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  • Finally it is possible that also species

    2020-03-25

    Finally, it is possible that also species differences (rat primary preadipocytes vs mouse 3T3-L1 cells) could contribute to these discrepancies. A recent study suggested that 3T3-L1 rotenone may contain adipocyte precursors with multiple cell lineage characteristics which may contribute to different biological responses to hormonal stimuli as compared to white primary preadipocytes [23]. Proliferation and differentiation of preadipocytes is controlled by multiple protein kinases [24]. There is strong evidence showing that stimuli of AKT phosphorylation enhance preadipocyte growth and induce differentiation of fat precursor cells into mature adipocytes [25,26]. Interestingly, previous data showed that PNX can enhance AKT phosphorylation in the heart of rats [11]. Since AKT signalling is implicated in controlling cell proliferation and differentiation, we studied phosphorylation of AKT in 3T3-L1 cells exposed to PNX. Our data show that PNX is unable to affect AKT phosphorylation in cells incubated with or without differentiation medium. Furthermore, the blocker of AKT phosphorylation LY294002 [27], had no effects neither on PNX-induced cell proliferation nor on PparĪ³ expression. These data suggest that PNX modulates preadipocyte proliferation and differentiation independently on AKT signalling. It is well known that stimuli of cAMP synthesis can enhance cellular differentiation processes [28]. Notably, previously published observations showed that in the mHypoA-GnRH/GFP cell model, PNX stimulates GnRH and kisspeptin mRNA expression via cAMP-dependent mechanisms [4]. Therefore, to study mechanisms by which PNX modulates adipogenesis, we assessed the effects of PNX on cAMP production. Our data clearly show that PNX increases cAMP synthesis in 3T3-L1 cells. However, these effects were detected only in the presence of serum and a differentiation mix containing insulin, dexamethasone and IBMX. Taken together, these data suggest that PNX alone is not able to increase cAMP production. Nevertheless, the effects of PNX on cAMP need to be studied in a more detailed fashion. Studies addressing the role of cAMP signalling in controlling adipogenesis implicated its role in the maturation of adipocytes. It was found that cAMP modulates differentiation process through an interaction with PKA and Epac [28]. However, others showed that activation of Epac enhances differentiation of preadipocytes [29], whereas PKA suppresses this process [30]. Therefore, to study whether PNX potentiates differentiation of 3T3-L1 preadipocytes via a cAMP/Epac-dependent mechanism, we utilized an Epac inhibitor ESI09 [31]. Incubation of 3T3-L1 preadipocytes with ESI09 attenuated PNX-induced PparĪ³ expression. By contrast, ESI09 failed to block PNX-stimulated cell proliferation. Therefore, these data suggest that PNX stimulates differentiation of preadipocytes via a cAMP/Epac-dependent mechanism. However, further studies are required to elucidate mechanism by which PNX may trigger cell growth. Little is known about the role of PNX in controlling energy balance and metabolism. Nevertheless, very recent data revealed that centrally administrated PNX stimulates food intake in rats [32]. Taking into account that adipose tissue is the main energy storage; stimulation of fat tissue formation by PNX seems to be physiologically rational. Interestingly, the recent study in rats showed that at normal diet post-prandial blood levels of PNX were higher when compared to pre-prandial levels [11]. Furthermore, hormonal stimuli of appetite such as ghrelin or orexin were found to induce preadipocytes proliferation or/and differentiation into adipocytes and lipid accumulation [[33], [34], [35]]. Nevertheless, it must be pointed out that adipogenic actions of PNX render this peptide potentially relevant in the context of pathophysiology of overweight/obesity. Interestingly, a previous study showed a positive correlation between PNX levels and BMI in women with polycystic ovary syndrome and with mild cognitive impairment [10]. Positive correlation between BMI and PNX levels was also reported in other patients with mild cognitive impairment [9]. Further studies are needed to elucidate the role and expression of PNX in obese individuals, who do not suffer from any other diseases.