br Introduction Phoenixin PNX is a

Introduction Phoenixin (PNX) is a recently discovered peptide produced mainly in hypothalamus by proteolytic cleavage of a small integral membrane protein 20 (Smim20) [1]. The most important endogenous active isoforms of PNX are amided peptides, composed of 14 and 20 Mycophenolic acid [2]. Previous data provided evidence that biological effects of PNX are mediated via GPR173 receptor activation [3,4]. Initially, PNX was characterized as a reproductive peptide which regulates pituitary gonadotropin secretion as well as GnRH expression in rats [1]. Further studies showed that PNX is involved in regulation of anxiety, itching sensation, pain, as well as memory [2,[5], [6], [7]]. There is strong evidence indicating that PNX is expressed outside of the central nervous system, such as in the gastrointestinal tract, heart, kidney and pancreatic islets [1,8]. Furthermore, PNX peptide was detected in the humans and rats circulation [[9], [10], [11]]. Interestingly, in humans PNX serum levels positively correlated with BMI [9,10]. These results collectively suggest that PNX may contribute to the regulation of energy homeostasis, thereby influencing body weight. It is well known that the adipose tissue is one of the most pivotal organs involved in regulation of energy homeostasis and body composition. Adipocytes store energy and produce numerous hormones, and metabolites which modulate energy balance as well as glucose and lipid metabolism [12]. Increased number and size of adipocytes which originate from preadipocytes [13], and particularly, their dysfunction, are hallmarks of obesity [14].
Materials and methods
Results
Discussion Our results provide evidence that PNX stimulates adipogenesis in 3T3-L1 and rat primary preadipocytes. First of all, we demonstrated that the PNX precursor Smim20 and the putative receptor - Gpr173 are expressed at mRNA levels in 3T3-L1 as well as in rat primary preadipocytes. Expression levels of Smim20 and Gpr173 mRNA increase during differentiation process, indicating their roles in the physiology of adipogenesis. Furthermore, we found that PNX is secreted from 3T3-L1 and rat primary adipocytes. Using Western blot we confirmed that PNX-14 amide is produced in both tested cells. It should be noticed that in addition to predicted PNX signal, several additional bands were detected. However, very similar Western blot data were recently published by Prinz et al. [8] who studied PNX protein production in the amygdala and hypothalamus and the nucleus of the solitary tract. Using the same antibody, the authors detected PNX peptide, however they also reported detection of several additional protein signals. The authors of this work suggested that anti-PNX-14 antibody may bind to a precursor form of PNX-14 or stable multimers, which may explain additional protein bands. Notably, preincubation of antibody with PNX-14 amide peptide caused a loss of any protein detection which supports this speculation. To address the question about the role of PNX in regulating differentiation of preadipocytes, we assessed the effects of PNX on mRNA expression of several adipogenic transcription factors (Pparγ, C/ebpβ) as well as on Fabp4 (surrogate marker of preadipocytes differentiation into adipocytes) [20]. We found that PNX strongly increased expression of all three genes suggesting its ability to enhance differentiation of 3T3-L1 preadipocytes into mature fat cells. In support of this statement, enhanced expression of proadipogenic genes was accompanied by increased intracellular lipid content in PNX-treated 3T3-L1 preadipocytes. To confirm the physiological relevance of our findings, we studied the effects of PNX on adipogenesis in rat primary preadipocytes. We found that PNX stimulates cell proliferation, lipid accumulation as well as the expression of two differentiation markers (Pparγ, Fabp4) in rat preadipocytes. It should be pointed out that adipogenic effects of PNX in rat primary preadipocytes were less pronounced as compared to that observed in 3T3-L1 preadipocytes. It is difficult to speculate about the potential reason of these discrepant observations. It is well known that C/ebpβ strongly contributes to differentiation of preadipocytes into mature adipocytes [21]. However, expression of C/ebpβ was not altered in PNX-treated rat preadipocytes. A possible explanation regarding the expression of C/ebpβ could be differences in the composition of differentiation medium. Due to well-established protocol [22] rat primary preadipocytes were continuously differentiated in the presence of insulin, T3 and dexamethasone without FCS. By contrast, 3T3-L1 preadipocytes during the last three days were differentiated in the presence of 10% FCS without any additive factors. Therefore, it cannot be excluded that the presence of these three differentiation factors (insulin, T3 and dexamethasone) without FCS in rat preadipocytes or the presence of FCS in the absence of insulin, T3 and dexamethasone in 3T3-L1 cells could impact the effects of PNX on C/ebpβ mRNA levels.