide the basis for a new strategy for diagnosis and treatment of endometriosis.

The Role of Peptide Hormones Discovered in the 21st Century in the Regulation of Adipose Tissue Functions.

Peptide hormones play a prominent role in controlling energy homeostasis and metabolism. They have been implicated in controlling appetite, the function of the gastrointestinal and cardiovascular systems, energy expenditure, and reproduction. Furthermore, there is growing evidence indicating that peptide hormones and their receptors contribute to energy homeostasis regulation by interacting with white and brown adipose tissue. In this article, we review and discuss the literature addressing the role of selected peptide hormones discovered in the 21st century (adropin, apelin, elabela, irisin, kisspeptin, MOTS-c, phoenixin, spexin, and neuropeptides B and W) in controlling white and brown adipogenesis. Furthermore, we elaborate how these hormones control adipose tissue functions in vitro and in vivo.

Phoenixin: More than Reproductive Peptide.

Phoenixin (PNX) neuropeptide is a cleaved product of the Smim20 protein. Its most common isoforms are the 14- and 20-amino acid peptides. The biological functions of PNX are mediated via the activation of the GPR173 receptor. PNX plays an important role in the central nervous system (CNS) and in the female reproductive system where it potentiates LH secretion and controls the estrus cycle. Moreover, it stimulates oocyte maturation and increases the number of ovulated oocytes. Nevertheless, PNX not only regulates the reproduction system but also exerts anxiolytic, anti-inflammatory, and cell-protective effects. Furthermore, it is involved in behavior, food intake, sensory perception, memory, and energy metabolism. Outside the CNS, PNX exerts its effects on the heart, ovaries, adipose tissue, and pancreatic islets. This review presents all the currently available studies demonstrating the pleiotropic effects of PNX.

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.

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.

Adropin as A Fat-Burning Hormone with Multiple Functions-Review of a Decade of Research.

Adropin is a unique hormone encoded by the energy homeostasis-associated (Enho) gene. Adropin is produced in the liver and brain, and also in peripheral tissues such as in the heart and gastrointestinal tract. Furthermore, adropin is present in the circulatory system. A decade after its discovery, there is evidence that adropin may contribute to body weight regulation, glucose and lipid homeostasis, and cardiovascular system functions. In this review, we summarize and discuss the physiological, metabolic, and pathophysiological factors regulating Enho as well as adropin. Furthermore, we review the literature addressing the role of adropin in adiposity and type 2 diabetes. Finally, we elaborate on the role of adropin in the context of the cardiovascular system, liver diseases, and cancer.

Effects of adropin on proliferation and differentiation of 3T3-L1 cells and rat primary preadipocytes.

Adropin is a protein encoded by Energy Homeostasis Associated (Enho) gene which is expressed mainly in the liver and brain. There is evidence that biological effects of adropin are mediated via GPR19 activation. Animal studies showed that adropin modulates adiposity as well as lipid and glucose homeostasis. Adropin deficient animals have a phenotype closely resembling that of human metabolic syndrome with are obesity dyslipidemia and impaired glucose production. Animals treated with exogenous adropin lose weight, in addition to having reduced expression of lipogenic genes in the liver and fat tissue. While it was shown that adropin may contribute to energy homeostasis and body weight regulation, the role of this protein in controlling fat tissue formation is largely unknown. Thus, in the present study we investigated the effects of adropin on adipogenesis using 3T3-L1 cells and rat primary preadipocytes. We found a low Enho mRNA expression in 3T3-L1 cells and rat primary preadipocytes. Adropin stimulated proliferation of 3T3-L1 cells and rat primary preadipocytes. Stimulation of 3T3-L1 cell proliferation was mediated via ERK1/2 and AKT. Adropin reduced lipid accumulation as well as expression of proadipogenic genes in 3T3-L1 cells and rat preadipocytes, suggesting that this protein attenuates differentiation of preadipocytes into mature fat cells. In summary, these results show that adropin modulates proliferation and differentiation of preadipocytes.

Phoenixin-14 stimulates proliferation and insulin secretion in insulin producing INS-1E cells.

Phoenixin (PNX) is a recently discovered neuropeptide which modulates appetite, pain sensation and neurons of the reproductive system in the central nervous system. PNX is also detectable in the circulation and in peripheral tissues. Recent data suggested that PNX blood levels positively correlate with body weight as well as nutritional status suggesting a potential role of this peptide in controlling energy homeostasis. PNX is detectable in endocrine pancreas, however it is unknown whether PNX regulates insulin biosynthesis or secretion. Using insulin producing INS-1E cells and isolated rat pancreatic islets we evaluated therefore, whether PNX controls insulin expression, secretion and cell proliferation. We identified PNX in pancreatic alpha as well as in beta cells. Secretion of PNX from pancreatic islets was stimulated by high glucose. PNX stimulated insulin mRNA expression in INS-1E cells. Furthermore, PNX enhanced glucose-stimulated insulin secretion in INS-1E cells and pancreatic islets in a time-dependent manner. Stimulation of insulin secretion by PNX was dependent upon cAMP/Epac signalling, while potentiation of cell growth and insulin mRNA expression was mediated via ERK1/2- and AKT-pathway. These results indicate that PNX may play a role in controlling glycemia by interacting with pancreatic beta cells.

Phoenixin-14 stimulates differentiation of 3T3-L1 preadipocytes via cAMP/Epac-dependent mechanism.

Phoenixin-14 (PNX) is a newly discovered peptide produced by proteolytic cleavage of the small integral membrane protein 20 (Smim20). Previous studies showed that PNX is involved in controlling reproduction, pain, anxiety and memory. Furthermore, in humans, PNX positively correlates with BMI suggesting a potential role of PNX in controlling fat accumulation in obesity. Since the influence of PNX on adipose tissue formation has not been so far demonstrated, we investigated the effects of PNX on proliferation and differentiation of preadipocytes using 3T3-L1 and rat primary preadipocytes. We detected Smim20 and Gpr173 mRNA in 3T3-L1 preadipocytes as well as in rat primary preadipocytes. Furthermore, we found that PNX peptide is produced and secreted from 3T3-L1 and rat primary adipocytes. PNX increased 3T3-L1 preadipocytes proliferation and viability. PNX stimulated the expression of adipogenic genes (Pparγ, C/ebpß and Fabp4) in 3T3-L1 adipocytes. 3T3-L1 preadipocytes differentiated in the presence of PNX had increased lipid content. Stimulation of cell proliferation and differentiation by PNX was also confirmed in rat preadipocytes. PNX failed to induce AKT phosphorylation, however, PNX increased cAMP levels in 3T3-L1 cells. Suppression of Epac signalling attenuated PNX-induced Pparγ expression without affecting cell proliferation. Our data show that PNX stimulates differentiation of 3T3-L1 and rat primary preadipocytes into mature adipocytes via cAMP/Epac-dependent pathway. In conclusion our data shows that phoenixin promotes white adipogenesis, thereby may be involved in controlling body mass regulation.

Changes in obestatin gene and GPR39 receptor expression in peripheral tissues of rat models of obesity, type 1 and type 2 diabetes.

BACKGROUND: Obestatin has a role in regulating food intake and energy expenditure, but the roles of obestatin and the GPR39 receptor in obesity and type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively) are not well understood. The aim of the present study was to investigate changes in obestatin and GPR39 in pathophysiological conditions like obesity, T1DM, and T2DM. METHODS: Using rat models of diet-induced obesity (DIO), T1DM and T2DM (n = 14 per group), obestatin, its precursor protein preproghrelin, and GPR39 expression was investigated in tissues involved in glucose and lipid homeostasis regulation. Furthermore, serum obestatin and ghrelin concentrations were determined. RESULTS: Serum obestatin concentrations were positively correlated with glucagon (r = 0.6456; P < 0.001) and visfatin (r = 0.5560; P < 0.001), and negatively correlated with insulin (r = -0.4362; P < 0.05), adiponectin (r = -0.3998; P < 0.05), and leptin (r = -0.4180; P < 0.05). There were differences in GPR39 and preproghrelin expression in the three animal models. Hepatic GPR39 and preproghrelin mRNA expression was greater in T1DM, T2DM, and obese rats than in lean controls, whereas pancreatic GPR39 mRNA and protein and preproghrelin mRNA expression was decreased in T1DM, T2DM, and DIO rats. Higher GPR39 and preproghrelin protein and mRNA levels were found in adipose tissues of T1DM compared with control. In adipose tissues of T2DM and DIO rats, GPR39 protein levels were lower than in lean or T1DM rats. Preproghrelin mRNA was higher in adipose tissues of T1DM, T2DM, and DIO than lean rats. CONCLUSION: We hypothesize that changes in obestatin, GPR39, and ghrelin may contribute to metabolic abnormalities in T1DM, T2DM, and obesity.