Phoenixin (PNX) is a highly conserved, novel hormone with diverse functions, including hypothalamic control of reproduction, appetite modulation, and regulation of energy metabolism and inflammation. While some functions appear conserved across vertebrates, additional research is required to fully characterize these complex pleiotropic effects. For instance, very little is known about transcriptome level changes associated with PNX exposure, including responses in the hypothalamic–pituitary–gonadal (HPG) axis, which is critical in vertebrate reproduction. In addition, the PNX system may be especially complex in fish, where an additional receptor is likely present in some species. The purpose of this study was to assess hypothalamic and ovarian transcriptomes after PNX-14 administration in female vitellogenic green-spotted puffer (Dichotomyctere nigroviridis). Steroid-related changes were also assessed in the liver and blood plasma. Hypothalamic responses included pro-inflammatory signals such as interleukin 1β, possibly related to gut–brain axis functions, as well as suppression of cell proliferation. Ovarian responses were more widely downregulated across all identified pathways, which may reflect progression to a less transcriptionally active state in oocytes. Both organs shared regulation in transforming growth factor-β and extracellular matrix remodeling (periostin) pathways. Reproductive processes were in general downregulated, but both inhibiting (bone morphogenetic protein 15 and follistatin) and promoting (17-hydroxyprogesterone) factors for oocyte maturation were identified. Select genes involved in reproduction (vitellogenins, estrogen receptors) in the liver were unresponsive to PNX-14 and higher doses may be needed to induce reproductive effects in D. nigroviridis. These results reinforce the complexity of PNX actions in diverse tissues and highlight important roles for this hormone in regulating the immune response, energy metabolism, and cell growth.

Butyrate, a short-chain fatty acid, is utilized by the gut epithelium as energy and it improves the gut epithelial barrier. More recently, it has been associated with beneficial effects on immune and cardiovascular homeostasis. Conversely, tumor necrosis factor alpha (TNFα) is a pro-inflammatory and pro-hypertensive cytokine. While butyrate and TNFα are both linked with hypertension, studies have not yet addressed their interaction in the colon. Here, we investigated the capacity of butyrate to modulate a host of effects of TNFα in primary rodent colonic cells in vitro. We measured ATP levels, cell viability, mitochondrial membrane potential (MMP), reactive oxygen species (ROS), mitochondrial oxidative phosphorylation, and glycolytic activity in colonocytes following exposure to either butyrate or TNFα, or both. To address the potential mechanisms, transcripts related to oxidative stress, cell fate, and cell metabolism (Pdk1, Pdk2, Pdk4, Spr, Slc16a1, Slc16a3, Ppargc1a, Cs, Lgr5, Casp3, Tnfr2, Bax, Bcl2, Sod1, Sod2, and Cat) were measured, and untargeted liquid chromatography–tandem mass spectrometry (LC-MS/MS) was employed to profile the metabolic responses of colonocytes following exposure to butyrate and TNFα. We found that both butyrate and TNFα lowered cellular ATP levels towards a quiescent cell energy phenotype, characterized by decreased oxygen consumption and extracellular acidification. Co-treatment with butyrate ameliorated TNFα-induced cytotoxicity and the reduction in cell viability. Butyrate also opposed the TNFα-mediated decrease in MMP and mitochondrial-to-intracellular calcium ratios, suggesting that butyrate may protect colonocytes against TNFα-induced cytotoxicity by decreasing mitochondrial calcium flux. The relative expression levels of pyruvate dehydrogenase kinase 4 (Pdk4) were increased via co-treatment of butyrate and TNFα, suggesting the synergistic inhibition of glycolysis. TNFα alone reduced the expression of monocarboxylate transporters slc16a1 and slc16a3, suggesting effects of TNFα on butyrate uptake into colonocytes. Of the 185 metabolites that were detected with LC-MS, the TNFα-induced increase in biopterin produced the only significant change, suggesting an alteration in mitochondrial biogenesis in colonocytes. Considering the reports of elevated colonic TNFα and reduced butyrate metabolism in many conditions, including in hypertension, the present work sheds light on cellular interactions between TNFα and butyrate in colonocytes that may be important in understanding conditions of the colon.

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