Regulation of the myo-inositol pathway during salinity stress
Many teleosts, including O. niloticus , rely onmyo -inositol as a compatible osmolyte during salinity stress. The link between myo -inositol synthesis and environmental salinity is well established in Oreochromis species (Cramb et al., 2013; Gardell et al., 2013; Kalujnaia, McVee, Kasciukovic, Stewart, & Cramb, 2010; Sacchi, Gardell, Chang, & Kültz, 2014; Sacchi, Li, Villarreal, Gardell, & Kültz, 2013). Myo -inositol is an organic osmolyte which is used to maintain osmotic pressure within the cell to overcome the negative effects of small and charged inorganic ions. Our data are in agreement with the previous literature as they indicate up-regulation of renal myo -inositol-3-phospate synthase (MIPS) during BW acclimation of fish (FC=3.35, p=.042). Interestingly, in O. mossambicus myo -inositol production is consistently increased through up-regulation of IMPase1.1, which is not differentially regulated in O. niloticus kidney (FC=1.001, p=.99, Supplementary Table 2). On the other hand, one of the most highly significant proteins in O. niloticus kidney is down-regulated myo -inositol oxygenase (MIOX, FC=-6.22, p=.0058), which is a key enzyme for the degradation of myo -inositol. MIOX has been shown to be important in determining myo -inositol levels in vivo (Arner et al., 2001). Transcripts of MIOX have been found to be differentially regulated in response to salinity acclimation in ayu (Plecoglossus altivelis ) larvae (Lu, Zhang, Yang, Li, & Chen, 2016) and turbot (Scophthalmus maximus ) (Cui et al., 2020). Nonetheless, MIOX regulation has not been previously noted in Oreochromis species.
The most highly regulated protein in our study is major facilitator superfamily domain-containing protein 4A (MFSD4A) (FC=8.44, p=.001), a transmembrane glucose transporter. Glucose, in addition to being an important source of energy, is the substrate of MIPS (in the form of glucose 6-phosphate), indicating that upregulation of MFSD4A facilitatesmyo -inositol production. Another indication that MFSD4A is linked to myo -inositol production is that it is one of the few proteins that has good correlation between protein and mRNA regulation, along with the other important myo -inositol related proteins MIPS and MIOX (Figure 3).
Our network and pathway analysis of DIA assay data demonstrates that MIOX is connected to many of the other highly salinity-regulated proteins. The most highly enriched KEGG pathway in BW acclimated fish containing significantly regulated proteins is the ascorbate and alderate metabolism pathway. This finding indicates the compensatory upregulation of UGT and ALDH enzymes to replace the products ofmyo -inositol degradation by MIOX. MIOX and ALDH3 (FC=4.65, p=.024) were both found in STRING network 3, and MIOX was also connected to a (non-significant) isoform of UGT in network 2 (Figure 6). ALDH and UGT also function in antioxidant metabolism and detoxification of xenobiotics during environmental pollution.